US 2012O141590A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0141590 A1 Malakhov et al. (43) Pub. Date: Jun. 7, 2012

(54) TECHNOLOGY FOR THE PREPARATION OF C07D 307/937 (2006.01) MCROPARTICLES COSB 37/02 (2006.01) COSB 37/6 (2006.01) (76) Inventors: Michael Malakhov, San Francisco, C07C 227/00 (2006.01) CA (US); Fang Fang, San Diego, C07C32.3/58 (2006.01) CA (US) A63L/7088 (2006.01) A6IP3/02 (2006.01) (21) Appl. No.: 13/250,653 A6IP37/02 (2006.01) A6II 47/36 (2006.01) (22) Filed: Sep. 30, 2011 CI2N 9/24 (2006.01) CI2N 9/76 (2006.01) Related U.S. Application Data CI2N 9/36 (2006.01) (63) Continuation of application No. 12/179.520, filed on CI2N 9/22 (2006.01) Jul. 24, 2008, now abandoned. CI2N 7/00 (2006.01) s s CI2N 7/02 (2006.01) (60) Provisional application No. 60/961,872, filed on Jul. C07K I4/765 (2006.01) 24, 2007. (52) U.S. Cl...... 424/490; 530/363; 530/385; 530/350; 530/367:530/322:536/16.8; 536/13.7: 540/336; Publication Classification 536/24.5; 530/328; 530/311; 549/510; 549/465; (51) Int. Cl. 530/354; 536/112:536/103,562/575; 562/573; A 6LX 9/50 (2006.01) 548/497; 562/559; 562/443; 514/44 R: 514/44 A: 514/777; 435/200; 435/213; 435/206: 435/199; C07K I4/435 (2006.01) 435/235.1435/239 C07K I4/8 (2006.01) • us C07K I4/77 (2006.01) (57) ABSTRACT C07K 9/00 (2006.01) C7H 5/234 (2006.01) Microspheres are produced by contacting a solution of a CO7D 499/68 (2006.01) macromolecule or Small molecule in a solvent with an anti C7H 2L/02 (2006.01) Solvent and a counterion, and chilling the solution. The A6 IK 38/08 (2006.01) microspheres are useful for preparing pharmaceuticals, A6 IK 38/3 (2006.01) nutraceuticals, cosmetic products and the like of defined (2006.01) dimensions. US 2012/O 141590 A1 Jun. 7, 2012

TECHNOLOGY FOR THE PREPARATION OF an antisolvent, can generate microparticles in an uncontrolled MICROPARTICLES manner that results in uneven-sized and/or aggregated micro particles. RELATED APPLICATIONS 0006. Accordingly, there is a need for a method for pro 0001. This application claims priority under 35 U.S.C. ducing protein and other macromolecular microparticles, and S119(e) to U.S. provisional application Ser. No. 60/961,872, Small-molecule microparticles, which does not require com entitled TECHNOLOGY FOR THE PREPARATION OF plex or specialized equipment and that produces uniform MICROPARTICLES” to Fang et al. filed Jul. 24, 2007. The sized microparticles for delivery. There further is a need for a Subject matter of the provisional application is incorporated method of producing microparticles of a compound that con in its entirety by reference herein. This application also is tain high concentrations of the compound relative to other related to International PCT Application No. (Attorney Dkt. components of the microparticles, that are stable and main No. 21865-005WO1/6505PC) filed on the same day here tain their activity for long periods of time when stored at with. The subject matter of the PCT application is incorpo ambient temperature, and that do not contain a significant rated by reference herein. amount of inactive compound. There also is a need for a 0002 This application is related to International PCT method of producing microparticles of compounds where Application Serial No. (Attorney Docket No. 21865 Substantially all of the compound present in the starting mate 004W01/6504PC, filed Jan. 24, 2007), and to U.S. applica rial (e.g., a solution of the compound) is recovered in the tion Ser. No. 1 1/657,812, filed Jan. 24, 2007 (Attorney microparticle formulation, with minimal loss. There also is a Docket No. 21865-004001/6504). This application also is need for microparticles containing these properties for related to published U.S. applications Serial Nos. administration, for example, as a therapeutic or nutritional US2005.0004020A1 and US2005O112751 A1. Each of these Supplement, or in a cosmetic product. applications is incorporated by reference herein in its entirety. SUMMARY INCORPORATION BY REFERENCE OF 0007. The methods of making a microparticle, the micro SEQUENCE LISTING FILED particles themselves, combinations, and articles of manufac ELECTRONICALLY ture provided below are characterized by a variety of compo nent ingredients, steps of preparation, and biophysical, 0003. An electronic version of the Sequence Listing is physical, biochemical and chemical parameters. As would be filed herewith, the contents of which are incorporated by apparent to one of skill in the art, the compositions and reference in their entirety. The computer-readable file, cre methods provided herein include any and all permutations ated on Jul. 24, 2008, is 46 kilobytes in size and titled and combinations of the ingredients, steps and/or parameters 21865.005001 SeqList.txt. described below. 0008 Provided herein are methods for producing micro BACKGROUND particles of a compound, which do not require complex or 0004. The preparation and delivery of compounds of inter specialized equipment and that produce uniform-sized micro est in powder or particle form is an area of concentrated particles for delivery. Also provided herein are methods of research and development activity in a variety of industries, producing microparticles of a compound that contain high including the pharmaceutical, nutraceutical and cosmetic concentrations of the compound relative to other components industries. For optimal efficacy, it is desirable to have a uni of the microparticles, that are stable and maintain their activ form formulation of the compound, whether it is a small ity for long periods of time when stored at ambient tempera molecule. Such as a steroid hormone or penicillin antibiotic, ture, and that do not contain a significant amount of inactive or a macromolecule. Such as a protein or nucleic acid. For compound. Also provided are methods of producing micro example, for pulmonary administration of a compound. Such particles of compounds where substantially all of the com as atherapeutic protein, antibiotic or chemotherapeutic agent, pound present in the starting material is recovered in the the compound ideally should be prepared in the form of microparticle formulation, with minimal loss. Also provided discrete microspheres, which are solid or semi-solid particles are methods of producing microparticle containing a carrier having a diameter of between 0.5 and 5.0 microns. It also is that facilitates the formation of microspheres containing the desirable for the microparticles to have as high a content of molecule that is the active agent or therapeutic agent of inter the compound as possible, in a form that maintains its activity est, or promotes stability of the resulting microspheres, or for concentrated delivery and therapeutic efficacy. facilitates transportation of the resulting microsphere to the 0005 Previous methods of producing microparticles or target (cells, tissues, etc.) of interest. In some embodiments, nanoparticles of compounds have involved complex steps, the carrier can be a material. Such as gelatin or dextran, which Such as blending with organic polymers and/or forming a is capable of forming a hydrogel. Further, provided herein are lattice array with polymers; spray drying, spray freeze-drying microparticles containing these properties for administration, or Supercritical fluid antisolvent techniques that use special for example, as a therapeutic or nutritional Supplement, as a ized and complex equipment; or lyophilization followed by diagnostic or in a cosmetic product. pulverization or milling that often results in non-uniform 0009. The methods of making the microparticles of the particles that must further be sorted. Often such methods compounds, including macromolecular microparticles and include processing steps, such as heating, that inactivate the Small-molecule microparticles, the compositions themselves, compounds and compromise their activity (e.g., denaturation combinations and articles of manufacture provided below are of a protein). In addition, Some methods do not provide a characterized by a variety of component ingredients, steps of quantitative recovery of the compound from Solution into the preparation, and biophysical, physical, biochemical and solid microparticle formulation. Other methods, such as chemical parameters. As would be apparent to one of skill in directly precipitating a compound out of Solution by adding the art, the compositions and methods provided herein US 2012/O 141590 A1 Jun. 7, 2012

include any and all permutations and combinations of the about or at 1000 or 1000 to about or at 100,000 or 100,000 ingredients, steps and/or parameters described below. Daltons; about or at 1000 or 1000 to about or at 50,000 or 0010. The methods provided herein can include the steps 50,000 Daltons; about or at 1000 or 1000 to about or at 25,000 of: or 25,000 Daltons; about or at 1000 or 1000 to about or at 0.011 a) adding a counterion to a solution containing the 15,000 or 15,000 Daltons; about or at 1000 or 1000 to about compound in a solvent; or at 10,000 or 10,000 Daltons; about or at 1000 or 1000 to 0012 b) adding an antisolvent to the solution; and about or at 5,000 or 5,000 Daltons; about or at 1000 or 1000 0013 c) gradually cooling the solution to a temperature to about or at 3,000 or 3000 Daltons; or about or at 1000 or below about 25° C., whereby a composition containing 1000 to about or at 2,000 or 2000 Daltons. microparticles of the compound is formed. In the method, 0018. The macromolecule can be a polynucleotide, a steps a), b) and c) can be performed simultaneously, sequen nucleic acid, a polypeptide, a glycopeptide, a protein, a car tially, intermittently, or in any order. bohydrate, a lipid, a fatty acid, a polysaccharide, carbohy 0014. In some examples, the counterion is not a polymer. drate- or polysaccharide-protein conjugate, virus, virus par In further examples, the antisolvent is not a polymer. The ticle, viroid, prion or mixture thereof. In other examples, the temperature at which the steps are performed also can be macromolecule is a hormone, prostaglandin, antibiotic, che altered. In some embodiments, the compound is dissolved in motherapeutic agent, hematopoietic, anti-infective agent, the solvent at a temperature of about or at 30° C. or below antiulcer agent, antiallergic agent, antipyretic, analgesic, prior to step a). In other embodiments, the compound is anti-inflammatory agent, antidementia agent, antiviral agent, dissolved in the solvent at a temperature of about or at 25°C. antitumor agent, antidepressant, psychotropic agents, cardio or below. In one aspect, none of the solutions of steps a)-c) are tonics, diuretic, antiarrhythmic agent, vasodilator, antihyper heated and/or maintained at a temperature above about or at tensive agent, antidiabetic agent, anticoagulant, or choles 30°C. In some examples, the compound in these methods is terol lowering agent. not a protein or polypeptide. 0019. In one embodiment, the macromolecule is conju 0015. In some embodiments, the compounds can be gated to a small molecule. In some embodiments, the Small heated to temperatures of above ambient temperature to dis molecule has a molecular weight of about or at 50 to about or Solve the compound in the solvent/antisolvent system, then at 1000 Daltons. The small molecule can be selected from cooled to a temperature at which microspheres are formed. among haptens, hormones, prostaglandins, antibiotics, che For example, for Some macromolecules and Small molecules, motherapeutic agents, hematopoietics, anti-infective agents, the compound can be heated in solution to about or at 35°C., antiulcer agents, antiallergic agents, antipyretics, analgesics, 37° C., 40° C. 450 C., 50° C., 60° C., 650 C., 70° C., 75° C., anti-inflammatory agents, antidementia agents, antiviral 80° C., 85°C., 90° C.,950 C., 100° C. 1250 C., 150° C. 1750 agents, antitumor agents, antidepressants, psychotropic C., 200° C. or greater, then cooled to a temperature of, for agents, cardiotonics, diuretics, antiarrhythmic agents, vasodi example, about or at 190° C., 170° C., 150° C., 125° C., 100° lators, antihypertensive agents, antidiabetic agents, antico C., 80° C., 75° C., 60° C., 50° C., 40° C., 30° C., 20° C. 150 agulants, and cholesterol lowering agents. For example, the C. or lower, at which the microspheres are formed. Small molecule can be an antibiotic, and can be selected from 0016. The order in which the steps are performed can be among aminoglycosides, ansamycins, carbacephem, carbap varied. For example, steps a) and b) can be performed simul enems, cephalosporins, macrollides, penicillins, quinolones, taneously, sequentially, intermittently, or in any order, fol Sulfonamides and tetracyclines. In instances where the anti lowed by step c). In other examples, steps b) and c) are biotic is an aminoglycoside, the aminoglycoside can be kana performed simultaneously, sequentially, intermittently, or in mycin or tobramycin. In instances where the Small molecule any order, preceded by step a). In a further example, steps a) is an antiviral agent, the antiviral agent can be for treatment of and c) are performed simultaneously. In other embodiments, influenza, parainfluenza or respiratory syncytial virus-medi steps a), b) and c) are performed sequentially in the order: a). ated infections. In some examples, the antiviral agent is Zan thenb), then c). In some embodiments, the counterion and the amivir or oseltamivir phosphate. In embodiments where the compound are identical to one another. In other embodi Small molecule is a chemotherapeutic agent, the chemothera ments, the compound and the counterion are different from peutic agent can be selected from among alkylating agents, one another. In other examples, the counterion and the anti anthracyclines, cytoskeletal disruptors, epothilones, inhibi Solvent are identical to one another. tors of topoisomerase II, nucleotide analogs, platinum-based 0017. The compound can be a small molecule or a mac agents, retinoids and Vinca alkaloids. In some examples, the romolecule. In instances where the compound is a macromol chemotherapeutic agent is a cytoskeletal disruptor, and the ecule, the macromolecule can have a molecular weight of cytoskeletal disruptor is paclitaxel. I other examples, the about or at 1000 or 1000 to about or at five billion or five Small molecule is a prostaglandin. billion Daltons; about or at 1000 or 1000 to about or at one 0020. In some embodiments, the macromolecule in the billion or one billion Daltons; about or at 1000 or 1000 to methods presented herein is a nucleic acid. The nucleic acid about or at 50 million or 50 million Daltons; about or at 1000 can be selected from among DNA, RNA and PNA. In or 1000 to about or at 20 million or 20 million Daltons; about instances where the nucleic acid is RNA, the RNA can be or at 1000 or 1000 to about or at 15 million or 15 million siRNA, snRNA, tRNA or a ribozyme. In some examples, the Daltons; about or at 1000 or 1000 to about or at 10 million or macromolecule is a virus, and the virus is tobacco mosaic 10 million Daltons; about or at 1000 or 1000 to about or at 5 virus. In other embodiments, the macromolecule is a glyco million or 5 million Daltons; about or at 1000 or 1000 to about peptides, and the glycopeptide is Vancomycin. In further or at one million or one million Daltons; about or at 1000 or embodiments, the macromolecule is a peptide. For example, 1000 to about or at 500,000 or 500,000 Daltons; about or at the peptide can be leuprolide or somatostatin. 1000 or 1000 to about or at 300,000 or 300,000 Daltons; about 0021. The solvent used in the methods provided hereincan or at 1000 or 1000 to about or at 200,000 or 200,000 Daltons; be miscible or partially miscible with the antisolvent. The US 2012/O 141590 A1 Jun. 7, 2012

methods provided herein also can contain a further process of 30% to about 50% or 50%, w/w; or about 80% or 80% to separating the microparticles from the solution to remove greater than about 99% or 99%, w/w. components other than the microparticles after step c). In one 0027. The temperature at which the solution is gradually aspect, the composition of this method can consistessentially cooled to can be between about or at 4°C. to about orat-200° of the microparticles containing the compound. In one C.; between about or at 2°C. to about or at -180°C.; between embodiment, the separation is effected by sedimentation or about or at 2°C. to about or at -170° C.; or between about 0° by filtration. In another embodiment, the separation is C. or 0° C. to about -2°C. or -2°C. to from about -150° C. effected by freeze-drying. or -150° C. to about -165° C. or -165° C. 0022. The antisolvent used in the methods provided herein 0028. In some aspects, the resulting composition has a can be selected from among water, buffered solutions, ali shelflife of from about or at one week to about or at 1 month, phatic alcohols, aromatic alcohols, chloroform, polyhydric from about or at 1 month to about or at six months, from about Sugar alcohols, aromatic hydrocarbons, aldehydes, ketones, or at six months to about or at one year, from about or at 1 year esters, ethers, dioxanes, alkanes, alkenes, conjugated dienes, to about or at 2 years, or from about or at 2 years to about or dichloromethane, carbon tetrachloride, dimethylformamide at 5 years at a temperature of about or at 55° C., 50° C., 45° C., (DMF), dimethyl sulfoxide (DMSO), acetonitrile, ethyl 44° C., 42°C., 40°C., 39°C., 38°C., 37° C. or below. acetate, polyols, polyimides, polyimines, polyesters, polyal 0029. In some embodiments, the solution and/or the dehydes and mixtures thereof. For example, the antisolvent is resulting composition further includes an active agent. In an aliphatic alcohol or an aromatic alcohol. In instances embodiments where the resulting composition further where the antisolvent is an aliphatic alcohol, the aliphatic includes an active agent, the active agent can be selected from alcohol can be isopropanol. among antibiotics, chemotherapeutic agents, antidiabetics, 0023. In examples, the counterion used in the methods anticonvulsants, analgesics, antiparkinsons, anti-inflamma provided herein is selected from among an anionic com tories, calcium antagonists, anesthetics, antimicrobials, anti pound, a cationic compound and a Zwitterionic compound. In malarials, antiparasitics, antihypertensives, antihistamines, examples, where the counterion is an anionic compound, the antipyretics, alpha-adrenergic agonists, alpha-blockers, bio anionic compound can be sodium citrate, Sodium sulfate, Zinc cides, bactericides, bronchial dilators, beta-adrenergic block Sulfate, magnesium sulfate, potassium sulfate or calcium Sul ing drugs, contraceptives, cardiovascular drugs, calcium fate. In one aspect, the anionic compoundis Sodium Sulfate. In channel inhibitors, depressants, diagnostics, diuretics, elec other examples, the counterion is selected from among citric trolytes, enzymes, hypnotics, hormones, hypoglycemics, acid, itaconic acid and pivalic acid. In further examples, the hyperglycemics, muscle contractants, muscle relaxants, neo counterion is an amino acid, such as, for example, glycine or plastics, glycoproteins, nucleoproteins, lipoproteins, oph arginine. thalmics, psychic energizers, sedatives, steroids, sympatho 0024. In some embodiments of the methods of making mimetics, parasympathomimetics, tranquilizers, urinary tract microparticles provided herein, the counterion is a polymer, drugs, vaccines, vaginal drugs, nonsteroidal anti-inflamma and the macromolecule is selected from among a polynucle tory drugs, angiotensin converting enzymes, polynucleotides, otide, a nucleic acid, a carbohydrate, a lipid, a fatty acid, a polypeptides, polysaccharides, enzymes, hormones, Vita polysaccharide, carbohydrate- or polysaccharide-protein mins, minerals, and nutritional Supplements. conjugates, a virus, virus particles, viroids, prions and mix 0030 The moisture content of the microparticles formed tures thereof. In one aspect, the polymer is the counterion and in the methods provided herein can be adjusted whereby at the antisolvent. The polymer can be, for example, polyethyl least about 90% or 90% of the activity of the compound is ene glycol (PEG) or polyethyleneimine (PEI). The counterion retained after storage for about six months to about 1 year at used in the methods provided herein also can be a polymer. In a temperature of about 25°C. In other examples, the moisture one example, the polymer is the counterion and the antisol content of the microparticles is adjusted whereby at least vent. The polymer can be, for example, polyethylene glycol about 90% of the microparticles are not aggregated after (PEG) or polyethyleneimine (PEI). storage for about six months to about 1 year at a temperature 0025. The pH of the solution used in the methods provided of about 25°C. In some aspects, the moisture content of the herein can be from about 4.0 or 4.0 to about 9.0 or 9.0; from microparticles is from about or at 0.01% to about or at 20%: about 4.0 or 4.0 to about 8.0 or 8.0; from about 4.5 or 4.5 to from about or at 0.05% to about or at 15%; from about or at about 7.5 or 7.5; or from about 5.0 or 5.0 to about 7.0 or 7.0. 0.1% to about or at 10%; from about or at 0.2% to about or at 0026. The microparticles formed in the methods provided 5%; from about or at 6% to about or at 12%; or from about or herein can be obtained by precipitation, by phase separation at 7% to about or at 10.5%. or by colloid formation. The resulting microparticle compo 0031. In some embodiments of the methods provided sition can further contain acid-resistant coating agents, pro herein, the concentration of counterion added to the Solution tease-resistant coating agents, enteric coating agents, bulking is from about or at 0 mM or 0 mM to about or at 100 mM or agents, excipients, inactive ingredients, stability enhancers, 100 mM; from about or at 0 mMor0mMto about or at 50 mM taste and/or odor modifiers or masking agents, vitamins, Sug or 50 mM; from about or at 0 mM or 0 mM to about or at 20 ars, therapeutic agents, anti-oxidants, immuno-modulators, mM or 20 mM, from about or at 0 mM or 0 mM to about or at trans-membrane transport modifiers, anti-caking agents, chi 10 mM or 10 mM, about or at 1 mM or 1 mM to about or at 5 tosans or flowability enhancers. In some examples, the mM or 5 mM, or is about or at 2 mM. amount of compound in the microparticles relative to the total 0032. The gradual cooling of the solution in the methods amount of compound in the solution of step a) is about 5% or provided herein can be effected by chilling. In other embodi 5% to greater than about 99% or 99%, w/w; is about 5% or 5% ments, the gradual cooling is by an endothermic reaction. In to about 20% or 20%, w/w; about 10% or 10% to about 85% Some aspects, the gradual cooling is at a rate of from about or or 85%, w/w; about 20% or 20% to about 60% or 60%, w/w: at 0.01° C./min or 0.01° C./min to about or at 20° C./min or about 25% or 25% to about 55% or 55%, w/w; about 30% or 20°C/min: from about or at 0.05°C/min or about or at 0.1° US 2012/O 141590 A1 Jun. 7, 2012

C./min to about or at 10° C./min or about or at 15° C./min: antitumor agents, antidepressants, psychotropic agents, car about or at 0.2° C./min to about or at 5°C/min: about or at diotonics, diuretics, antiarrhythmic agents, vasodilators, anti 0.5°C./minto about or at 2°C./min: or at a rate of about or at hypertensive agents, antidiabetic agents, anticoagulants, and 1° C./min. cholesterol lowering agents. 0033. In one embodiment, the size of the size of the micro 0037. In one aspect, the small compound is selected from particles is from about or at 0.001 um or 0.001 um to about or among hormones, prostaglandins, antibiotics, chemothera at 50 um or 50 um; about or at 0.3 um or 0.3 um to about or at peutic agents, hematopoietics, anti-infective agents, antiulcer 30 um or 30 um; about or at 0.5um or 0.5um to about or at 10 agents, antiallergic agents, antipyretics, analgesics, anti-in um or 10 um; about or at 0.5 um or 0.5 um to about or at 5.0 flammatory agents, antidementia agents, antiviral agents, um or 5.0 um; about or at 1.0 um or 1.0 um to about or at 5.0 antitumor agents, antidepressants, psychotropic agents, car um or 5.0 um; or from about or at 1.0 um to about or at 2.0, 3.0, diotonics, diuretics, antiarrhythmic agents, vasodilators, anti 4.0 or 5.0 um. hypertensive agents, antidiabetic agents, anticoagulants, and 0034. Also provided herein are compositions containing cholesterollowering agents. In embodiments where the Small microparticles of a compound and a counterion, wherein the molecule is an antibiotic, the antibiotic can be selected from compound and the counterion are different from one another. among aminoglycosides, ansamycins, carbacephem, carbap In some embodiments, the compound is a macromolecule enems, cephalosporins, macrollides, penicillins, quinolones, with a molecular weight of about or at 1000 or 1000 to about Sulfonamides and tetracyclines. For example, the antibiotic is or at five billion or five billion Daltons; about or at 1000 or a penicillin or a tetracycline. In other examples, the antibiotic 1000 to about oratone billion or one billion Daltons; about or is an aminoglycoside, such as, for example, kanamycin or at 1000 or 1000 to about or at 50 million or 50 million tobramycin. In other aspects, the compound is an antiviral Daltons; about or at 1000 or 1000 to about or at 20 million or agent. In Such instances, the antiviral agent can be for treat 20 million Daltons; about or at 1000 or 1000 to about or at 15 ment of influenza, parainfluenza, or respiratory syncytial million or 15 million Daltons; about or at 1000 or 1000 to virus-mediated infections. For example, the antiviral agent about or at 10 million or 10 million Daltons; about or at 1000 can bezanamivir or oseltamivir phosphate. In further embodi or 1000 to about or at 5 million or 5 million Daltons; about or ments, the compound is a chemotherapeutic agent. Where the at 1000 or 1000 to about or at one million or one million compound is a chemotherapeutic agent, the chemotherapeu Daltons; about or at 1000 or 1000 to about or at 500,000 or tic agent can be selected from among alkylating agents, 500,000 Daltons; about or at 1000 or 1000 to about or at anthracyclines, cytoskeletal disruptors, epothilones, inhibi 300,000 or 300,000 Daltons; about or at 1000 or 1000 to about tors of topoisomerase II, nucleotide analogs, platinum-based or at 200,000 or 200,000 Daltons; about or at 1000 or 1000 to agents, retinoids and Vinca alkaloids. In some examples, the about or at 100,000 or 100,000 Daltons; about or at 1000 or chemotherapeutic agent is a cytoskeletal disruptor, Such as, 1000 to about or at 50,000 or 50,000 Daltons; about or at 1000 for example, paclitaxel. In still further examples, the com or 1000 to about or at 25,000 or 25,000 Daltons; about or at pound is a prostaglandin. 1000 or 1000 to about or at 15,000 or 15,000 Daltons; about 0038. In some embodiments, the macromolecule in the or at 1000 or 1000 to about or at 10,000 or 10,000 Daltons; compositions provided herein is a nucleic acid. The nucleic about or at 1000 or 1000 to about or at 5,000 or 5,000 Daltons; acid can be selected from, for example, among DNA, RNA about or at 1000 or 1000 to about or at 3,000 or 3000 Daltons; and PNA. In instances where the nucleic acid is RNA, the or about or at 1000 or 1000 to about or at 2,000 or 2000 RNA can be selected from among siRNA, snRNA, tRNA and Daltons. ribozymes. In some examples, the RNA is siRNA. In other 0035. In some examples, the compound in the composi embodiments, the macromolecule in the compositions pro tion is a small molecule. The Small molecule can have vided herein is a virus. For example, the macromolecules can molecular weight of about or at 50 to about or at 1000 Dal be a tobacco mosaic virus. In other aspects, the macromol tons. In examples where the compound in the composition is ecule is a glycopeptides, such as, for example, Vancomycin. a macromolecule, the macromolecule can selected from In further aspects, the macromolecule is a peptide. The pep among a polynucleotide, a nucleic acid, a polypeptide, a tide can be, for example, leuprolide or somatostatin. glycopeptide, a protein, a carbohydrate, a lipid, a fatty acid, a 0039. The compound in the compositions provided herein polysaccharide, carbohydrate- or polysaccharide-protein can be water-insoluble. The counterion can be selected from conjugates, virus, virus particles, viroids, prions and mixtures among an anionic compound, a cationic compound and a thereof. In some embodiments, the macromolecule is selected Zwitterionic compound. In instances where the counterion is from among hormones, prostaglandins, antibiotics, chemo an anionic compound, the anionic compound can be sodium therapeutic agents, hematopoietics, anti-infective agents, citrate, sodium sulfate, Zinc sulfate, magnesium sulfate, antiulcer agents, antiallergic agents, antipyretics, analgesics, potassium sulfate and calcium sulfate. In some examples, the anti-inflammatory agents, antidementia agents, antiviral anionic compound is sodium sulfate. In other examples, the agents, antitumor agents, antidepressants, psychotropic counterion is selected from among citric acid, itaconic acid agents, cardiotonics, diuretics, antiarrhythmic agents, vasodi and pivalic acid. In a further aspect, the counterion is an lators, antihypertensive agents, antidiabetic agents, antico amino acid, such as, for example, glycine or arginine. In other agulants, and cholesterol lowering agents. aspects, the counterion is polyethylene glycol (PEG) or poly 0036. In some embodiments, the macromolecule in the ethyleneimine (PEI). composition is conjugated to a small molecule. In Such 0040. The resulting microparticle compositions provided instances, the Small molecule is selected from among hap herein can further contain acid-resistant coating agents, pro tens, hormones, prostaglandins, antibiotics, chemotherapeu tease-resistant coating agents, enteric coating agents, bulking tic agents, hematopoietics, anti-infective agents, antiulcer agents, excipients, inactive ingredients, stability enhancers, agents, antiallergic agents, antipyretics, analgesics, anti-in taste and/or odor modifiers or masking agents, vitamins, Sug flammatory agents, antidementia agents, antiviral agents, ars, therapeutic agents, anti-oxidants, immuno-modulators, US 2012/O 141590 A1 Jun. 7, 2012

trans-membrane transport modifiers, anti-caking agents, chi um to about 5.0 um or 5.0 um; from about 1.0 um or 1.0 um to tosans or flowability enhancers. In some aspects, the compo about 5.0 um or 5.0 um; or from about 1.0 um to about 2.0, sition has a shelflife of from about or at one week to about or 3.0, 4.0 or 5.0 um. at 1 month, from about or at 1 month to about or at six months, 0046. Also provided herein are articles of manufacture from about or at six months to about oratone year, from about containing the composition provided herein, a packaging or at 1 year to about or at 2 years, or from about or at 2 years material for the composition and a label that indicates that the to about or at 5 years at a temperature of about or at 55°C., 50° composition is for a therapeutic, nutraceutical or cosmetic C., 45° C., 44° C., 42°C., 40° C., 39° C., 38°C., 37° C. or indication. In some examples, the composition used in the below. article is for a therapeutic indication, Such as, for example, cancer, influenza, parainfluenza or respiratory disorders. The 0041. The compositions provided herein also can contain article of can further contain an inhaler for pulmonary admin an active agent. The active agent can be selected from among istration of the composition. In some embodiments, the antibiotics, chemotherapeutic agents, antidiabetics, anticon inhaler is a dry powder inhaler, a metered dose inhaler or an Vulsants, analgesics, antiparkinsons, anti-inflammatories, electrostatic delivery device. calcium antagonists, anesthetics, antimicrobials, antimalari 0047 Provided herein are methods of preventing or treat als, antiparasitics, antihypertensives, antihistamines, anti ing an infectious disease, by administering a therapeutically pyretics, alpha-adrenergicagonists, alpha-blockers, biocides, effective amount of the composition provided herein to a bactericides, bronchial dilators, beta-adrenergic blocking Subject. In some aspects, the infectious disease is selected drugs, contraceptives, cardiovascular drugs, calcium channel from among arboviral infections, botulism, brucellosis, can inhibitors, depressants, diagnostics, diuretics, electrolytes, didiasis, campylobacteriosis, chickenpox, chlamydia, chol enzymes, hypnotics, hormones, hypoglycemics, hyperglyce era, coronovirus infections, staphylococcus infections, cox mics, muscle contractants, muscle relaxants, neoplastics, gly sackie virus infections, Creutzfeldt-Jakob disease, coproteins, nucleoproteins, lipoproteins, ophthalmics, psy cryptosporidiosis, cyclospora infection, cytomegalovirus chic energizers, sedatives, Steroids, sympathomimetics, infections, Epstein-Barr virus infection, dengue fever, diph parasympathomimetics, tranquilizers, urinary tract drugs, theria, ear infections, encephalitis, influenza virus infections, vaccines, vaginal drugs, nonsteroidal anti-inflammatory parainfluenza virus infections giardiasis, gonorrhea, Haemo drugs, angiotensin converting enzymes, polynucleotides, philus influenzae infections, hantavirus infections, viral hepa polypeptides, polysaccharides, enzymes, hormones, Vita titis, herpes simplex virus infections, HIV/AIDS, helico bacter infection, human papillomavirus (HPV) infections, mins, minerals, and nutritional supplements. infectious mononucleosis, legionellosis, leprosy, leptospiro 0042. The amount of compound in the microparticles of sis, listeriosis, lyme disease, lymphocytic choriomeningitis, the compositions provided herein can be from about or at malaria, measles, marburg hemorrhagic fever, meningitis, 0.1% to about or at 99% or greater, w/w; from about or at monkeypox, mumps, mycobacteria infection, mycoplasma 0.2% to about or at 95% or greater, w/w; from about or at infection, norwalk virus infection, pertussis, pinworm infec 0.5% to about or at 90% or greater, w/w; from about or at 1% tion, pneumococcal disease, Streptococcus pneumonia infec to about or at 85% or greater, w/w; from about or at 2% to tion, Mycoplasma pneumoniae infection, Moraxella about or at 80% or greater, w/w; from about or at 5% to about catarrhalis infection, Pseudomonas aeruginosa infection, or at 75% or greater, wfw; from about 65% to about 90% w/w: rotavirus infection, psittacosis, rabies, respiratory syncytial from about 70% to about 85%, 86%, 87%, 88%, 89% or 90% virus infection, (RSV), ringworm, rocky mountain spotted w/w; or from about 90% to about 99% w/w. fever, rubella, salmonellosis, SARS, scabies, sexually trans 0043. In some aspects, the moisture content of the micro mitted diseases, shigellosis, shingles, sporotrichosis, Strepto particles is adjusted whereby at least about 90% or 90% of the coccal infections, syphilis, tetanus, trichinosis, tuberculosis, activity of the compound is retained after storage for about or tularemia, typhoid fever, viral meningitis, bacterial meningi at six months to about or at 1 year at a temperature of about tis, west nile virus infection, yellow fever, adenovirus-medi 25°C. In some embodiments, the amount of counterion in the ated infections and diseases, retrovirus-mediated infectious microparticles is from about 0.01% or 0.01% to about 60% or diseases and yersiniosis Zoonoses. For example, the infec 60% w/w; from about 0.5% or 0.5% to about 50% or 50% tious disease can be influenza, parainfluenza, respiratory syn w/w; from about 1% or 1% to about 2% or 2% w/w; from cytial virus. about 0.01% or 0.01% to about 20% or 20% w/w; from about 0048. The methods of treatment can be administered by 0.05% or 0.05% to about 15% or 15% w/w; from about 0.1% oral, intravenous, intranasal, parenteral, Subcutaneous, trans or 0.1% to about 10% or 10% w/w; or from about 0.2% or dermal, topical, intraarticular, intramuscular or inhalation 0.2% to about 5% or 5% w/w. administration of the composition. 0044. In one aspect, the moisture content of the micropar 0049 Provided herein also are methods making micropar ticles is from about 6% or 6% to about 12% or 12%. In another ticles of siRNA, which includes the steps of: aspect, the moisture content of the microparticles is from 0050 (a) adding an antisolvent to a solution of siRNA in about 7% or 7% to about 10.5% or 10.5%. an aqueous solvent; and 0045. The compositions provided herein can be for inges 0051 (b) gradually cooling the solution to a temperature tion, inhalation, oral administration, intravenous, intranasal, below about 25° C., whereby a composition containing parenteral, pulmonary, Subcutaneous, ophthalmic or intra microparticles of siRNA is formed, and steps (a) and (b) are muscular administration. In one aspect, the size of the micro performed simultaneously, sequentially, intermittently, or in particles of the compositions provided herein is from about any order. 0.001 um or 0.001 um to about 50 um or 50 um; from about 0.052 The method can further include a step (c), adding a 0.3 um or 0.3 um to about 30 um or 30 um; from about 0.5um counterion, where steps (a), (b) and (c) are performed simul or 0.5um to about 10um or 10 um; from about 0.5um or 0.5 taneously, sequentially, intermittently, or in any order. US 2012/O 141590 A1 Jun. 7, 2012

0053. In some examples, the antisolvent used in the meth portions by weight, and is characterized by a chemical for ods making microparticles of siRNA is isopropanol. In some mula. The compound can be an inorganic compound, which examples, the solvent is water. as used herein is a compound that generally does not contain 0054 Also provided herein are compositions that include carbon-carbon bonds, or the compound can be an organic microparticles of siRNA. In some examples, the composition compound, which generally is characterized by the presence also contains a counterion. of carbon and hydrogen, and can additionally contain hetero 0055 Also provided herein are methods of making micro atoms, such as nitrogen, oxygen, halogens and other Such particles of a virus, which includes the steps of: atoms. Examples of inorganic compounds, discussed else 0056 (a) adding an antisolvent to a solution of virus in an where herein, include alkali and alkaline earth metal com aqueous solvent; and pounds and salts and other derivatives thereof, transition 0057 (b) gradually cooling the solution to a temperature metal compounds, including coordination compounds and below about 25° C., whereby a composition containing salts and other derivatives thereof inorganic polymers. Such microparticles of a virus is formed, where steps (a) and (b) are as polysiloxanes, and other Such compounds known to those performed simultaneously, sequentially, intermittently, or in of skill in the art. Examples of organic compounds, discussed any order. elsewhere herein, include aliphatic, aromatic and alicyclic 0058. The method also can include a step (c), adding a alcohols, aldehydes, carboxylic acids, esters, ketones, ethers, counterion, where steps (a), (b) and (c) are performed simul amines, amides, lactams, polymers thereof, and other Such taneously, sequentially, intermittently, or in any order. In compounds known to those of skill in the art. Some examples, the antisolvent used in the methods making 0067. The term compound, as used herein, also refers to microparticles of a virus is isopropanol. assemblies of inorganic and/or organic compounds, including 0059 Also provided herein are methods of making micro macromolecular assemblies Such as phages and viruses. particles of a virus, which includes the steps of: 0068 A compound as used herein, whether inorganic or 0060 (a) adding a counterion to a solution of virus in an organic, can be a macromolecule or a small molecule. The aqueous solvent; and term "macromolecule' is used herein in the sense that is 0061 (b) gradually cooling the solution to a temperature understood by those of skill in the art, and generally refers to below about 25°C., a naturally occurring or chemically synthesized organic or 0062 whereby a composition containing microparticles inorganic molecule that is greater than or equal to about a of a virus is formed, where steps (a) and (b) are performed 1000 Daltons to about or greater than 1, 2, 3, 5, 7, 10 or more simultaneously, sequentially, intermittently, or in any order. trillion Daltons. A "macromolecule' as used herein includes 0063. The method also can include a step (c), adding an a molecule containing two or more monomeric Subunits, or antisolvent, wherein steps (a), (b) and (c) are performed derivatives thereof, which are linked by a covalent bond, an simultaneously, sequentially, intermittently, or in any order. ionic bond, or other chemical interactions, such as hydrogen In some embodiments, the antisolventis isopropanol. In other bonding, ionic pairing, base pairing or pairing between embodiments, the solvent is water. charges formed by charge polarization. The monomeric Sub 0064. Also provided herein are compositions containing units can be different from one another, or identical to one microparticles of a virus. Such compositions also can contain another, and, in some embodiments, can form a polymer. The a counterion. In some aspects, the virus is tobacco mosaic polymers can be inorganic polymers, such as silicones, pol virus. ysilanes, polygermanes, polyStannanes or polyphospa haZenes, organic polymers. Such as polyethylene or poly DETAILED DESCRIPTION thene, polypropylene, nylon, teflon, polystyrene, polyesters, polymethylmethacrylate, polyvinylchloride or polyisobuty A. Definitions lene, or biological polymers, such as polysaccharides, poly 0065. Unless defined otherwise, all technical and scien nucleotides and polypeptides. A macromolecule also refers to tific terms used herein have the same meaning as is commonly a molecule that, regardless of whether it has more than one understood by one of skill in the art to which the invention(s) Subunit and/or is a polymer, can form tertiary and/or quater belong. All patents, patent applications, published applica nary structure. Examples of macromolecules include a poly tions and publications, Genbank sequences, websites and nucleotide, a nucleic acid molecule including DNA, RNA, other published materials referred to throughout the entire including siRNA, snRNA, tRNA, antisense RNA, and disclosure herein, unless noted otherwise, are incorporated by ribozymes, peptide nucleic acid (PNA), a polypeptide. Such reference in their entirety. In the event that there area plurality as leuprolide and somatostatin, glycopeptides, such as van of definitions for terms herein, those in this section prevail. comycin, a protein, a carbohydrate, or a lipid, orderivatives or Where reference is made to a URL or other such identifier or combinations thereof, for example, a nucleic acid molecule address, it understood that such identifiers can change and containing a peptide nucleic acid portion or a glycoprotein, particular information on the internet can come and go, but respectively. Examples of macromolecules further include equivalent information can be found by searching the inter macromolecular assemblies, for examples, viruses, virus par net. Reference thereto evidences the availability and public ticles, phages, viroids, prions and combinations and conju dissemination of Such information. gates thereof. 0066. The term “molecule' is used interchangeably herein 0069. The term “macromolecule' as used herein also is with “compound, and refers to a naturally occurring or intended to encompass all molecules that are within the scope chemically synthesized entity containing two or more atoms of the description above and have a function, including mac or ions linked by a covalent bond. The atoms or ions can romolecules having a biological function, such as a nucleic belong to the same chemical element, or they can belong to acid, peptide, protein, hormone, cytokine, chemokine, etc., different elements. A molecule or compound as used herein macromolecules having a therapeutic function, Such as a contains the composite elements in definite, unvarying pro drug, macromolecules having a nutraceutical function, Such US 2012/O 141590 A1 Jun. 7, 2012

as a nutritional Supplement, and macromolecules having a molecule containing two or more monomeric Subunits. Such cosmetic formulation, Such as a Soap or a skin cream. For as a dipeptide or dinucleotide, and generally is understood to example, a compound can be a macromolecule and also can refer to molecules that are about or at 1000 Daltons or below belong to one or more of the classes of compounds selected in molecular weight. from among hormones, prostaglandins, antibiotics, chemo 0072 Examples of small molecules include, but are not therapeutic agents, hematopoietics, anti-infective agents, limited to, inorganic molecules Such as, but not limited to, antiulcer agents, antiallergic agents, antipyretics, analgesics, carbon monoxide, carbon dioxide, metal (alkali metal, alka anti-inflammatory agents, antidementia agents, antiviral line earth metal, transition metal, e.g.) carbonates, cyanides, agents, antitumor agents, antidepressants, psychotropic cyanates, carbides, halides, thiocyanates, oxides, hydroxides, agents, cardiotonics, diuretics, antiarrhythmic agents, vasodi Sulfides and hydrozide, coordination compounds, e.g., the lators, antihypertensive agents, antidiabetic agents, antico cobalt salt Co(NH) Cls, and organometallic compounds, agulants, cholesterol lowering agents and nutritional Supple e.g. Fe(C5H5). Small molecules that are organic compounds ments. The methods, compositions, combinations, kits and include, for example, nucleotides, amino acids, pteridines articles of manufacture provided herein, described with ref Such as Furterene and Triamterene; purines such as Acefyl erence to some macromolecules, such as proteins, peptides, line, 7-Morpholinomethyltheophylline, Pamabrom, Protheo nucleic acids and viruses, can be adapted for use with other bromine and Theobromine; sterols such as cholesterol and macromolecules as defined and/or provided herein. lanosterol, Steroids Such as estrogen, testosterone, canrenone, 0070 The term “polymer as used herein includes any of oleandrin and spironolactone; penicillins, tetracyclines, Sul numerous natural and synthetic compounds containing two or fonamide derivatives Such as Acetazolamide, Ambuside, more repeat units of molecules linked together, generally AZosemide, Bumetanide. Butazolamide, Diphenylmethane about or at 5, 10, 15, 20, hundreds, thousands, up to millions 4.4'-disulfonamide, Disulfamide, Furosemide, uracils such as of repeating units. Each repeating unit generally is under Aminometradine and Amisometradine, and the like, and stood by those of skill in the art as a monomer. A polymer can prostaglandins. have identical repeating units, or more than one type of 0073. The term “small molecule' as used herein also is repeating unit. Exemplary repeating monomeric units intended to encompass all molecules that are within the scope include, for example, nucleotides or nucleotide derivatives of the description above and have a function, including a such as those found in deoxyribonucleic acid (DNA), ribo biological function, such as a hormone, a therapeutic func nucleic acid (RNA), and mixed DNA or RNA derivatives, or tion, such as a drug, a nutraceutical function, such as a nutri peptide nucleic acids (PNA). Other monomer units can tional Supplement, and a cosmetic formulation, Such as a Soap include, Such as those found in synthetic organic polymers, or a skin cream. For example, a compound can be a small include, but are not limited to, acrylamides, styrenes, alkyl molecule and also belong to one or more of the classes of Substituted Styrenes, acrylates, methacrylates, acrylic acid, compounds selected from among hormones, prostaglandins, methacrylic acid, vinyl chloride, vinyl acetate, butadiene, antibiotics, chemotherapeutic agents, hematopoietics, anti isoprene, ethylene glycol and ethyleneimine. Exemplary infective agents, antiulcer agents, antiallergic agents, anti organic or inorganic polymers, natural and synthetic poly pyretics, analgesics, anti-inflammatory agents, antidementia mers, include, but are not limited to, agarose, cellulose, nitro agents, antiviral agents, antitumor agents, antidepressants, cellulose, cellulose acetate, other cellulose derivatives, dext psychotropic agents, cardiotonics, diuretics, antiarrhythmic ran, dextran-derivatives and dextran co-polymers, other agents, vasodilators, antihypertensive agents, antidiabetic polysaccharides, glass, silica gels, gelatin, polyethylene gly agents, anticoagulants, cholesterol lowering agents and nutri cols, polyethyleneimines, polyethyleneimides, polyvinyl tional Supplements. The methods, compositions, combina pyrrolidone, rayon, nylon, polyethylene, polypropylene, tions, kits and articles of manufacture provided herein, exem polybutylene, polycarbonate, polyesters, polyamides, vinyl plified for Some types of Small molecules Such as polymers, polyvinylalcohols, polystyrene and polystyrene aminoglycosides, penicillins, amplicillins and prostaglandins, copolymers, polystyrene cross-linked with divinylbenzene or can be adapted for use with other small molecules as defined the like, acrylic resins, acrylates and acrylic acids, acryla and/or provided herein. mides, polyacrylamides, polyacrylamide blends, co-poly 0074 The term "conjugate' as used herein refers to a mers of vinyl and acrylamide, methacrylates, methacrylate chemical linkage or interaction. A conjugate can be a covalent derivatives and the like. orionic chemical linkage between two or more atoms, ions, or (0071. The term “small molecule' is used herein in the compounds, or can be formed by other chemical interactions, sense that is understood by those of skill in the art, and Such as hydrogen bonding, ionic pairing, base pairing or generally refers to a naturally occurring or chemically Syn pairing between charges formed by charge polarization. thesized organic or inorganic molecule that is less than about Exemplary conjugation means include Streptavidin- or avi 1000 Daltons, from about or at 1000 Daltons to about or at din- to biotin interaction; hydrophobic interaction; magnetic 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, interaction (e.g., using functionalized magnetic beads), polar 375, 350, 325, 300, 275,250, 225, 200, 175, 150, 125, 100, interactions, such as wetting associations between two polar 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less Surfaces or between oligo/polyethylene glycol; formation of a Daltons. A small molecule as understood by those of skill in covalent bond, such as an amide bond, disulfide bond, thio the art and used herein is a term that evolved to differentiate ether bond, or via crosslinking agents, or via acid-labile or traditional drugs, such as the penicillin antibiotics, from the photocleavable linkers. new class of drugs based on developments in genetic engi (0075. The term “substantially” or “substantial” as used neering and biotechnology, Such as proteins, nucleic acids herein generally means at least about 60% or 60%, about 70% and the like. A small molecule is understood to mean any or 70%, or about or at 75%, 80%, 85%, 90%, 95%, 96%, 97%, molecule that is not a macromolecule. Such as a protein or 98%, 99% or higher relative to a reference such as, for nucleic acid. A 'small molecule' as used herein can include a example, a nucleic acid or protein sequence or the original US 2012/O 141590 A1 Jun. 7, 2012

composition of an entity. Thus, a composition containing strength, pH, dielectric constant, counterion concentration, microparticles separated from “substantially' all other con organic solvent concentration, the addition of polyelectro taminants and/or ingredients including counterions, salts and lytes or polymers, Surfactants, detergents, or a combination Solvents from the cocktail solution means that at least about thereof. 60% or 60%, about 70% or 70%, or about or at 75%, 80%, 0080. The term “phase separation' as used herein refers to 85%, 90%. 95%, 96%, 97%, 98%, 99% or higher amounts of the transformation of a single homogeneous phase, Such as a contaminants and/or reagents have been removed from the Solution, into two or more phases, such as a suspension of a cocktail solution in which the microparticles are formed. The term “substantially identical' or “substantially homologous Solid particle in a solvent or solution. or similar varies with the context as understood by those 0081. The term "sedimentation” as used herein refers to skilled in the relevant art and generally means at least about the motion of particles, such as microparticles, which are in a 60% or 60%, about 70% or 70%, or about or at 75%, 80%, Suspension in a liquid or which are formed in a solution in 85%, 90%. 95%, 96%, 97%, 98%, 99% or higher identity. response to an external force Such as gravity, centrifugal force 0076. The term “consists essentially of or “consisting or electric force. essentially of as used herein refers to an entity from which I0082. The term “solution' is used interchangeably with Substantially all other components/ingredients that are not "cocktail Solution' herein and refers to a homogeneous mix associated with the entity or its properties have been removed ture of two or more ingredients in a single phase, Solid, liquid, or separated from the entity. Thus, a composition “consisting orgas, where the distinct ingredients only are recognizable at essentially of microparticles means that all other ingredients the molecular level. The solution can be a liquid in which one Such as contaminants and solvents have Substantially been or more solutes, such as salts, are dissolved in a solvent. Such removed from the solution/suspension containing the micro as water or alcohol, or dissolved in a mixture of miscible particles. solvents, such as a mixture of water and ethyl alcohol. The 0077. The term “microparticle' as used herein is inter Solution also can be a frozen form of a liquid solution. changeable with “microsphere' and refers to particles in the 0083. The term “miscible' as used herein refers to the size range (average length, width or diameter) of about or at ability of one or more components, such as liquids, Solids and 0.001 micron (um) to about or at 500 microns that contain a gases, to mix together to form a single, homogeneous phase. compound of interest. The compound of interest can be a Thus, two liquids are miscible if they can be mixed to form a macromolecule or a small molecule, an organic compound or single, homogenous liquid whose distinct components are an inorganic compound. The compound of interest can be an recognized only at the molecular level. When components are active agent, or the microparticle can in addition contain an “partially miscible, it means that they can be mixed to form active agent. The compound of interest that forms the micro a single homogenous phase in a certain concentration range, particle, e.g., a macromolecule including a protein, nucleic but not at other concentration ranges. As used herein, when a acid, lipid or polysaccharide, or a small molecule including a solvent is “partially miscible” with another solvent, it means sterol or steroid hormone, can be a carrier for the active agent, that it is miscible at a concentration of about or at 50%, 45%, Such as a drug or a nutritional Supplement. The microparticles 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, also can contain synthetic macromolecules including poly 5%, 4%, 3%, 2%, 1%, 0.5% or below volume/volume (v/v), mers, such as polyethylene glycol (PEG), polylactic acid when mixed with the other solvent. (PLA), polylactic-co-glycolic acid (PLGA), and natural poly 0084 As used herein, “immiscible” means that when two mers such as albumin, gelatin, chitosan and dextran. The or more components, such as liquids, Solids or gases are “microparticles' as described herein can contain and can be mixed, they form more than one phase. For example, when an made from a particular natural or synthetic compound alone, organic solvent is immiscible with an aqueous solvent (e.g., or from more than one type of the same natural or synthetic hexane and water), the organic solvent is visible as a distinct compound (e.g., more than one type of protein), or from layer that does not mix with the layer of aqueous solvent. combinations of more than one different type of natural or I0085. As used herein, the term “polypeptide.” means at synthetic compound (e.g., an antibiotic and a leuprolide pep least two amino acids, or amino acid derivatives, including tide). mass modified amino acids and amino acid analogs, that are 0078. The term “microparticle' as used herein also gener linked by a peptide bond, which can be a modified peptide ally refers to a particle that is not a solid form of the entire bond. The terms “polypeptide.” “peptide' and “protein’ are Solution from which it is produced, although frozen and/or used essentially synonymously herein, although the skilled dried particles of a solution containing macromolecules also artisan will recognize that peptides generally contain fewer are contemplated herein. Rather, the microparticle as used than about fifty to about one hundredamino acid residues, and herein generally is an assembly of a fraction of the compo that proteins often are obtained from a natural source and can nents of a solution, including salts, counterions, solvents and contain, for example, post-translational modifications. other ingredients, that is formed by a process including, but I0086 A polypeptide or protein can be translated from a not limited to, precipitation, sedimentation, phase separation polynucleotide, which can include at least a portion of a and colloid formation. coding sequence, or a portion of a nucleotide sequence that is 0079. The term “precipitation” as used herein refers to a not naturally translated due, for example, to it being located in process whereby a solute or Solutes of interest in a solution, a reading frame other than a coding frame, or it being an Such as the components of a microparticle, no longer stay in intron sequence, a 3' or 5' untranslated sequence, a regulatory solution and form a phase that is distinct from the solvent or sequence Such as a promoter, or the like. A polypeptide also solvents that were used to form the solution. Precipitation of can be chemically synthesized and can be modified by chemi a microparticle and controlling the size of the precipitated cal or enzymatic methods following translation or chemical microparticle can be accomplished by a variety of means synthesis. A polypeptide can be post-translationally modified including, but not limited to, adjusting temperature, ionic by phosphorylation (phosphoproteins), glycosylation (glyco US 2012/O 141590 A1 Jun. 7, 2012 proteins, proteoglycans), and the like, which can be per ing a deoxyribonucleic acid (DNA), a ribonucleic acid formed in a cell or in a reaction in vitro. (RNA), and a DNA or RNA derivative containing, for 0087 As used herein, the term “fusion protein’ refers to a example, a nucleotide analog or a “backbone' bond other protein that is a conjugate of domains obtained from more than a phosphodiester bond, for example, a phosphotriester than one protein or polypeptide. A domain can be a polypep bond, a phosphoramidate bond, a phosphorothioate bond, a tide tag, such as a His tag. The conjugates can be prepared by thioester bond, or a peptide bond (peptide nucleic acid). The linking the domains by chemical conjugation, recombinant term "oligonucleotide' also is used herein essentially syn DNA technology, or combinations of recombinant expression onymously with “polynucleotide, although those in the art and chemical conjugation. will recognize that oligonucleotides, for example, PCR prim 0088 A variety of chemical linkers are known to those of ers, generally are less than about fifty to one hundred nucle skill in the art and include, but are not limited to, amino acid otides in length. and peptide linkages, typically containing between one and (0093. As used herein, the term “flowability characteristic” about 60 amino acids, more generally between about 10 and refers to a property that renders the ability to “flow, where 30 amino acids, heterobifunctional cleavable cross-linkers, “flow” is a property that can permit a substance to be poured including but are not limited to, N-Succinimidyl (4-io and to assume the shape of a container that it is poured into, doacetyl)-aminobenzoate, Sulfo Succinimidyl (4-iodoacetyl)- without hindrance due to, for example, aggregation. Fluids aminobenzoate, 4-Succinimidyl-oxycarbonyl-a-(2-py generally have the property of “flow,” which generally ren ridyldithio)toluene, SulfoSuccinimidyl-6-a-methyl-a- ders them deformable, i.e., they can change their shape. The (pyridyldithiol)-toluamidohexanoate, N-succinimidyl-3-(- term “fluid as used herein encompasses colloids containing 2-pyridyldithio)-propionate, Succinimidyl 63(-(-2- liquids, including emulsions, aerosols and gases. Liquids, pyridyldithio)-propionamidohexanoate, SulfoSuccinimidyl aerosols and gases with Suspensions of solid particles, such as 63(-(-2-pyridyldithio)-propionamidohexanoate, 3-(2-py microparticles, also are considered “fluid as defined herein. ridyldithio)-propionylhydrazide, Ellman's reagent, dichloro 0094. As used herein, an emulsion is defined as a colloid of triazinic acid, and S-(2-thiopyridyl)-L-cysteine. two immiscible liquids, a first liquid and a second liquid, 0089. The term “sialidase fusion protein’ as used herein where the first liquid is dispersed in the second liquid. refers to a fusion protein in which one or more domains is a 0.095 As used herein, surfactants (or “surface-active sialidase or a portion thereofthat retains at least about 60% or agents') are chemical or naturally occurring entities which, 60%, about 70% or 70%, or about or at 75%, 80%, 85%, 90%, when dissolved in an aqueous solution, reduce the Surface 95%, 96%,97%.98%, 99% or more of its catalytic activity. A tension of the solution or the interfacial tension between two sialidase fusion protein as used herein also can refer to a or more phases in Solution. The Surfactant molecules gener fusion protein that contains a protein or polypeptide that is ally are amphiphilic and contain hydrophilic head groups and Substantially homologous to a sialidase and possesses the hydrophobic tails. The Surfactant molecules can act as stabi enzymatic activity of a sialidase. lizers and/or improve flowability characteristics of the micro 0090 The term “catalytic domain” of a protein as used particles provided herein. herein refers to a protein or polypeptide in which the only 0096. As used herein, a combination refers to any associa portion of the sequence that is substantially homologous to a tion between two or among more items for a purpose. For sialidase is a sequence of amino acid residues that includes example, a combination of microparticles and an inhaler can the domain responsible for the catalytic activity of the protein be used for pulmonary delivery of a therapeutic agent. (e.g., residues 274-666 of SEQID NO: 1 are identified as the 0097. As used herein, a composition refers to any mixture. catalytic domain of Actinomyces viscosus sialidase) or cata It can be a solution, a Suspension, liquid, powder, a paste, lytically active fragments thereof. The catalytic domain or aqueous, non-aqueous or any combination thereof. catalytically active fragment thereof retains at least about 0098. As used herein, a kit refers to a combination in 60% or 60%, about 70% or 70%, or about or at 75%, 80%, which components are packaged optionally with instructions 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the cata for use and/or reagents and apparatus for use with the com lytic activity of the protein. bination. 0.091 As used herein, the term “nucleic acid refers to 0099. As used herein, the term “enzyme” means a protein single-stranded and/or double-stranded polynucleotides Such that catalyzes a chemical reaction or biological process. as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) Enzymes generally facilitate and/or speed up Such reactions as well as analogs or derivatives of either RNA or DNA. Also and processes. In addition, enzymes generally are specific for included in the term “nucleic acid are analogs of nucleic a particular reaction or process, converting a specific set of acids such as peptide nucleic acid (PNA), phosphorothioate reactants into specific products. DNA, siRNA, snRNA, tRNA, ribozymes and other such ana 0100. As used herein, the term “colloid” refers to a disper logs and derivatives or combinations thereof. Nucleic acid sion of solid particles, such as microparticles, in a liquid. Such can refer to polynucleotides Such as deoxyribonucleic acid as the solution in which the microparticles are formed. The (DNA) and ribonucleic acid (RNA). The term also includes, term “colloidal stability” refers to a colloid in which the as equivalents, derivatives, variants and analogs of either particles are not substantially aggregated. For example, a RNA or DNA made from nucleotide analogs, single (sense or stable colloid is one in which about 30%, 25%, 20%, 15%, antisense) and double-stranded polynucleotides. Deoxyribo 10%.5%, 4%,3%, 2%, 1%, 0.5% or less of the solid particles, nucleotides include deoxyadenosine, deoxycytidine, deox Such as microparticles, have formed aggregates. yguanosine and deoxythymidine. For RNA, the uracil base is 0101 The term 'agglomerates’ refers to the association of uridine. one or more particles, such as microspheres, loosely held 0092. As used herein, the term "oligonucleotide' or “poly together by van der Waals forces or surface tension or elec nucleotide' refers to an oligomer or polymer containing at trostatic or combinations thereof. In some instances, associa least two linked nucleotides or nucleotide derivatives, includ tions held by electrostatic forces can be defined as “Floccu US 2012/O 141590 A1 Jun. 7, 2012 lates.” For the purposes herein, Agglomerates' also tion, heat, chemicals, enzyme action, exposure to acids or encompass "Flocculates’. Agglomerates can generally alkalis, and ion-exchange and any combinations thereof. readily be broken apart by shear forces within the air or liquid. Denaturation of a protein generally results in diminishing all The term “disperse' or “dispersivity” refers to the ability of or some, generally more than 50% and at least about 70%, the particles to “flow, i.e., the extent to which the movement 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, is not impeded by the presence of for example, aggregates. 98% or 99%, of the original properties including activity and 0102 The term “aggregates' or "clumps' refers to the function of the protein in its native or non-denatured State. association of one or more particles, such as microspheres, 0107 As used herein, the term “nutritional supplement' amorphous precipitates, crystal- or glass-like particles or means a Substance or composition that provides nutrients, combinations thereof. Aggregates generally are not easily including vitamins, minerals, fatty acids, amino acids, carbo broken apart which inhibits their ability to disperse or form hydrates, enzymes, proteins, biochemicals and their metabo homogeneous Suspensions or to form aerosols with desirable lites, herbs and plants, to a host, Such as an animal, including properties. a human being. Nutrients that are Supplied to the host through 0103. The term “non-denatured’ as used herein is in ref nutritional Supplements can include nutrients essential for erence to proteins and means a conformation of a protein, i.e., Survival, good health, curing disease or preventing disease its secondary structure, tertiary structure, quaternary struc that are missing or deficient in a host’s diet, and nutrients that ture or combinations thereof, which essentially is unaltered are believed to augment good health, prevent disease or cure from the protein in its naturally occurring state. The terms disease but are not considered essential for Survival or good “non-denatured and “native' are used interchangeably health. herein and mean a protein that retains all or at least about 0108. As used herein, “hydrophobic” refers to a substance 50%, 60%, 70%, 80%, 85%, 90° k 91%, 92%, 93%, 94%, that is not charged or charge-polarized, or is not sufficiently 95%, 96%, 97%, 98% or 99% of its length and/or natural charged or charge-polarized to bond with water or other polar conformation. The terms “non-denatured’ or “native' as used solvents, as understood by those of skill in the art. Hydropho interchangeably herein include the natural state of a protein in bic ligands can associate with each other or with other non a cell. Such as it's length and conformation including second polar molecules or solvents in the presence of water or a polar ary, tertiary and quaternary structures. As defined herein, the solvent, through hydrophobic interactions. A hydrophobic “non-denatured’ or “native' proteins including those in the ligand generally also is more soluble in non-polar solvents compositions provided herein generally retain all or at least than in polar solvents. Examples of non-polar solvents about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, include alkanes such as hexane, alkyl ethers such as diethyl 94%. 95%, 96%, 97%, 98% or 99% of the normal activity or ether, aromatic hydrocarbons such as benzene and alkyl function of the proteins in their natural state, e.g., as a nutrient halides such as methylene chloride and carbon tetrachloride, to provide amino acid building blocks, an antioxidant, an mono-, di- and triglycerides, fatty acids, Such as oleic, enzyme, an antibody, a regulator of gene expression, a scaf linoleic, palmitic, Stearic, conjugated forms thereof and their fold, etc. esterS. 0104. As used herein, the terms “activity” or “function' 0109 The term “water-insoluble' compound is used inter are interchangeable with “biological activity” and refer to the changeably herein with “hydrophobic' compound and refers in vivo activities of a compound, such as a protein, Vitamin, to a compound that has a greater Solubility in non-aqueous mineral or drug, orphysiological responses that result upon in Solvents than in aqueous solvents. For example, a “water Vivo administration of a compound, composition or other insoluble' compound is a compound that is fully or partly— mixture. Activity, thus, encompasses therapeutic effects and about or equal to 20, 25, 30, 35, 40, 45, 50, 55,60, 65,70, 75, pharmaceutical activity of compounds, compositions and 80, 85,90, 95, 96, 97,98, 99 or 100%, insoluble in solutions mixtures. Biological activities also can be observed in in vitro that contain about or equal to 20, 25, 30,35, 40, 45,50,55, 60, systems designed to test or use Such activities. 65, 70, 75,80, 85,90, 95, 96, 97,98, 99 or 100% by volume 0105. As used herein, “functional activity” also is inter of water or aqueous solution, such as a buffer. changeable with “activity.” “biological activity” or “func 0110. As used herein, a “hydrophilic' or “polar ligand is tion' and refers to a compound that displays one or more a ligand that has a charge or is charge-polarized. A hydro activities associated with its natural state, or with the class of philic ligand as used herein has either a charged functional compounds to which it belongs. For example, an aminogly group, such as a carboxylate or ammonium, or a charge coside that is an antibiotic is exhibiting the functional activity polarized bond, such as hydroxyl or sulfhydryl that provides of several compounds of its class. Similarly, a polypeptide or a charge to the ligand. Hydrophilic ligands can bond with portion thereofthat displays one or more activities associated water and other polar solvents including alcohols, amines, with the native or non-denatured protein is functionally amides, acids, carboxylic acids, esters, nitriles, ketones, gly active. Functional activities include, but are not limited to, cols and glycol ethers, through hydrogen bonds orionic inter therapeutic efficacy, biological in vivo activity, catalytic or actions. A hydrophilic ligand also has greater solubility in enzymatic activity, antigenicity (ability to bind to or compete polar solvents than in non-polar solvents. with a polypeptide for binding to an anti-polypeptide anti 0111. As used herein, the term “therapeutic agent’ means body), immunogenicity, ability to form multimers, and the an agent which, upon administration to a host, including ability to specifically bind to a receptor or ligand for the humans, effectively ameliorates or eliminates symptoms or polypeptide. manifestations of an inherited or acquired disease or that 0106. The term “denatured’ as used herein refers to a cures said disease. Exemplary therapeutic agents include, for protein that is altered from its native or non-denatured con example, chemical compounds for cancer therapy, e.g., che formation, i.e., its secondary, tertiary or quaternary structure motherapeutic agents, chemical compounds directed against or combinations thereof. The altered conformation generally bacterial infections, e.g., antibiotics, antiviral compounds occurs by processing steps that include pasteurization, radia and the like, as understood by those of skill in the art. US 2012/O 141590 A1 Jun. 7, 2012

0112. As used herein, the term “carrier or “micro-carrier' lites, isomers, fragments, analogs, and the like. When the refers to a molecule that facilitates the formation of micro terms “biologically active agent,” “biological agent” and spheres containing the molecule that is the active agent or "agent” are used, then, or when a particular active agent is therapeutic agent of interest, or promotes Stability of the specifically identified, it is intended to include the active resulting microspheres, or facilitates transportation of the agent perse as well as pharmaceutically acceptable, pharma resulting microsphere to the target (cells, tissues, etc.) of cologically active salts, esters, amides, prodrugs, active interest. In some embodiments, carriers can be employed to metabolites, isomers, fragments and analogs. impart stability to the microspheres. In embodiments where 0116. As used herein, a “subject' is defined as an animal, the therapeutic agent or active agent of interest contained in including a mammal, typically a human. the microspheres has a high potency and is incorporated at a 0117. As used herein, “therapeutically effective amount relatively low concentration (generally, about or at 0.001%. refers to an amount of the active agent for a desired therapeu 0.005%, 0.01%, 0.02%, 0.05, 0.1%, 0.2%, 0.5%, 1%, 2%, tic, prophylactic, or other biological effect or response when 3%. 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or a composition is administered to a subject in a single dosage 50%, or within a range of about or at 0.001% to about or at form. The particular amount of active agent in a dosage will 50%), carriers can stabilize microsphere formulations that vary widely according to conditions such as the nature of the might otherwise be readily degraded. Examples of high active agent, the nature of the condition being treated, the age potency compounds can include cytotoxic anti-cancer agents and size of the subject. or nucleic acids such as siRNA. Exemplary carriers include 0118. As used herein, “pharmaceutically acceptable amino acids, carboxylic acids (e.g. citric acid, maleic acid), derivatives” of a compound include Salts, esters, enol ethers, polymers including proteins and nucleic acids, materials enol esters, acids, bases, Solvates, hydrates or prodrugs capable of forming hydrogels including gelatin and various thereof. Such derivatives can be readily prepared by those of polysaccharides, and their combinations. In some embodi skill in this art using known methods for Such derivatization. ments, active agents that are proteins or nucleic acids such as The compounds produced can be administered to animals or tRNA and siRNA are incorporated into microspheres that are humans without substantial toxic effects and either are phar stabilized using polysaccharides such as dextran or proteins maceutically active or are prodrugs. Pharmaceutically Such as gelatin as micro-carriers. acceptable salts include, but are not limited to, amine salts, 0113 Molecules used as carriers generally have demon such as but not limited to N,N'-dibenzylethylenediamine, strated safety and stability. For a given active agent or thera chloroprocaine, choline, ammonia, diethanolamine and other peutic agent, carrier systems can be optimized in a high hydroxyalkylamines, ethylenediamine, N-methylglucamine, throughput manner. procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2- 0114. As used herein, “shelf life” or “stability” refers to pyrrolidin-1-ylmethylbenzimidazole, diethylamine and the time after preparation of the microparticle composition other alkylamines, piperazine and tris(hydroxymethyl)ami that the composition retains at least about or 70%, 80%, 85%, nomethane; alkali metal salts, such as but not limited to 90%, 91%, 92%, 93%, 94%, 95%,96%.97%, 98% or 99% of lithium, potassium and sodium; alkali earth metal salts. Such the initial protein activity that is present in the composition as but not limited to barium, calcium and magnesium; tran and other general physical characteristics of microspheres sition metal salts, such as but not limited to Zinc, and other Such as size, shape, and aerodynamic particle size distribu metal salts, such as but not limited to Sodium hydrogen phos tion. Thus, for example, a composition that is stable for or has phate and disodium phosphate; and also including, but not a shelf life of 30 days at room temperature, defined herein as limited to, salts of mineral acids, such as but not limited to range of between about 18°C. to about 25°C., 26°C., 27°C. hydrochlorides and Sulfates; and salts of organic acids. Such or 28°C., would have at least about 70%, 80%, 85%, 90% as but not limited to acetates, lactates, malates, tartrates, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the citrates, ascorbates. Succinates, butyrates, Valerates and initial amount of the activity of protein present in the compo fumarates. Pharmaceutically acceptable esters include, but sition at 30 days following storage at 18°C. to about 25°C., are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, 26°C., 27°C. or 28°C. The shelf life of the microparticle aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of compositions provided herein generally is at least about 10 acidic groups, including, but not limited to, carboxylic acids, days at 55°C., at least about 2-3 weeks at 42°C., and at least phosphoric acids, phosphinic acids, Sulfonic acids, Sulfinic about eight months or greater at 25°C., however, micropar acids and boronic acids. ticles compositions of any length of shelflife at any tempera 0119. As used herein, “treatment’ means any manner in ture that are produced by the methods provided herein are which one or more of the symptoms of a condition, disorder contemplated herein. or disease are ameliorated or otherwise beneficially altered. 0115. As used herein, “a biologically active agent, “an Treatment also encompasses any pharmaceutical use of the active agent.” “a biological agent or “an agent is any Sub compositions herein, such as use for treating influenza. stance which when introduced into the body causes a desired 0.120. As used herein, “organic solvent refers to a solvent biological response, Such as altering body function at the that is an organic compound, which is any member of a large cellular, tissue or organ level and/or altering cosmetic appear class of chemical compounds whose molecules contain car ance. Such as body weight and shape. Such substance can be bon and hydrogen. Such solvents can include, for example, any synthetic or natural element or compound, protein, cell, compounds from the following classes: aliphatic or aromatic or tissue including a pharmaceutical, drug, therapeutic, nutri alcohols, polyols, aldehydes, alkanes, alkenes, alkynes, tional Supplement, herb, hormone, or the like, or any combi amides, amines, aromatics, azo compounds, carboxylic acids, nations thereof. The terms also encompass pharmaceutically esters, dioxanes, ethers, haloalkanes, imines, imides, ketones, acceptable, pharmacologically active derivatives of those nitriles, phenols and thiols. active agents specifically mentioned herein, including, but I0121. As used herein, an “aqueous solvent” refers to not limited to, salts, esters, amides, prodrugs, active metabo water, or a mixture of solvents that contains at least about 50% US 2012/O 141590 A1 Jun. 7, 2012

or 50%, at least about 60% or 60%, at least about 70% or 70%, I0127. Whether a charged molecule is a counterion can be or about or at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, determined empirically based on parameters including, but 99% or higher amounts of water. The term “aqueous solvent' not limited to, the type of molecule to be formulated into a as used herein also refers to solutions containing water as a microsphere, the pH, the ionic strength, the type of solvent/ Solvent, such as buffers, salt solutions, solutions containing antisolvent system used, and the presence of salts and addi counterions, and other solutes that are soluble in water. tional ingredients such as active agents. As provided and 0122. As used herein, “antisolvent’ means a solvent described herein, counterions can be anionic or having a net which, when added to a solution of the microparticle-forming negative charge or charge-polarizable group(s), cationic or compound of interest, lowers the solubility of the compound having a net positive charge or charge-polarizable group(S), in the resulting mixture (i.e., the “cocktail solution' from or Zwitterionic and possessing both negative and positive which the microparticles are eventually obtained). The anti charged or charge-polarizable groups. Solvent generally is added in an amount that retains the com I0128. A compound can sometimes be its own counterion, pound in solution until the microparticles are formed by a step facilitating the formation of microparticles in the absence of of gradual chilling followed by microparticle recovery, e.g., any additional counterion. For example, under certain condi by lyophilization. Thus, the antisolvent is added to the solu tions, Small molecule compounds such as tetracycline, kana tion of the compound in an amount that is insufficient to mycin and amplicillin, and macromolecules Such as siRNA precipitate the compound out of Solution at the temperature and tobacco mosaic virus, can form microparticles in the (generally, ambient temperature) used to prepare the cocktail absence of added counterion. Other counterions, such as solution. The antisolvent can be miscible or partially miscible polyethyleneimine (PEI) and Na-acetate/Na-sulfate buffer, with the solvent in which the compound is dissolved, or the which are capable of forming microparticles on their own, in Solvent/counterion solution, or the solvent/counterion/com the absence of a compound of interest, can facilitate forma pound solution. For example, an organic Solvent Such as tion and/or nucleation of microparticles of the compound of isopropanol can be an antisolvent for compounds that are interest by acting as “carriers' or “seeds.” water-soluble, and water or an aqueous buffer can be an I0129. As used herein, the term “cooling” refers to a low antisolvent for compounds that are water-insoluble. Both sol ering of temperature to a desired temperature for obtaining vent and antisolvent, however, can be organic solvents. Some microparticles or, once the microparticles of desired dimen antisolvents and solvents can also serve as counterions. For sions are obtained, further lowering the temperature to a example, aqueous buffered solutions can be a counterion and desired temperature for obtaining dry preparations of the a solvent or antisolvent. Similarly, a polymer, such as poly microparticles by volatilizing solvents (e.g., for freeze-dry ethylene glycol (PEG) or polyethyleneimine (PEI), can be an ing). The term 'gradual cooling' or 'gradually cooling” or antisolvent and a counterion. “gradually cooled as used herein means that the lowering of 0123. As used herein, the term “solvent/antisolvent sys temperature to a desired temperature from ambient tempera tem’ means a mixture of Solvents in which a compound that ture (about or at 15° C. to about or at 50°C., generally about can form a microsphere is soluble at ambient temperature, but or at 18°C. to about or at 30°C.) for microparticle formation forms microspheres upon chilling of the mixture, generally in occurs at a rate or for an amount of time that is suitable for the presence of a counterion, to temperatures below ambient generating microparticles in a solution before the Solution temperature. As noted above, a solvent and/or an antisolvent becomes frozen. Thus gradual cooling is different from, for can also be a counterion and eliminate the need for an addi example, Snap freezing, spray drying or spray freeze-drying, tional counterion. The solvent and the antisolvent generally whereby the entire solution is converted to a solid form with are miscible or partially miscible with one another, although out the generation of distinct microparticles. Solvent/antisolvent systems in which the solvent and antisol 0.130. The rate of gradual cooling is empirically deter vent are immiscible also can be used. mined based on the type of macromolecule, solvents, coun 0.124. As used herein, the term “pl” or “isoelectric point' terions and other ingredients as well as the method of cooling refers to the pH at which there is no net charge on a protein or (e.g., an endothermic reaction, a heat exchanger, refrigerator polypeptide. or freezer or freeze-dryer) and can vary, for example, for an 0125. As used herein, the term “counterion” refers to a amount of time for microparticle formation of between about charged or charge-polarizable molecule that can initiate for or at 1 min, 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 mation of a microparticle from a macromolecule. Such as a min, 25 min, 30 min, 1 h, 2h, 5hor 10h to about or at 1.5 min, protein, nucleic acid, lipid or oligosaccharide, or from a small 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20 min, 25 min, molecule. Such as a tetracycline or prostaglandin. A counte 30 min, 1 h, 2 h, 5h, 10 h or 15 h. rion can be a polymer, Such as polyethylene glycol (PEG) or I0131 Microparticles of desired size also can be formed, polyethyleneimine (PEI). for example, by rapidly chilling the cocktail (e.g. using a heat 0126 The choice of counterion can empirically be deter exchanger) and allowing the Suspension of microparticles to mined for each compound (macromolecule or Small mol be maintained for a certain period of time without significant ecule) of interest. For example, in the case of the DAS181 temperature changes, then Snap freezing the cocktail. fusion protein (SEQID NO:17), sodium sulfate is a counte 0.132. The temperature at which microparticles are formed rion because it can initiate the formation of microparticles in also is empirically determined based on the type of macro the methods provided herein, whereas glycine, sodium chlo molecule or Small molecule, solvents, counterions and other ride or sodium acetate generally are not suitable as counteri ingredients as well as the method and uniformity of cooling ons for DAS181. For kanamycin, itaconic and citric acids can and can vary from about or at 15° C., 10°C., 8°C.,5°C., 4 C, serve as Suitable counterions because they can initiate the 3°C., 2°C., 1° C., -2°C, -5°C., -7.5°C., -10°C., -15°C., formation of microparticles of kanamycin in the methods -20°C., -25°C., -30°C., -35°C., -40°C.,-45° C., -50° C., provided herein, whereas arginine generally is not suitable as -55° C., -60° C., -70° C., -80° C., -85°C., -90° C., -100° a counterion for kanamycin. C., -110° C., -115° C., -120° C., -125° C., -135° C. - 145° US 2012/O 141590 A1 Jun. 7, 2012

C., -150° C., -160° C., -165° C., -170° C., -175° C., -180° 0.138. The microspheres obtained by the methods pro C., -185° C. - 190° C. - 195°C., or -200° C. vided herein are useful as prophylactic, therapeutic or diag 0133. The term "ambient temperature' is sometimes used nostic agents for treating or diagnosing disease states in a interchangeably herein with “room temperature' and refers to subject in vivo or in vitro. The sizes of the microspheres the temperature of air or other media in the environment of the obtained by the methods provided herein can be controlled by designated area in which the cocktail reactions are mixed adjusting parameters including type and concentration of and/or are maintained prior to the initiation of microsphere antisolvent, types and relative concentrations of Solvent and formation. Ambient temperature as used herein can be from antisolvent in the solvent/antisolvent system, macromolecule about or at 15° C. to about or at 50° C., generally about or at or Small molecule concentration, ionic strength, counterion 18°C. to about or at 30° C., or about or at 25°C. to about or type and concentration, rate and time of cooling, to provide at 30° C. microspheres in a wide range of sizes, from 0.001 micron to 0134. As used herein, an “endothermic reaction' is any 50 microns or greater, that can deliver therapeutic agents via chemical reaction that absorbs heat from its environment, a desired route including pulmonary (exemplary sizes can e.g., in Solution, thus cooling the Surrounding environment or include, but are not limited to, 1 micron to 5 micron particles Solution. For example, the addition of ammonium Sulfate or for delivery to the throat, trachea and bronchi for treatment of acetonitrile to water results in an endothermic reaction; these influenza and other respiratory infections), Subcutaneous, compounds, therefore, can serve as counterion and antisol intramuscular, intravenous and other routes (using particles vent, respectively, and also facilitate chilling to form micro that can include, but are not limited to, particles that are tens particles. Other examples of endothermic reactions include, of microns in size). but are not limited to, dissolving ammonium chloride in 0.139. The compositions provided herein can be formu water, mixing water and ammonium nitrate, mixing water lated for a variety of modes of administration. For example, with potassium chloride, and reacting ethanoic acid with the compositions can be orally e.g. by ingestion, intrave Sodium carbonate. nously, intranasally, parenterally, Subcutaneously, transder 0135. As used herein, the term “spray drying refers to a mally, topically, cutaneously, intraarticularly or intramuscu process wherein a solution containing a molecule. Such as a larly administered. The compositions also can be formulated protein or Small molecule, is transformed into a dry particu for pulmonary or ophthalmic administration. In a certain late form by atomizing into a hot drying medium, generally aspect, the composition provided herein is for inhalation. for a period of about a few milliseconds to 1-2 seconds to a 0140. The compositions provided herein can be formu few tens of seconds. The term “spray freeze-drying as used lated as tablets, caplets, capsules, gels, vials, pre-filled herein refers to a process wherein a solution containing a Syringes, inhalers, electrostatic devices and other devices for macromolecule. Such as a protein, is atomized into a cryo delivery. The delivery dosage of the compositions can be from genic medium, Such as liquid nitrogen, to obtain frozen drop between about or at 0.01 mg to about or at 0.1 mg; about or at lets of solution that can then be dried by lyophilization. The 0.1 mg compound per dose to about or at 1000 mg compound term "snap freezing” or “rapid freezing” or "quick freezing per dose, or about or at 0.2 mg, 0.3 mg, 0.5 mg, 0.6 mg, 0.75 or “flash freezing” as used interchangeably herein refers to mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg. freezing a solvent or Solution, including solutions containing 30 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65mg, 70 mg, 75 macromolecules. Such as proteins, by immersing the con mg, 80 mg. 85 mg. 90 mg, 95 mg, 100 mg, 150 mg, 200 mg. tainer with good heat transfer properties (e.g. thin-wall glass 250 mg. 300 mg. 350 mg, 400 mg. 450 mg, 500 mg, 600 mg. or plastic or metal test tube) holding the solvent or solution in 700 mg. 800 mg, 900 mg or about or at 1000 mg compound liquid nitrogen or pouring the Solution directly into liquid per dose. The frequency of administration of a dose, for nitrogen. "Snap freezing” and “rapid freezing’ generally example, for the treatment or prophylaxis of influenza, can be occur within a period of about a few milliseconds to 1-2 from three or more times a day, to two times a day, to once a seconds to a few tens of seconds. day, to two times a week, to once a week, to once every two 0136. The term “lyophilize” or “lyophilization” as used weeks or less frequent than once every two weeks. For pro herein is synonymous with “freeze drying and refers to a phylaxis, the administration generally can be of the order of process wherein a solution, including an emulsion, colloid or about once every two weeks or less frequent, such as once Suspension, is frozen and the solvents are volatilized (Subli every three weeks or once every four weeks or longer. mated) directly into the vapor state, leaving behind the solid 0.141. The compositions formulated according to the components. methods provided herein can be used for the prevention, prophylaxis and/or treatment of diseases and disorders. B. Methods for Preparing Microparticle Accordingly, provided herein are methods of prevention, pro Compositions phylaxis or treatment of a disease by administering a thera 0.137 Provided herein are methods of making micro peutically effective amount of microspheres of a compound spheres having a high content of a compound. The compound of interest. The diseases and disorders can include, but are not can be macromolecule. Such as a protein, or a small molecule, limited to neural disorders, respiratory disorders, immune Such as a prostaglandin. The microspheres provided herein system disorders, muscular disorders, reproductive disorders, are prepared by controlled precipitation in the presence of a gastrointestinal disorders, pulmonary disorders, digestive counterion and an antisolvent. The microspheres are Suitable disorders, metabolic disorders, cardiovascular disorders, for preparing pharmaceutical, diagnostic, nutraceutical or renal disorders, proliferative disorders, cancerous diseases cosmetic compositions that can be delivered to Subjects by a and inflammation. variety of delivery routes, including pulmonary, Subcutane 0.142 For example, the microparticles provided hereincan ous, transdermal, intramuscular, parenteral and oral adminis be used in methods of treating Infectious diseases, such as tration routes. The method also can be performed in a batch or arboviral infections, botulism, brucellosis, candidiasis, continuous mode, for increased efficiency and production. campylobacteriosis, chickenpox, chlamydia, cholera, coro US 2012/O 141590 A1 Jun. 7, 2012

novirus infections, staphylococcus infections, coxsackie 0.148. The above steps of the method can be performed virus infections, Creutzfeldt-Jakob disease, cryptosporidi sequentially, intermittently or simultaneously in any order, osis, cyclospora infection, cytomegalovirus infections, although one of the skill in the art would understand that the Epstein-Barr virus infection, dengue fever, diphtheria, ear step of dehydration to separate the solvent from the micro infections, encephalitis, influenza virus infections, parainflu spheres can occur simultaneously with, or following, micro enza virus infections giardiasis, gonorrhea, Haemophilus sphere formation, but not prior to microsphere initiation and/ influenzae infections, hantavirus infections, viral hepatitis, or formation. In one embodiment, the counterion and the herpes simplex virus infections, HIV/AIDS, helicobacter antisolvent are added simultaneously or sequentially in any infection, human papillomavirus (HPV) infections, infec order to the solution containing the compound, followed by chilling. In other embodiments, the same Substance serves as tious mononucleosis, legionellosis, leprosy, leptospirosis, the counterion and the antisolvent (for example, a polymer listeriosis, lyme disease, lymphocytic choriomeningitis, Such as polyethylene glycol or polyethyleneimine). In yet malaria, measles, marburg hemorrhagic fever, meningitis, other embodiments, the Solution containing the compound monkeypox, mumps, mycobacteria infection, mycoplasma can be pre-chilled to a temperature suitable for microsphere infection, norwalk virus infection, pertussis, pinworm infec formation, prior to adding the counterion and antisolvent. tion, pneumococcal disease, Streptococcus pneumonia infec Pre-chilling can be performed using a device, such as a refrig tion, Mycoplasma pneumoniae infection, Moraxella erator or freezer, or by endothermic reaction. For example, a catarrhalis infection, Pseudomonas aeruginosa infection, pre-chilled aqueous solution of a compound can beformed by rotavirus infection, psittacosis, rabies, respiratory syncytial adding ammonium Sulfate and acetonitrile, whose dissolution virus infection, (RSV), ringworm, rocky mountain spotted proceeds via an endothermic reaction, prior to or simulta fever, rubella, salmonellosis, SARS, scabies, sexually trans neously with forming microspheres. mitted diseases, shigellosis, shingles, sporotrichosis, Strepto 014.9 The resulting suspension of microparticles can be coccal infections, syphilis, tetanus, trichinosis, tuberculosis, converted into a dry powder by further cooling to a tempera tularemia, typhoid fever, viral meningitis, bacterial meningi ture below freezing point and subsequent removal of volatiles tis, west nile virus infection, yellow fever, adenovirus-medi (solvent, antisolvent and, where desired, the counterion) by, ated infections and diseases, retrovirus-mediated infectious for example, Sublimation using a standard freeze dryer. diseases, yersiniosis Zoonoses, and any other infectious res 0150. In some embodiments, the addition of a counterion piratory, pulmonary, dermatological, gastrointestinal and uri is not necessary. For example, under certain conditions, some nary tract diseases. molecules in Solution with a Suitable solvent can form micro 0143. Other diseases and conditions that can be treated by particles in the presence of an antisolvent and no added coun administering a therapeutically effective amount of micro terion. Without being bound by any theory, it is possible that spheres of a compound of interest can include arthritis, the molecules can act as counterions to themselves, or other asthma, allergic conditions, Alzheimer's disease, cancers, components in the resulting cocktail solution or combinations cardiovascular disease, multiple sclerosis (MS), Parkinson's thereof, such as the solvent, antisolvent. Several such mol disease, cystic fibrosis (CF), diabetes, non-viral hepatitis, ecules are exemplified herein, including siRNA, tobacco hemophilia, bleeding disorders, blood disorders, genetic dis mosaic virus, tetracycline, kanamycin and ampicillin. Thus, orders, hormonal disorders, drug addictions and dependen also provided herein is a method of making microparticles by: cies, pain, kidney disease, liver disease, angiogenesis, pulmo (a) adding an antisolvent to a solution of a compound in an a nary arterial hypertension, neurological disorders, metabolic Solvent; and diseases, skin conditions, thyroid disease, osteoporosis, obe (b) gradually cooling the Solution to a temperature below sity, stroke, anemia, inflammatory diseases and autoimmune about 25°C., whereby a composition containing micropar diseases. ticles of the compound is formed, wherein steps (a) and (b) are 0144. The steps of the method provided herein include: performed simultaneously, sequentially, intermittently, or in combining a solution containing the compound with a coun any order. terion and an antisolvent, and gradually cooling the resulting 0151. In other embodiments, the microparticles can be Solution to a temperature whereby microparticles are formed. formed in the absence of antisolvent. Thus, also provided In one embodiment, the steps can be described as follows: herein is a method of making microparticles, by: (a) adding a 0145 1) To a solution containing a compound dissolved in counterion to a solution of a compound in a solvent; and (b) a Suitable solvent, adding a counterion and an antisolvent at gradually cooling the solution to a temperature below about concentrations that do not cause precipitation of the com 25°C., whereby a composition containing microparticles of pound at ambient temperature; the compound is formed, wherein steps (a) and (b) are per 0146 2) Precipitation: chilling the compound/counterion/ formed simultaneously, sequentially, intermittently, or in any antisolvent cocktail solution, via methods including chilling order. (heat-exchange) and endothermic reactions, to initiate forma 0152. In one embodiment, the microspheres formed by tion of microspheres; and contacting the compound with a counterion and antisolvent 0147 3) Dehydration: freezing of the microsphere suspen and exposed to low temperature, are separated from the Sus sion and removal of antisolvent and water by sublimation pension by methods including sedimentation or filtration (freeze-drying, e.g., at a temperature of about or at -5°C. to techniques. After separation from the original precipitation about or at -200° C.; or to about or at -20° C. to about or at mix, the microspheres can be washed and/or combined with -200° C., or about or at -30° C. to about or at -200° C., or other materials that improve and/or modify characteristics of about or at -40°C. to about or at -180°C., or about or at -45° the compounds and/or the microspheres. C. to about or at -180°C., or about-65°C. to about -175°C., 0153. In another embodiment, the microspheres prepared or about -80° C. to about or at -120° C., or about or at-65° by the methods provided herein do not have a direct thera C. to about or at -100° C.). peutic effect, but serve as micro-carriers for other therapeutic US 2012/O 141590 A1 Jun. 7, 2012 agent(s) or active agent(s), including diagnostic markers and agents, enteric coating agents, agents that confer acid resis nutritional Supplements. The additional agents can be added tance, such as against the acids of the digestive system, agents at the time of precipitation or can be added to the Suspension that confer protease resistance, chitosans, polymers, and of formed microspheres prior to lyophilization. Alternatively, flowability enhancers. the additional agents can be blended into dry powder contain (0160 The formation and characteristics of the micro ing microspheres. spheres produced by the methods provided herein can empiri 0154 Without being bound by any theory, in one aspect, cally be determined by varying parameters, including: nature the methods provided herein can permit the formation of and concentration of the compound, pH of the cocktail solu microspheres by: (1) neutralization of charges on the Surface tion, nature and concentration of the counterion, nature and of the compound by the counterion and (2) decreased solu concentration of the antisolvent, ionic strength and the cool bility of the compound in the solvent, caused by the combined ingrate by which gradual cooling is effected. The steps of the effects of added antisolvent and gradual cooling. methods provided herein render the method amenable to 0155 By choosing a suitable pH that is empirically deter high-throughput Screening, Such as in a microplate format, mined and can be in the range of, for example, about or at 1.0 for determining suitable combinations of compound, antisol to about or at 14.0, generally about or at pH 2.0 to about or at vent, counterion, pH, ionic strength and cooling ramp for the 10.5 or greater, depending on the compound, counterion, and generation of microspheres. antisolvent, in the presence of a suitable amount of the coun (0161 Molecules terion, a Substantial number of the charged groups, in some 0162 Any naturally occurring or synthetic molecule or embodiments all charged groups, on the Surface of the com compound that can form microparticles when in Solution in pound can become neutralized. A decrease in the polarity of the presence of one or more of a counterion and an antisol the solution by adding a suitable antisolvent can then initiate vent, is contemplated for use in the methods provided herein. the formation of microspheres by precipitation, phase sepa The compound can be an inorganic compound, including ration, colloid formation, or other such method. alkali and alkaline earth metal compounds and salts and other 0156 Alternatively, without being bound by any theory, in derivatives thereof, transition metal compounds, including Some embodiments, the observed phenomenon of the precipi coordination compounds and salts and other derivatives tation of microspheres also can be explained by the kosmo thereof, inorganic polymers. Such as polysiloxanes, and other tropic (structure forming) effect of counterions and antisol Such compounds known to those of skill in the art. Examples vents due to interactions with the solvent containing the of inorganic compounds include Some compounds that con compound at low temperatures. Regardless of the underlying tain carbon, but generally no carbon-carbon bonds; for mechanism, in the methods provided herein, the addition of example, carbon monoxide, carbon dioxide, carbonates, cya relatively small amounts of antisolvent and counterion to a nides, cyanates, carbides, and thiocyanates. Other inorganic Solution containing a compound of interest (aqueous or polar compounds include compounds formed from elements of the Solvent for polar compounds; non-polar or organic solvent for periodic table other than carbon. For example, any metal water-insoluble compounds) and cooling of the resulting (alkali metal, alkaline earth metal, transition metal, e.g.) car cocktail solution results in the production of compositions bonates, cyanides, cyanates, carbides, halides (F, Cl, Br, I), containing microspheres of the compounds. thiocyanates, selenocyanate, azides, oxides, hydroxides, Sul 0157. In one embodiment, gradual cooling chilling of the fides and hydrozides, coordination compounds, organometal cocktail solution can be performed by passing the cocktail lic compounds, and other such compounds as understood by Solution through a heat exchanger. The temperature of the those of skill in the art. heat exchanger and the flow rate of the cocktail through the 0163. Other classes of inorganic compounds are studied heat exchanger can be adjusted so that the cocktail is either and developed by chemists trained in materials Science, for pre-chilled prior to formation of the microspheres, or is example, polymeric and/or refractory materials such as sili chilled to a temperature whereby microspheres are formed. con and gallium arsenide, yttrium barium copper oxide, poly 0158. In another embodiment, the microspheres formed mers such silicones, polysilanes, polygermanes, polystan by the methods provided herein are concentrated or separated names and polyphospahaZenes. from the Suspension by methods such as sedimentation or 0164. The compound can be an organic compound, filtration techniques. Upon formation of the microspheres, including aliphatic, aromatic and alicyclic alcohols, alde their growth (size) can be controlled by adjusting the ionic hydes, carboxylic acids, esters, ketones, ethers, amines, strength, polarity, pH, or other parameters of the Suspension. amides, lactams, polymers thereof, and other such com The separation of microspheres from the liquid phase of the pounds known to those of skill in the art. Examples of organic cocktail solution can be performed by centrifugation, filtra compounds, which can be aliphatic, aromatic oralicyclic, can tion (hollow fiber, tangential flow, etc.), or other techniques. be any of the following, and similar classes of compounds The resulting microspheres or concentrated Suspensions known and understood by those of skill in the art: thereof can be lyophilized or air dried. 0.165 Alkyl refers to straight or branched chain substi 0159. In some embodiments, the microspheres separated tuted or unsubstituted hydrocarbon groups, generally from from the original precipitation mix or the dried microspheres about 1 to 40 carbonatoms, 1 to 20 carbon atoms, or 1 to 10 can be reconstituted prior to administration as a therapeutic carbon atoms. "Lower alkyl generally is an alkyl group of 1 agent or a carrier, or can be suspended in Solutions that con to 6 carbon atoms. An alkyl group can be a “saturated alkyl tain agents that modify characteristics of the microspheres. meaning that it does not contain any alkene or alkyne groups, The modifying agents can include but are not limited to or an alkyl group can be an "unsaturated alkyl meaning that bulking agents, excipients, inactive ingredients, stability it contains at least one alkene or alkyne group. An alkyl group enhancers, taste and/or odor modifiers or masking agents, that includes at least one carbon-carbon double bond (C—C) Vitamins, Sugars, therapeutic agents, anti-oxidants, immuno is referred to by the term “alkenyl, and an alkyl group that modulators, trans-membrane transport modifiers, anti-caking includes at least one carbon-carbon triple bond (C=C) is US 2012/O 141590 A1 Jun. 7, 2012

referred to by the term “alkynyl.” and in certain embodiments, azolyl), 1.2-oxazolyl (isoxazolyl), oxadiazolyl, 1.3-thiazolyl alkynyl groups are optionally Substituted. Alkyls include, but (thiazolyl), 1,2-thiazolyl (isothiazolyl), tetrazolyl pyridinyl are not limited to, methyl, ethyl, propyl, isopropyl, butyl, (pyridyl)pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, bute 1,2,4-triazinyl, 1.3.5-triazinyl, 1,2,4,5-tetrazinyl, indazolyl, nyl, hexenyl, ethynyl, propynyl, butynyl, hexynyl, haloalkyl indolyl, benzothiophenyl, benzofuranyl, benzothiazolyl, and heteroalkyl. benzimidazolyl, benzodioxolyl, acridinyl, quinolinyl, iso 0166 “Cycloalkyl. i.e. a saturated mono- or multicyclic quinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, ring system where each of the atoms forming a ring is a thienothiophenyl, 1.8-naphthyridinyl, other naphthyridinyls, carbon atom. Cycloalkyls can be formed by three, four, five, pteridinyl or phenothiazinyl. The heteroaryl compounds can six, seven, eight, nine, or more than nine carbon atoms. The be in the form of bicyclic radicals, and/or can optionally be ring system generally includes about 3 to about 12 carbon substituted. Examples of substituents include halo, hydroxy, atoms. The term "cycloalkyl includes rings that contain one amino, cyano, nitro, alkylamido, acyl, C-alkoxy, C-hy or more unsaturated bonds, and those that are Substituted. droxy-alkyl, C-aminoalkyl, C-alkylamino, alkylsulfe Examples of cycloalkyls include, but are not limited to, cyclo nyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluorom propane, cyclobutane, cyclopentane, cyclopentene, cyclo ethyl. Examples of heteroaryl groups include, but are not pentadiene, cyclohexane, cyclohexene, 1.3-cyclohexadiene, limited to, unsubstituted and mono- or di-substituted deriva 1,4-cyclohexadiene, cycloheptane and cycloheptene. tives of furan, benzofuran, thiophene, benzothiophene, pyr 0167 “Heterocyclic' compounds, which are rings where role, pyridine, indole, oxazole, benzoxazole, isoxazole, ben at least one atom forming the ring is a carbonatom and at least Zisoxazole, thiazole, benzothiazole, isothiazole, imidazole, one atom forming the ring is a heteroatom. benzimidazole, pyrazole, indazole, tetrazole, quinoline, iso 0168 “Bicyclic ring, which refers to two rings that are quinoline, pyridazine, pyrimidine, purine and pyrazine, fura fused. Bicyclic rings include, for example, decaline, pental Zan, 1.2.3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, ene, naphthalene, aZulene, heptalene, isobenzofuran, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, chromene, indolizine, isoindole, indole, purine, indoline, benzopyrazole, quinolizine, cinnoline, phthalazine, quinaZo indene, quinolizine, isoquinoline, quinoline, phthalazine, line and quinoxaline. Substituents can be, for example, halo, naphthyrididine, quinoxaline, cinnoline, pteridine, isochro hydroxy, cyano, O—C-alkyl, C-alkyl, hydroxy-C- man, chroman and various hydrogenated derivatives thereof. alkyl and amino C-alkyl. Bicyclic rings can be optionally Substituted. Each ring is 0172 “Non-aromatic heterocycle', i.e., a non-aromatic independently aromatic or non-aromatic. ring wherein one or more atoms forming the ring is a heteroa 0169. Aromatic' compounds, such as phenyl, naphthale tom. Non-aromatic heterocyclic rings can beformed by three, nyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl four, five, six, seven, eight, nine, or more than nine atoms. and indanyl. Aromatic compounds include benzenoid groups, Non-aromatic heterocycles can be optionally substituted. connected via one of the ring-forming carbon atoms, and Examples of non-aromatic heterocycles include, but are not optionally carrying one or more Substituents selected from an limited to, lactams, lactones, cyclic imides, cyclic thioimides, aryl, a heteroaryl, a cycloalkyl, a non-aromatic heterocycle, a cyclic carbamates, tetrahydrothiopyran, 4H-pyran, tetrahy halo, a hydroxy, an amino, a cyano, a nitro, an alkylamido, an dropyran, piperidine, 1,3-dioxin, 1.3-dioxane, 1,4-dioxin, acyl, a C- alkoxy, a C- alkyl, a C- hydroxyalkyl, a C 1,4-dioxane, piperazine, 1.3-oxathiane, 1,4-oxathin, 1.4-OX aminoalkyl, a C- alkylamino, an alkylsulfenyl, an alkyl athiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, Sulfinyl, an alkylsulfonyl, an Sulfamoyl, or a trifluoromethyl. Succinimide, barbituric acid, thiobarbituric acid, dioxopip An aromatic group can be substituted at one or more of the erazine, hydantoin, dihydrouracil, morpholine, trioxane, para, meta, and/or ortho positions. Examples of aromatic hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofu groups containing Substitutions include, but are not limited ran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyra to, phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, Zoline, pyrazolidine, imidazoline, imidazolidine, 1,3-diox 4-hydroxy-phenyl, 3-aminophenyl, 4-aminophenyl, 3-meth ole, 1,3-dioxolane, 1,3-dithiole, 1,3-dithiolane, isoxazoline, ylphenyl, 4-methylphenyl, 3-methoxyphenyl, 4-methox isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazo yphenyl, 4-trifluoromethoxyphenyl, 3-cyano-phenyl, 4-cy line, thiazolidine and 1.3-oxathiolane. anophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl, 0173 Arylalkyl compounds refer to an alkyl substituted hydroxymethyl-phenyl, (trifluoromethyl)phenyl, alkoxyphe with an aryl that can be optionally substituted. nyl, 4-morpholin-4-ylphenyl, 4-pyrrolidin-1-ylphenyl, 0.174 “Heteroarylalkyl compounds to an alkyl substi 4-pyrazolylphenyl, 4 triazolylphenyl and 4-(2-oxopyrrolidin tuted with a heteroaryl that can be optionally substituted. 1-yl)phenyl. 0.175. The substituent groups on organic compounds can 0170 Aryl compounds, which are monocyclic, bicyclic be one of several, including: “Amino” compounds refer to or tricyclic aromatic systems that contain no ring heteroat those containing a group of NH; “Hydroxy” refers to a oms. Examples of aryl include phenyl, naphthyl, anthracyl, group of —OH: “Nitro” refers to a group of —NO: “O indanyl, 1,2-dihydro-naphthyl, 1,4-dihydronaphthyl, inde carboxy' refers to a group of formula RC(=O)C)—: “C car nyl, 1.4-naphthoguinonyl and 1.2.3,4-tetrahydronaphthyl. boxy' refers to a group of formula C(=O)CR; “alkoxy” 0171 “Heteroaryl' compounds, which refer to an aro refers to a group of formula—OR; “acetyl' or “acyl refers to matic ring in which at least one atom forming the aromatic a group of formula C(=O)CH: “cyano” refers to a group of ring is a heteroatom. Such groups include oxazolyl, isox formula CN; “nitrile' refers to a compound having the struc azolyl, oxadiazolyl, thiazolyl, isothiazolyl pyridinyl, ture RC=N; "isocyanato” refers to a group of formula NCO: pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, “thiocyanato” refers to a group of formula CNS: “isothiocy quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, pyrro anato’ refers to a group of formula NCS: “Camido” refers to lyl, furanyl (furyl), thiophenyl (thienyl), imidazolyl pyra a group of formula C(=O)NR: “Namido” refers to a group Zolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1.3-oxazolyl (OX of formula RC(=O)NR; “sulfenyl refers to a group of for US 2012/O 141590 A1 Jun. 7, 2012

mula—SR: “sulfinyl refers to a group of formula—S(=O) bromine and Theobromine; sterols such as cholesterol and R; “sulfonyl refers to a group of formula—S(=O).R; “sul lanosterol, Steroids Such as estrogen, testosterone, canrenone, famoyl refers to a group of formula —S(=O)NR; oleandrin and spironolactone; penicillins, tetracyclines, Sul “sulfonyl halide” refers to compound of formula X S(=O) fonamide derivatives Such as Acetazolamide, Ambuside, R, where X is halo: “ester” refers to a group of formula AZosemide, Bumetanide. Butazolamide, Diphenylmethane RC(=O)CR', where R'zH; “amide” refers to a group of for 4.4'-disulfonamide, Disulfamide, Furosemide, uracils such as mula RC(=O)NR'. Aminometradine and Amisometradine, and the like, and (0176) Macromolecules and Small Molecules prostaglandins. 0177. The compounds used to form microparticles accord 0.179 The macromolecules and small molecules can fur ing to the methods provided herein can be macromolecules, ther be inorganic compounds or organic compounds, as dis or small molecules. The term "macromolecule' is understood cussed above, or combinations thereof. In addition, the mac by those of skill in the art, and generally refers to a naturally romolecules and Small molecules can have a variety of occurring or chemically synthetized organic or inorganic functional applications, such as therapeutic agents, diagnos molecule whose molecular weight is greater than or equal to tic agents, nutritional Supplements and other active agents. about a 1000 Daltons to about or greater than 1, 2, 3, 5, 7, 10 Macromolecule and Small molecule agents that can be for or more trillion Daltons, about 1000 or 1000 to about five mulated into microparticles according to the methods pro billion or five billion, about 1000 or 1000 to about one billion vided herein include, for example, antibiotics, chemothera or one billion, about 1000 or 1000 to about 50 million or 50 peutic agents, vaccines, hematopoietics, anti-infective million, about 1000 or 1000 to about 20 million or 20 million, agents, antiulcer agents, antiallergic agents, antipyretics, about 1000 or 1000 to about 15 million or 15 million, about analgesics, anti-inflammatory agents, antidementia agents, 1000 or 1000 to about 10 million or 10 million, about 1000 or antiviral agents, antitumoral agents, antidepressants, psycho 1000 to about 5 million or 5 million, about 1000 or 1000 to tropic agents, cardiotonics, antiarrhythmic agents, vasodila about one million or one million, about 1000 or 1000 to about tors, antihypertensive agents, antidiabetic agents, anticoagul 500,000 or 500,000, about 1000 or 1000 to about 300,000 or lants, cholesterol lowering agents, diagnostic markers, and 300,000, about 1000 or 1000 to about 200,000 or 200,000, nutritional Supplements, including herbal Supplements. about 1000 or 1000 to about 100,000 or 100,000, about 1000 0180. The macromolecule and small molecule agents or 1000 to about 50,000 or 50,000, about 1000 or 1000 to additionally can be selected frominorganic and organic drugs about 25,000 or 25,000, about 1000 or 1000 to about 15,000 including, but not limited to drugs that act on the peripheral or 15,000, about 1000 or 1000 to about 10,000 or 10,000, nerves, adrenergic receptors, cholinergic receptors, nervous about 1000 or 1000 to about 5,000 or 5,000, about 1000 or system, skeletal muscles, cardiovascular system, Smooth 1000 to about 3,000 or 3000, or about 1000 or 1000 to about muscles, blood circulatory system, synaptic sites, neuro-ef 2,000 or 2000 Daltons. Examples of macromolecules include fector junctional sites, endocrine system, hormone systems, proteins, peptides, nucleic acids, including DNA, RNA, immunological system, reproductive system, skeletal system, siRNA, snRNA, antisense RNA, and ribozymes, carbohy autocoid systems, alimentary and excretory systems, hista drates, lipids, fatty acids, polysaccharides, protein conju mine systems, and the like. The active agents that can be gates, viruses, virus particles, hormones, carbohydrate- or delivered using the compositions provided herein include, but polysaccharide-protein conjugates, viroids, prions and mix are not limited to, anticonvulsants, analgesics, antiparkin tures thereof. Sons, anti-inflammatories, calcium antagonists, anesthetics, antimicrobials, antimalarials, antiparasitics, antihyperten 0.178 The term “small molecule' is used herein in the sives, antihistamines, antipyretics, alpha-adrenergicagonists, sense that is understood by those of skill in the art, and alpha-blockers, biocides, bactericides, bronchial dilators, generally refers to a naturally occurring or chemically Syn beta-adrenergic blocking drugs, contraceptives, cardiovascu thetized organic or inorganic molecule that is less than about lar drugs, calcium channel inhibitors, depressants, diagnos 1000 Daltons, from about or at 1000 Daltons to about or at tics, diuretics, electrolytes, enzymes, hypnotics, hormones, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, hypoglycemics, hyperglycemics, muscle contractants, 375, 350, 325, 300, 275,250, 225, 200, 175, 150, 125, 100, muscle relaxants, neoplastics, glycoproteins, nucleoproteins, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less lipoproteins, ophthalmics, psychic energizers, sedatives, Ste Daltons. A Small molecule is understood to mean any mol roids, sympathomimetics, parasympathomimetics, tranquil ecule that is not a macromolecule. Such as a protein or nucleic izers, urinary tract drugs, vaccines, vaginal drugs, vitamins, acid, nor a macrmolecular assembly, such as a virus. A 'small minerals, nonsteroidal anti-inflammatory drugs, angiotensin molecule' as used herein can include a molecule containing converting enzymes, polynucleotides, polypeptides and two or more monomeric Subunits, such as a dipeptide or polysaccharides. dinucleotide, and generally is understood to refer to mol ecules that are about or at 1000 Daltons or below in molecular 0181 Exemplary agents that are macromolecules or small weight. Examples of Small molecules include, but are not molecules that can be used to form microparticles according limited to, inorganic molecules such as, but not limited to, to the methods provided herein include: carbon monoxide, carbon dioxide, metal (alkali metal, alka line earth metal, transition metal, e.g.) carbonates, cyanides, Exemplary Active Agent Categories for Macromolecules and cyanates, carbides, halides, thiocyanates, oxides, hydroxides, Small Molecules Sulfides and hydrozide, coordination compounds, e.g., the 0182 C-Adrenergic agonists such as Adrafinil, Adre cobalt salt Co(NH) Cls, and organometallic compounds, nolone, Amidephrine, Apraclonidine, Budralazine, Cloni e.g. Fe(C5H5). Small molecules that are organic compounds dine, Cyclopentamine, Detomidine, Dimetofrine, Dipivefrin, include, for example, nucleotides, amino acids, pteridines Ephedrine, Epinephrine, Fenoxazoline, Guanabenz, Guanfa Such as Furterene and Triamterene; purines such as Acefyl cine, Hydroxyamphetamine, Ibopamine, Indanazoline, line, 7-Morpholinomethyltheophylline, Pamabrom, Protheo Isometheptene, Mephentermine, Metaraminol, Methoxam US 2012/O 141590 A1 Jun. 7, 2012

ine Hydrochloride, Methylhexaneamine, Metizolene, Mido Phenazocine, Pheoperidine, Piminodine, Piritramide, Pro drine, Naphazoline, Norepinephrine, Norfenefrine, Octo heptazine, Promedol, Properidine, Propiram, Propoxyphene, drine, Octopamine, Oxymetazoline, Phenylephrine Sufentanil and Tilidine; Hydrochloride, Phenylpropanolamine Hydrochloride, Phe 0191 Analgesics (non-narcotic) Such as Acetaminophen, nylpropylmethylamine, Pholedrine, Propylhexedrine, Pseu Acetaminosalol, Acetanilide, Acetylsalicylsalicylic Acid, doephedrine, Rilmenidine, Synephrine, Tetrahydrozoline, Alclofenac. Alminoprofen, Aloxiprin, Aluminum Bis(acetyl Tiamenidine, TramaZoline, Tuaminoheptane, Tymazoline, salicylate), Aminochlorthenoxazin, 2-Amino-4-picoline, Tyramine and Xylometazoline; Aminopropylori, Aminopyrine, Ammonium Salicylate, Anti pyrine, Antipyrine Salicylate, Antrafenine, ApaZone, Aspirin, 0183 B-Adrenergic agonists such as Albuterol, Bam Benorylate, Benoxaprofen, BenZpiperylon, BenZydamine, buterol, Bitolterol, Carbuterol, Clenbuterol, Clorprenaline, p-Bromoacetanilide, 5-Bromosalicylic Acid Acetate, Buce Denopamine, Dioxethedrine, Dopexamine, Ephedrine, Epi tin, Bufexamac, Bumadizon, Butacetin, Calcium Acetylsali nephrine, Etafedrine, Ethylnorepinephrine, Fenoterol, For cylate, Carbamazepine, Carbetidine, Carbiphene, Carsalam, moterol, Hexoprenaline, Ibopamine, Isoetharine, Isoproter Chloralantipyrine, Chlorthenoxazin(e), Choline Salicylate, enal, Mabuterol, Metaproterenol, Methoxyphenamine, Cinchophen, Ciramadol, Clometacin, Cropropamide, Oxyfedrine, Pirbuterol, Prenalterol, Procaterol, Protokylol, Crotethamide, Dexoxadrol, Difenamizole. Diflunisal, Dihy Reproterol, Rimiterol, Ritodrine, Soterenol, Terbuterol and droxyaluminum Acetylsalicylate, Dipyrocetyl. Dipyrone, Xamoterol; Emorfazone, Enfenamic Acid, Epirizole, Etersalate, Ethen 0184 C.-Adrenergic blockers such as Amosulalol, Aroti Zamide, Ethoxazene, Etodolac, Felbinac, Fenoprofen, Floc nolol, Dapiprazole, Doxazosin, Ergoloid Mesylates, Fen tafenine, Flufenamic Acid, Fluoresone, Flupirtine, Flupro spiride, Indoramin, Labetalol, Nicergoline, PraZosin, Tera quaZone, Flurbiprofen, Fosfosal, Gentisic Acid, Glafenine, Zosin, Tolazoline, Trimazosin and Yohimbine; Ibufenac, Imidazole Salicylate, Indomethacin, Indoprofen, 0185 B-Adrenergic blockers such as Acebutolol, Alpre IsofeZolac, Isoladol, Isonixin, Ketoprofen, Ketorolac, p-Lac nolol, Amosulalol, Arotinolol, Atenolol, Befunolol, Betax tophenetide, Lefetamine, Loxoprofen, Lysine Acetylsalicy olol, Bevantolol, Bisoprolol, Bopindolol, Bucumolol. Befe late, Magnesium Acetylsalicylate, Methotrimeprazine, Meto tolol, Bufuralol, Bunitrolol, Bupranolol. Butidrine foline, Miroprofen, Morazone, Morpholine Salicylate, Hydrochloride. Butofilolol, Carazolol, Carteolol, Carvedilol, Naproxen, Nefopam, Nifenazone, 5' Nitro-2' propoxyaceta Celiprolol, Cetamolol, Cloranolol, Dilevalol, Epanolol, nilide, Parsalmide, Perisoxal, Phenacetin, Phenazopyridine Esmolol, Indenolol, Labetalol, Levobunolol, Mepindolol, Hydrochloride, Phenocoll, Phenopyrazone, Phenyl Acetyl Metipranalol, Metoprolol, Moprolol, Nadoxolol, Nifemalol, salicylate, Phenyl Salicylate, Phenyramidol, PipebuZone, Nipradillol, Oxprenolol, Penbutolol, Pindolol, Practolol. Piperylone, Prodilidine, Propacetamol, Propyphenazone, Proxazole, Quinine Salicylate, RamifenaZone, Rimazolium Pronethalol, Propranolol, Sotalol, Sulfinalol, Talinolol, Ter Metilsulfate, Salacetamide, Salicin, Salicylamide, Salicyla tatolol, Timolol, Toliprolol and Xibenolol; mide O-Acetic Acid, Salicylsulfuric Acid, Salsalte, Salver 0186 Alcohol deterrents such as Calcium Cyanamide Cit ine, Simetride, Sodium Salicylate, Sulfamipyrine, Suprofen, rated, Disulfuram, Nadide and Nitrefazole; Talniflumate, Tenoxicam, Terofenamate, Tetradrine, Tinori 0187 Aldose reductase inhibitors such as Epalrestat, dine, Tolfenamic Acid, Tolpronine, Tramadol, Viminol, Xen Ponalrestat, Sorbinil and Tolrestat; bucin and Zomepirac: 0188 Anabolics such as Androisoxazole, Androstenediol, 0.192 Androgens such as Androsterone, Boldenone, Bolandiol, Bolasterone, Clostebol, Ethylestrenol; Formyl Dehydroepiandrosterone, Fluoxymesterone, Mestanolone, dienolone, 4-Hydroxy-19-nortestosterone, Methandriol, Mesterolone, Methandrostenolone, 17-Methyltestosterone, Methenolone, Methyltrienolone, Nandrolone, Nandrolone 17C.-Methyltestosterone 3-Cyclopenty1 Enol Ether, Nore Decanoate, Nandrolone p-Hexyloxyphenylpropionate, Nan thandrolone, Normethandrone, Oxandrolone, Oxymester drolone Phenpropionate, Norbolethone, Oxymesterone, one, Oxymetholone, Prasterone, Stanlolone, Stanozolol, Tes Pizotyline, Quinbolone, Stenbolone and Trenbolone: tosterone, Testosterone 17-Chloral Hemiacetal, Testosterone 0189 Analgesics (dental) such as Chlorobutanol, Clove 17B-Cypionate, Testosterone Enanthate, Testosterone Nico and Eugenol; tinate, Testosterone Pheynylacetate, Testosterone Propionate 0.190 Analgesics (narcotic) such as Alfentanil, Allylpro and Tiomesterone; dine, Alphaprodine, Anileridine, Benzylmorphine, Bezitra 0193 Anesthetics such as Acetamidoeugenol, Alfadolone mide, Buprenorphine. Butorphanol, ClonitaZene, Codeine, Acetate, Alfaxalone, Amucaine. Amolanone, Amylocalne Codeine Methyl Bromide, Codeine Phosphate, Codeine Sul Hydrochloride, Benoximate, Benzocaine, Betoxycaine, fate, Desomorphine, Dextromoramide, DeZocine, Diampro Biphenamine, Bupivacaine. Butacaine, Butaben, Butanilic mide, Dihydrocodeine, Dihydrocodeinone Enol Acetate, aine, Burethamine. Buthalital Sodium, Butoxycaine, Cartic Dihydromorphine, Dimenoxadol, Dimepheptanol, Dimeth aine, 2-Chloroprocaine Hydrochloride, Cocaethylene, ylthiambutene, Dioxaphetyl Butyrate, Dipipanone, Eptazo Cocaine, Cyclomethycaine, Dibucaine Hydrochloride, cine, Ethoheptazine, Ethylmethlythiambutene, Ethylmor Dimethisoquin, Dimethocaine, Diperadon Hydrochloride, phine, Etonitazene, Fentanyl, Hydrocodone, Hydrocodone Dyclonine, Ecgonidine, Ecgonine, Ethyl Aminobenzoate, Bitartrate, Hydromorphone, Hydroxypethidine, Isometha Ethyl Chloride, Etidocaine, Etoxadrol, B-Eucaine, Euprocin, done, Ketobemidone, Levorphanol, Lofentanil, Meperidine, Fenalcomine, Fomocaine, Hexobarbital, Hexylcaine Hydro Meptazinol, Metazocine, Methadone Hydrochloride, Meto chloride, Hydroxydione Sodium, Hydroxyprocaine, Hydrox pon, Morphine, Morphine Derivatives, Myrophine, Nalbu ytetracaine, Isobutyl p-Aminobenzoate, Kentamine, Leuci phine, Narceline, Nicomorphine, Norlevorphanol, Normetha nocaine Mesylate, LeVoxadrol, Lidocaine, Mepivacaine, done, Normorphine, Norpipanone, Opium, Oxycodone, Meprylcaine Hydrochloride, Metabutoxycaine Hydrochlo Oxymorphone, Papavereturn, Pentazocine, Phenadoxone, ride, Methohexital Sodium, Methyl Chloride, Midazolam, US 2012/O 141590 A1 Jun. 7, 2012

Myrtecaine, Naepaine, Octacaine, Orthocaine, Oxethazaine, 0203 Antiandrogens such as Bifluranol, Cyoctol, Cypro Parethoxycaine, Phenacaine Hydrochloride, Phencyclidine, terone, Delmadinone Acetate, Flutimide, Nilutamide and Phenol, Piperocaine, Piridocaine, Polidocanol, Pramoxine, Oxendolone; Prilocalne, Procaine, Propanidid, Propanocaine, Propara 0204 Antianginals such as Acebutolol, Alprenolol, Amio caine, Propipocaine, Propofol, Propoxycaine Hydrochloride, darone, Amlodipine, Arotinolol, Atenolol, Bepridil, Bevan Pseudococaine, Pyrrocaine, Quinine Urea Hydrochloride, tolol, Bucumolol, Bufetolol, Bufuralol, Bunitrolol, Bupra Risocaine, Salicyl Alcohol, Tetracaine Hydrochloride. Thial nolol, Carozolol, Carteolol, Carvedilol, Celiprolol, Cinepazet barbital. Thimylal. Thiobutabarbital. Thiopental Sodium, Maleate, Diltiazem, Epanolol, Felodipine, Gallopamil, Imo Tolycaine, Trimecaine and Zolamine; lamine, Indenolol, Isosorbide Dinitrate, Isradipine, 0194 Anorexics such as Aminorex, Amphecloral, Limaprost, Mepindolol, Metoprolol, Molsidomine, Nadolol, Amphetamine, BenZaphetamine, Chlorphentermine, Nicardipine, Nifedipine, Nifenalol, Nilvadipine, Nipradilol, Clobenzorex, Cloforex, Clortermine, Cyclexedrine. Destro Nisoldipine, Nitroglycerin, Oxprenolol, Oxyfedrine, Oza amphetamine Sulfate, Diethylpropion, Diphemethoxidine, grel, Penbutolol, Pentaerythritol Tetranitrate, Pindolol, N-Ethylamphetamine, Fenbutrazate, Fenfluramine, Fenpro Pronethalol, Propranolol, Sotalol, Terodiline, Timolol, Tolip porex. Furfurylmethylamphetamine, Levophacetoperate, rolol and Verapamil: Mazindol, Mefenorex, Metamfeproamone, Methamphet 0205 Antiarrhythmics such as Acebutol, Acecaine, amine, Norpseudoephedrine, Phendimetrazine, Phendime Adenosine, Ajmaline, Alprenolol, Amiodarone, Amoproxan, trazine Tartrate, Phenmetrazine, Phenpentermine, Phenyl Aprindine, Arotinolol, Atenolol, Bevantolol, Bretylium Tosy propanolamine Hydrochloride and Picilorex: late, Bubumolol, Bufetolol, Bunaftine, Bunitrolol, Bupra 0.195 Anthelmintics (Cestodes) such as Arecoline, Aspi nolol. Butidrine Hydrochloride, Butobendine, Capobenic din, Aspidinol, Dichlorophen(e), Embelin, Kosin, Naptha Acid, Carazolol, Carteolol, Cifenline, Cloranolol, Disopyra lene, Niclosamide, Pellertierine, Pellertierine Tannate and mide, Encamide, Esmolol, Flecamide, Gallopamil, Hydro Quinacrine; quinidine, Indecamide, Indenolol, Ipratropium Bromide, 0196. Anthelmintics (Nematodes) such as Alantolactone, Lidocaine, Lorajmine, Lorcamide, Meobentine, Metipra Amoscanate, Ascaridole, Bephenium, Bitoscanate, Carbon nolol, Mexiletine, Moricizine, Nadoxolol, Nifemalol, Oxpre Tetrachloride, Carvacrol, Cyclobendazole, Diethylcarbam nolol, Penbutolol, Pindolol, Pirmenol, Practolol, Prajmaline, azine, Diphenane, Dithiazanine Iodide, Dymanthine, Gentian Procainamide Hydrochloride, Pronethalol, Propafenone, Violet, 4-Hexylresorcinol, Kainic Acid, Mebendazole, Propranolol, Pyrinoline, Quinidine Sulfate, Quinidine, 2-Napthol, Oxantel, Papain, piperazine, piperazine Adipate, Sotalol, Talinolol, Timolol, Tocamide, Verapamil, Vicquidil piperazine Citrate, piperazine Edetate Calcium, piperazine and Xibenolol; Tartrate, Pyrantel, Pyrvinium Pamoate, C.-Santonin, Stilba 0206 Antiarteriosclerotics such as Pyridinol Carbamate; Zium Iodide, Tetrachloroethylene, Tetramisole, thiabenda 0207 Antiarthritic/Antirheumatics such as Allocupreide Zole, Thymol, Thymyl N-Isoamylcarbamate, Triclofenol pip Sodium, Auranofin, Aurothioglucose, Aurothioglycanide, erazine and Urea Stibamine; Azathioprine, Calcium 3-Aurothio-2-propanol-1-sulfonate, 0.197 Anthelmintics (Onchocerca) such as Ivermectin and Celecoxib, Chloroquine, Clobuzarit, Cuproxoline, Diacerein, Suramin Sodium; Glucosamine, Gold Sodium Thiomalate, Gold Sodium Thio 0198 Anthelmintics (Schistosoma) such as Amoscanate, sulfate, Hydroxychloroquine, KebuZone, Lobenzarit, Melit Amphotalide, Antimony Potassium Tartrate, Antimony tin, Methotrexate, Myoral and Penicillamine: Sodium Gluconate, Antimony Sodium Tartrate, Antimony 0208 Antibacterial (antibiotic) drugs including: Ami Sodium Thioglycollate, Antimony Thioglycollamide, Becan noglycosides such as Amikacin, Apramycin, Arbekacin, thone, Hycanthone, Lucanthone Hydrochloride, Niridazole, Bambermycins, Butirosin, Dibekacin, Dihdrostreptomycin, Oxamniquine, Praziquantel, Stibocaptate, Stibophen and Fortimicin(s), Gentamicin, Ispamicin, Kanamycin, Microno Urea Stibamine; micin, Neomycin, Neomycin Undecylenate, Netilmicin, 0199 Anthelmintic (Trematodes) such as Anthiol imine Paromomycin, Ribostamycin, Sisomicin, Spectinomycin, and Tetrachloroethylene; Streptomycin, Streptonicozid, Vancomycin (also considered 0200 Antiacne drugs such as Adapelene, Algestone a glycopeptide) and Tobramycin; Acetophenide, AZelaic Acid, Benzoyl Peroxide, Cyoctol, 0209 Amphenicols such as AZidamfenicol, Chloram Cyproterone, Motretinide, Resorcinol, Retinoic Acid, Tetro phenicol, Chloramphenicol Palmitate, Chloramphenicol quinone and Tretinonine; Pantothenate, Florfenicol and Thiamphenicol; 0201 Antiallergics such as Amlexanox, Astemizole, AZelastine, Cromolyn, Fenpiprane. Histamine, Ibudilast, 0210 Ansamycins such as Rifamide, Rifampin, Rifamy Nedocromil, Oxatomide, Pentigetide, Poison Ivy Extract, cin and Rifaximin: Poison Oak Extract, Poison Sumac Extract, Repirinast, Tra 0211 B-Lactams, including: Carbapenems such as Imi nilast, TraxanoX and Urushiol: penem; 0202 Antiamebics such as Arsthinol, Bialamicol, Carbar 0212 Cephalosporins such as Cefactor, Cefadroxil, Cefa sone, Cephaeline, Chlorbetamide, Chloroquine, Chlorphe mandole, Cefatrizine, Cefazedone, Cefazolin, Cefixime, noxamide, Chlortetracycline, Dehydroemetime, Dibro Cefinenoxime, Cefodizime, Cefonicid, CefoperaZone, Cefo mopropamidine, Diloxanide, DephetarSone, Emetine, ranide, Cefotaxime, Cefotiam, Ce?pimizole, Ce?pirimide, Fumagillin, Glaucarubin, Glycobiarsol, 8-Hydroxy-7-iodo Cefpodoxime Proxetil, Cefroxadine, Cefsulodin, Ceftazi 5-quinolinesulfonic Acid, Iodochlorhydroxyquin, dime, Cefteram, Ceftezole, Ceftibuten, Ceftizoxime, Ceftri Iodoquinol, Paromomycin, Phanquinone, Phearsone Sul axone, Cefuroxime, CefuZonam, Cephacetrile Sodium, foxylate, Polybenzarsol, Propamidine, Quinfamide, Secnida Cephalexin, Cephaloglycin, Cephaloridine, Zole, Sulfarside, Teclozan, Tetracycline. Thiocarbamizine, 0213 Cephalosporin, Cephalothin, Cephapirin Sodium, Thiocarbarsone and Timidazole; Cephradine and Piveefalexin; US 2012/O 141590 A1 Jun. 7, 2012 20

0214) Cephamycins such as CefbuperaZone, Cefinetazole, sulfathiazole, Sulfabenzamide, Sulfacetamide, Sulfachlorpy Cefninox, Cefetan and Cefoxitin; ridazine, Sulfachrysoidine, Sulfacytine, Sulfadiazine, 0215 Monobactams such as Aztreonam, Carumonam and Sulfadicramide, Sulfadimethoxine, Sulfadoxine, Sulfaethi Tigemonam, dole, Sulfaguanidine, Sulfaguanol, Sulfalene, Sulfaloxic 0216 Oxacephems such as Flomoxef and Moxolactam; Acid, Sulfamerazine, Sulfameter, Sulfamethazine, Sulfame 0217 Penicillins such as Amidinocillin, Amdinocillin thizole, Sulfamethomidine, Sulfamethoxazole, Sul Pivoxil, Amoxicillin, Ampicillin, Apalcillin, Aspoxicillin, famethoxypyridazine, Sulfametrole, Sulfamidochrysoidine, Azidocillan, AZlocillan, Bacampicillin, Benzylpenicillinic Sulfamoxole, Sulfanilamide, Sulfanilamidomethanesulfonic Acid, Benzylpenicillin Sodium, Carbenicillin, Carfecillin Acid Triethanolamine Salt, 4-Sulfanilamidosalicylic Acid, Sodium, Carindacillin, Clometocillin, Cloxacillin, Cyclacil N-Sulfanilylsulfanilamide, Sulfanily lurea, N-Sulfanilyl-3,4- lin, Dicloxacillin, Diphenicillin Sodium, Epicillin, Fenbeni xylamide, Sulfanitran, Sulfaperine, Sulfaphenazole, Sul cillin, Floxicillin, Hetacillin, Lenampicillin, Metampicillin, faproxyline, Sulfapyrazine, Sulfapyridine, Sulfasomizole, Methicillin Sodium, Mezlocillin, Nafcillin Sodium, Oxacil Sulfasymazine, Sulfathiazole, Sulfathiourea, Sulfatolamide, lin, Penamecillin, Penethamate Hydriodide, Penicillin G Sulfisomidine and Sulfisoxazole; Benethamine, Penicillin G BenZathine, Penicillin G Benzhy 0227 Sulfones such as Acedapsone, Acediasulfone, drylamine, Penicillin G Calcium, Penicillin G Hydrabamine, Acetosulfone Sodium, Dapsone, Diathymosulfone, Gluco Penicillin G Potassium, Penicillin G Procaine, Penicillen N, sulfone Sodium, Solasulfone, Succisulfone, Sulfanilic Acid, Penicillin O, Penicillin V, Penicillin V BenZathine, Penicillin p-Sulfanilylbenzylamine, pp'-Sulfonyldianiline-N,N' diga V Hydrabamine, Penimepicycline, Phenethicillin Potassium, lactoside, Sulfoxone Sodium and Thiazolsulfone; and Piperacillin, Pivapicillin, Propicillin, Quinacillin, Sulbenicil 0228 others such as Clofoctol, Hexedine, Methenamine, lin, Talampicillin, Temocillin and Ticarcillin; Methenamine Anhydromethylene-citrate, Methenamine 0218 Lincosamides Such as Clindamycin and Lincomy Hippurate, Methenamine Mandelate, Methenamine Sul cin; fosalicylate, Nitroxoline and Xibornol: 0219 Macrollides such as Azithroimycin, Carbomycin, 0229 Anticholinergics such as Adiphenine Hydrochlo Clarithromycin, Erythromycin, Erythromycin Acistrate, ride, Alverine, Ambutonomium Bromide, Aminopentamide, Erythromycin Estolate, Erythromycin Glucoheptonate, Amixetrine, Amprotropine Phosphate, Anisotropine Methyl Erythromycin Lactobionate, Erythromycin Propionate, bromide, Apoatropine, Atropine, Atropine N-Oxide, Benac Erythromycin Stearate, Josamycin, Leucomycins, Mideca tyZine, Benapryzine, Benzetimide, Benzilonium Bromide, mycins, Miokamycin, Oleandomycin, Primycin, Rokitamy Benztropine Mesylate, Bevonium Methyl Sulfate, Biperiden, cin, Rosaramicin, Roxithromycin, Spiramycin and Trolean Butropium Bromide, N-Butylscopolammonium Bromide, domycin; Buzepide, Camylofine, Caramiphen Hydrochloride, Chlor 0220 Polypeptides such as Amphomycin, Bacitracin, benzoxamine, Chlorphenoxamine, Cimetropium Bromide, Capreomycin, Colistin, Enduracidin, Enviomycin, Clidinium Bromide, Cyclodrine, Cyclonium Iodide, Fusafungine, Gramicidin(s), Gramicidin S. Mikamycin, Cycrimine Hydrochloride, Deptropine, Dexetimide, Dibuto Polymyxin, Polymyxin B-Methanesulfonic Acid, Pristina line Sulfate, Dicyclomine Hydrochloride, Diethazine, Dife mycin, Ristocetin, Teicoplanin, Thiostrepton, Tuberactino merine, Dihexyverine, Diphemanil Methylsulfate, N-(1,2- mycin, Tyrocidine, Tyrothricin, Vancomycin, Viomycin, Vio Diphenylethyl)nicotinamide, Dipiproverine, Diponium mycin Pantothenate, Virginiamycin and Zinc Bacitracin; Bromide, Emepronium Bromide, Endobenzyline Bromide, 0221 Tetracyclines such as Apicycline, Chlortetracycline, Ethopropazine, Ethybenztropine, Ethylbenzhydramine, Eto Clomocycline, Demeclocycline, Doxycycline, Guamecy midoline, Eucatropine, Fenpiverinium Bromide, Fentonium cline, Lymecycline, Meclocycline, Methacycline, Minocy Bromide, Flutropium Bromide, Glycopyrrolate, Heteronium cline, Oxytetracycline, Penimepicycline, Pipacycline, Roli Bromide, Hexocyclium Methyl Sulfate, Homatropine, Hyos tetracycline, Sancycline, Senociclin and Tetracycline; and cyamine, Ipratropium Bromide, Isopropamide, Levomepate, 0222 other antibiotics such as Cycloserine, Mupirocin Mecloxamine, Mepenzolate Bromide, Metcaraphen, Meth and Tuberin; antheline Bromide, Methixene, Methscopolamine Bromide, 0223) Antibacterial drugs (synthetic), including: 2,4-Di Octamylamine, Oxybutynin Chloride, Oxyphencyclimine, aminopyrimidines Such as Brodimoprim, TetroXoprim and Oxyphenonium Bromide, Pentapiperide, Penthienate Bro Trimethoprim; mide, Phencarbamide, Phenglutarimide, Pipenzolate Bro 0224 Nitrofurans such as Furaltadone. Furazolium Chlo mide, Piperidolate, Piperilate, Poldine Methysulfate, Pridi ride, Nifuradene, Nifuratel, Nifurfoline, Nifurpirinol, Nifur nol, Prifinium Bromide, Procyclidine, Propantheline prazine, Nifurtoinol and Nitrofurantoin: Bromide, Propenzolate, Propyromazine, Scopolamine, Sco 0225. Quinolones and Analogs such as Amifloxacin, polamine N-Oxide, Stilonium Iodide, Stramonium, Sultropo Cinoxacin, Ciprofloxacin, Difloxacin, Enoxacin, Fleroxacin, nium, Thihexinol, Thiphenamil, Tiemonium Iodide, Time Flumequine, Lomefloxacin, Miloxacin, Nalidixic Acid, Nor pidium Bromide, Tiquizium Bromide, Tridihexethyl Iodide, floxacin, Ofloxacin, Oxolinic Acid, Pefloxacin, Pipemidic Trihexyphenidyl Hydrochloride, Tropacine, Tropenzile, Acid, Piromidic Acid, Rosoxacin, Temafloxacin and Tosu Tropicamide, Trospium Chloride, Valethamate Bromide and floxacin: Xenylropium Bromide: 0226 Sulfonamides such as Acetyl Sulfamethoxypyra 0230 Anticonvulsants such as Acetylpheneturide, Albu zine, Acetyl Sulfisoxazole, AZosulfamide, Benzylsulfamide, toin, Aloxidone, Aminoglutethimide, 4-Amino-3-hydroxy Chloramine-B, Chloramine-T, Dichloramine T. Formosul butyric Acid, Atrolactamide, Beclamide, Buramate, Calcium fathiazole, N. Formylsulfisomidine, N°-f-D-Glucosylsulfa Bromide, Carbamazepine, Cinromide, Clomethiazole, Clon nilamide, Mafenide, 4'-(Methylsulfamoyl)sulfanilanilide, azepam, Decimenide, Diethadione, Dimethadione, Doxeni p-Nitrosulfathiazole, Noprylsulfamide, Phthalylsulfaceta toin, Eterobarb, Ethadione, Ethosuximide, Ethotoin, Fluo mide, Phthalylsulfathiazole, Salazosulfadimidine, Succinyl resone, Garbapentin, 5-Hydroxytryptophan, Lamotrigine, US 2012/O 141590 A1 Jun. 7, 2012

Lomactil, Magnesium Bromide, Magnesium Sulfate, Mephe 0244 Antifungal drugs (antibiotics), including: Polyenes nyloin, Mephobarbital, Metharbital, Methetoin, Methsuxim Such as Amphotericin-B, Candicidin, Dermostatin, Filipin, ide, 5-Methyl-5-(3-phenanthryl)hydantoin, 3-Methyl-5-phe Fungichromin, Hachimycin, Hamycin, Lucensomycin, nylhydantoin, Narcobarbital, Nimetazepam, Nitrazepam, Mepartricin, Natamycin, Nystatin, Pecilocin and Perimycin; Paramethadione, Phenacemide, Phenetharbital, Pheneturide, and others such as AZaserine, Griseofulvin, Oligomycins, Phenobarbital, Phenobarbital Sodium, Phensuximide, Phe Neomycin Undecylenate, Pyrrolnitrin, Siccanin, Tubercidin nylmethylbarbituric Acid, Phenyloin, Phethenylate Sodium, and Viridin; Potassium Bromide, Pregabatin, Primidone, Progabide, 0245 Antifungal drugs (synthetic), including: Ally Sodium Bromide, Sodium Valproate, Solanum, Strontium lamines such as Naftifine and Terbinafine; Bromide, Suclofenide, Sulthiame, Tetrantoin, Tiagabine, Tri 0246. Imidazoles such as Bifonazole, Butoconazole, methadione, Valproic Acid, Valpromide, Vigabatrin and Chlordantoin, Chlormidazole, Cloconazole, Clotrimazole, Zonisamide: Econazole, Enilconazole, Fenticonazole, Isoconazole, Keto 0231. Antidepressants, including: Bicyclics such as conazole, Miconazole, Omoconazole, Oxiconazole, Nitrate, Binedaline, CaroXaZone, Citalopram, Dimethazan, Indal Sulconazole and Tioconazole; pine, Fencamine, Fluvoxamine Maleate, Indeloxazine 0247 Triazoles such as Fluconazole, Itraconazole and Hydrochcloride, Nefopam, Nomifensine, Oxitriptan, Terconazole; and Oxypertine, Paroxetine, Sertraline. Thiazesim, Trazodone, 0248 others such as Acrisorcin, Amorolfine, Biphe Venlafaxine and Zometapine; namine, Bromosalicylchloranilide, Buclosamide, Calcium 0232 Hydrazides/Hydrazines such as Benmoxine, Ipro Propionate, Chlophenesin, Ciclopirox, Cloxyquin, Coparaf clozide, Iproniazid, Isocarboxazid, Nialamide, Octamoxin finate, Diamthazole, Dihydrochloride. Exalamide, Flucy and Phenelzine; tosine, Halethazole, Hexetidine, Loflucarban, Nifuratel, 0233 Pyrrolidones such as Cotinine, Rolicyprine and Potassium Iodide, Propionic Acid, Pyrithione, Salicylanilide, Rolipram; Sodium Propionate, Sulbentine, Tenonitrozole, Tolciclate, 0234 Tetracyclics such as Maprotiline, Metralindole, Tolindate, Tolnaftate, Tricetin, Ujothion, Undecylenic Acid Mianserin and Oxaprotiline: and Zinc Propionate; 0235 Tricyclics such as Adinazolam, Amitriptyline, Ami 0249 Antiglaucoma drugs such as Acetazolamide, triptylinoxide. Amoxapine, Butriptyline, Clomipramine, Befunolol, Betaxolol, Bupranolol, Carteolol, Dapiprazoke. Demexiptiline, Desipramine, Dibenzepin, Dimetracrine, Dichlorphenamide, Dipivefrin, Epinephrine, Levobunolol, Dothiepin, Doxepin, Fluacizine, Imipramine, Imipramine Methazolamide, Metipranolol, Pilocarpine, Pindolol and N-Oxide, Iprindole, Lofepramine, Melitracen, Metapramine, Timolol; Nortriptyline, Noxiptilin, Opipramol. Pizotyline, 0250 Antigonadotropins such as Danazol, Gestrinone and Propizepine, Protriptyline, Quinupramine, Tianeptine and Paroxypropione; Trimipramine; and 0251 Antigout drugs such as Allopurinol, Carprofen, 0236 others such as Adrafinil, Benactyzine, Bupropion, Colchicine, Probenecid and Sulfinpyrazone: Butacetin, Deanol, Deanol Aceglumate, Deanol Acetamido 0252 Antihistamines, including: Alkylamine derivatives benzoate, Dioxadrol, Etoperidone, Febarbamate, Femoxet Such as Acrivastine, Bamipine, Brompheniramine, Chlorphe ine, Fenpentadiol, Fluoxetine, Fluvoxamine, Hematoporphy niramine, Dimethindene, Metron S. Pheniramine, Pyrrob rin, Hypercinin, Levophacetoperane, Medifoxamine, utamine. Thenaldine, Tolpropamine and Triprolidine; Minaprine, Moclobemide, Oxaflozane, Piberaline, Prolin 0253) Aminoalkyl ethers such as Bietanautine, Bromo tane, Pyrisuccideanol, Rubidium Chloride, Sulpiride, Sulto diphenhydramine, Carbinoxamine, Clemastine, Diphenly pride, Teniloxazine, ThoZalinone, Tofenacin, Toloxatone, pyraline, Doxylamine, Embrammine, Medrylamine, Tranylcypromine, L-Tryptophan, Viloxazine and Zimeldine; Mephenphydramine, p-Methyldiphenhydramine, 0237 Antidiabetics, including: Biguanides such as Orphenadrine, Phenyltoloxamine, Piprinhydrinate and Seta Buformin, Metformin and Phenformin; S1ne, 0238 Hormones such as Glucagon, Insulin, Insulin Injec 0254 Ethylenediamine derivatives such as Alloclamide, tion, Insulin Zinc Suspension, Isophane Insulin Suspension, p-Bromtripelennamine, Chloropyramine, Chlorothen, Protamine Zinc Insulin Suspension and Zinc Insulin Crystals: Histapyrrodine, Methafurylene, Methaphenilene, Methapy 0239 Sulfonylurea derivatives such as Acetohexamide, rilene, Phenbenzamine, Pyrilamine, Talastine, Thenyl 1-Butyl-3-metanily lurea, Carbutamide, Chlorpropamide, diamine, Thonzylamine Hydrochloride, Tripelennamine and Glibornuride, Gliclazide, Glipizide, Gliquidone, Glisoxepid, Zolamine; Glyburide, Glybuthiazol(e), Glybuzole, Glyhexamide, Gly 0255 Piperazines such as Cetirizine, Chlorcyclizine, Cin midine, Glypinamide, Phenbutamide, Tolazamide, Tolbuta narizine, Clocinizine and Hydroxy Zine; mide and Tolcyclamide; and 0256 Tricyclics, including: Phenothiazines such as 0240 others such as Acarbose, Calcium Mesoxalate and Ahistan, Etymemazine, Fenethazine, N-Hydroxyethyl Miglitol; promethazine Chloride, Isopromethazine, Mequitazine, 0241 Antidiarrheal drugs such as Acetyltannic Acid, Promethazine, Pyrathiazine and Thiazinamium Methyl Sul Albumin Tannate, Alkofanone, Aluminum Salicylates—Ba fate; and sic, Catechin, Difenoxin, Diphenoxylate, Lidamidine, Lop 0257 others such as AZatadine, Clobenzepam, Cyprohep eramide, Mebiquine, Trillium and Uzarin; tadine, Deptropine, Isothipendyl, Loratadine and Pro 0242 Antidiuretics such as Desmopressin, Felypressin, thipendyl; and Lypressin, Ornipressin, Oxycinchophen, Pituitary—Poste 0258 other antihistamines such as Antazoline, Astemi rior, Terlipressin and Vasopressin; Zole, AZelastine, Cetoxime, Clemizole, Clobenztropine, 0243 Antiestrogens such as Delmadinone Acetate, Diphenazoline, Diphenhydramine, Fluticasone Propionate, Ethamoxytriphetol, Tamoxifen and Toremifene: Mebhydroline, Phenindamine, Terfenadine and Tritoqualine: US 2012/O 141590 A1 Jun. 7, 2012 22

0259 Antihyperlipoproteinemics, including: Aryloxyal 0275 Reserpine derivatives such as Bietaserpine, Deser kanoic acid derivatives such as Beclorbrate, Bazafibrate, Bin pidine, Rescinnamine, Reserpine and Syrosingopine; ifibrate, Ciprofibrate, Clinofibrate, Clofibrate, Clofibric Acid, 0276 Sulfonamide derivatives such as Ambuside, Clopa Etonfibrate, Fenofibrate, Gemfibrozil, Nicofibrate, Pirifi mide, Furosemide, Indapamide, QuinethaZone, Tripamide brate, Ronifibrate, Simfibrate and Theofibrate; and Xipamide; and 0260 Bile acid sequesterants such as Cholestyramine 0277 others such as Ajmaline, Y-Aminobutyric Acid, Resin, Colestipol and Polidexide: Bufeniode, Candesartan, Chlorthalidone, C-cletaine, 0261 HMG CoA reductase inhibitors such as Fluvastatin, C-closidomine, Cryptenamine Tannates, Eprosartan, Lovastatin, Pravastatin Sodium and Simvastatin: Fenoldopam, Floseduinan, Indoramin, Irbesartan, Ket 0262 Nicotinic acid derivatives Aluminum Nicotinate, anserin, Losartan, Metbutamate, Mecamylamine, Methyl Acipimox, Niceritrol, Nicoclonate, Nicomol and Oxiniacic dopa, Methyl 4-Pyridyl Ketone Thiosemicarbarzone, Meto Acid; lazone, Minoxidil, Muzolimine, Pargyline, Pempidine, 0263 Thyroid hormones and analogs such as Etiroxate, Pinacidil, Piperoxan, Primaperone, Protoveratrines, Thyropropic Acid and Thyroxine; and Raubasine, Rescimetol, Rilmenidene, Saralasin, Sodium 0264 others such as Acifran, AZacosterol, Benfluorex, Nitroprusside, Ticrynafen, Trimethaphan Camsylate, Tyrosi B-Benzalbutyramide, Carnitine, Chondroitin Sulfate, Clom nase, Urapidil and Valsartan; estone, Detaxtran, Dextran Sulfate Sodium, 5,8,11,14, 17 0278 Antihyperthyroids such as 2-Amino-4-methylthiaz Eicosapentaenoic Acid, Eritadenine, Furazbol, Meglutol, ole, 2-Aminothiazole, Carbimazole, 3,5-Dibromo-L-ty Melinamide, Mytatrienediol, Ornithine, Y-Oryzanol, Pan rosine, 3,5-Diiodotyrosine, Hinderin, Iodine, lothiouracil, tethine, Penataerythritol Tetraacetate, C.-Phenylbutyramide, Methimazole, Methylthiouracil, Propylthiouracil, Sodium Pirozadil, Probucol, C-Sitosterol, Sultosilic Acid, piperazine Perchlorate. Thibenzazoline. Thiobarbital and 2-Thiouracil; Salt, Tiadenol, Triparanol and Xenbucin: 0279 Antihypotensive drugs such as Amezinium Methyl 0265 Antihypertensive drugs, including: Arylethanola Sulfate, Angiotensin Amide, Dimetofrine, Dopamine, Etife mine derivatives such as Amosulalol, Bufuralol, Dilevalol, lmin, Etilefrin, Gepefrine, Metaraminol, Midodrine, Norepi Labetalol, Pronethalol, Sotalol and Sulfinalol; nephrine, Pholedrinead and Synephrine; 0266 Aryloxypropanolamine derivatives such as Acebu 0280 Antihypothyroid drugs such as Levothyroxine tolol, Alprenolol, Arotinolol, Atenolol, Betaxolol, Bevan Sodium, Liothyronine, Thyroid, Thyroidin, Thyroxine, tolol, Bisoprolol, Bopindolol, Bunitrolol, Bupranolol. Buto Tiratricol and TSH: filolol, Carazolol, Cartezolol, Carvedilol, Celiprolol. 0281 Anti-Inflammatory (non-steroidal) drugs, includ Cetamolol, Epanolol, Indenolol, Mepindolol, Metipranolol, ing: Aminoarylcarboxylic acid derivatives such as Enfenamic Metoprolol, Moprolol, Nadolol, Nipradilol, Oxprenolol, Pen Acid, Etofenamate, Flufenamic Acid, Isonixin, Meclofe butolol, Pindolol, Propranolol, Talinolol, Tetraolol, Timolol namic Acid, Mefanamic Acid, Niflumic Acid, Talniflumate, and Toliprolol; Terofenamate and Tolfenamic Acid; 0267 Benzothiadiazine derivatives such as Althiazide, Bendroflumethiazide, Benzthiazide, Benzylhydrochlorothi 0282 Arylacetic acid derivatives such as Acemetacin, azide, Buthiazide, Chlorothiazide, Chlorthalidone, Cyclo Alclofenac, Amfenac, Bufexamac, Cinmetacin, Clopirac, penthiazide, Cyclothiazide, Diazoxide, Epithiazide, Ethiaz Diclofenac Sodium, Etodolac, Felbinac, Fenclofenac, Fen ide, Fenguizone, Hydrochlorothiazide, Hydroflumethiazide, clorac, Fenclozic Acid, Fentiazac, Glucametacin, Ibufenac, Methyclothiazide, Meticrane, Metolazone, Paraflutizide, Indomethacin, IsofeZolac, ISOXepac, Lonazolac, Metiazinic Polythiazide, Tetrachlormethiazide and Trichlormethiazide; Acid, Oxametacine, Proglumetacin, Sulindac, Tiaramide, 0268 N-Carboxyalkyl (peptide/lactam) derivatives such Tolimetin and Zomepirac: as Alacepril, Captopril, CilaZapril, Delapril, Enalapril, Enala 0283 Arylbutyric acid derivatives such as Bumadizon, prilat, Fosinopril, Lisinopril, Moveltipril, Perindopril, Butibufen, Fenbufen and Xenbucin; Quinapril and Ramipril; 0284 Arylcarboxylic acids such as Clidanac, Ketorolac 0269. Dihydropyridine derivatives such as Amlodipine, and Tinoridine; Felodipine, Isradipine, Nicardipine, Nifedipine, Nilvadipine, 0285) Arylpropionic acid derivatives such as Alminopro Nisoldipine and Nitrendipirne; fen, Benoxaprofen, Bucloxic Acid, Carprofen, Fenoprofen, 0270 Guanidine derivatives such as Bethanidine, Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam, Debrisoquin, Guanabenz, Guanacline, Guanadrel, GuanaZo Indoprofen, Ketoprofen, Loxoprofen, Miroprofen, dine, Guanethidine, Guanfacine, Guanochlor, Guanoxabenz Naproxen, Oxaprozin, Piketoprofen, Pirprofen, Pranoprofen, and Guanoxan; Protizinic Acid, Suprofen and Tiaprofenic Acid; 0271 Hydrazines and phthalazines such as Budralazine, 0286 Pyrazoles such as Difenamizole and Epirizole; Cadralazine, Dihydralazine, Endralazine, Hydracarbazine, 0287 Pyrazolones such as Apazone, BenZpiperylon, Hydralazine, Pheniprazine, Pildralazine and Todralazine; Feprazone, Mofebutazone, Morazone, Oxyphenbutazone, 0272 Imidazole derivatives such as Clonidine, Lofexi Phenylbutazone, Pipebuzone, Propyphenazone, Ramifena dine, Phentolamine, Phentolamine Mesylate, Tiamenidine Zone, SuxibuZone and Thiazolinobutazone; and Tolonidine; 0288 Salicylic acid derivatives such as Acetaminosalol, 0273 Quaternary ammonium compounds AZamethonium Aspirin, Benorylate, Bromosaligenin, Calcium Acetylsalicy Bromide, Chlorisondamine Chloride, Hexamethonium, Pen late. Diflunisal, Etersalate, Fendosal, Gentisic Acid, Glycol tacynium Bis(methyl sulfate), Pentamethonium Bromide, Salicylate, Imidazole Salicylate, Lysine Acetylsalicylate, Pentolinium Tartate, Phenactopinium Chloride and Trime Mesalamine, Morpholine Salicylate, 1-Naphthyl Salicylate, thidiunum Methosulfate; Olsalazine, Parsalmide, PhenylAcetylsalicylate, Phenyl Sali 0274 Quinazoline derivatives such as Alfuzosin, cylate, Salacetamide, Salicylamine O-Acetic Acid, Salicyl BunaZosin, Doxazosin, Prasosin, Terazosin and TrimaZosin; sulfuric Acid, Salsalate and Sulfasalazine; US 2012/O 141590 A1 Jun. 7, 2012

0289. Thiazinecarboxamides such as Droxicam, Isoxi 0302 Pyrimidine analogs such as Ancitabine, AZacitidine, cam, Piroxicam and Tenoxicam, and 6-AZauridine, Carmofur, Cytarabine, Doxifluridine, Enocit 0290 others such as (f-Acetamidocaproic Acid, S-Adeno abine, Floxuridine Fluoroouracil and Tegafur; Sylmethionine, 3-Amino-4-hydroxybutyric Acid, Amix 0303 Enzymes such as L-Asparaginase; and etrine, Bendazac, BenZydamine, Bucolome. Difempiramide, 0304 others such as Aceglatone, Amsacrine, Bestrabucil, Ditazol, EmorfaZone, GuaiaZulene, Nabumetone, Nime Bisantrene, Bryostatin 1, Carboplatin, Cisplatin, Defofa sulide, Orgotein, Oxaceprol, Paranyline, Perisoxal, Pifoxime, mide, Demecolcine, Diaziquone, Elformithine, Elliptinium ProquaZone, Proxazole and Tenidap: Acetate, Etoglucid, Etoposide, Gallium Nitrate, Hydrox 0291 Antimalarial drugs such as Acedapsone, Amodi yurea, Interferon-C. Interferon-?3, Interferon-Y, Interleukine aquin, Arteether, Artemether, Artemisinin, Artesunate, 2, Lentinan, Letrozole, Lonidamine, MitoguaZone, Mitox Bebeerine, Berberine, Chirata, Chlorguanide, Chloroquine, antrone, Mopidamol, Nitracrine, Pentostatin, Phenamet, Chlorproguanil, Cinchona, Cinchonidine, Cinchonine, Pirarubicin, Podophyllinicc Acid, 2-Ethylhydrazide, Polyni Cycloguanil, Gentiopicrin, Halofantrine, Hydroxychloro trocubanes, Procarbazine, PSK7, Razoxane, Sizofuran, quine, Mefloquine Hydrochloride, 3-Methylarsacetin, Pam Spirogermanium, Taxol. Teniposide, TenuaZonic Acid, Tri aquine, Plasmocid, Primaquine, Pyrimethamine, Quinacrine, aziquone, 2.2.2"-Trichlorotriethylamine, Urethan, Vinblas Quinine, Quinine Bisulfate, Quinine Carbonate, Quinine tine, Vincristine, Vindesine and Vinorelbine; Dihydrobromide, Quinine Dihydrochloride, Quinine Ethyl 0305 Antineoplastic (hormonal) drugs, including: Andro carbonate, Quinine Formate, Quinine Gluconate, Quinine gens such as Calusterone, Dromostanolone Propionate, Epi Hydriodide, Quinine Hydrochloride, Quinine Salicylate, tioStanol, Mepitiostane and Testolactone; Quinine Sulfate, Quinine Tannate, Quinine Urea Hydrochlo 0306 Antiadrenals such as Aminoglutethimide, Mitotane ride, Quinocide, Quinoline and Sodium Arsenate Diabasic; and Trilostane; 0292 Antimigraine drugs such as Alpiropride, Dihydro 0307 Antiandrogens such as Flutamide and Nilutamide: ergotamine, Eletriptan, Ergocornine, Ergocorninine, Ergoc and ryptine, Ergot, Ergotamine, Flumedroxone acetate, Fonazine, Lisuride, Methysergid(e), Naratriptan, Oxetorone, Pizoty 0308 Antiestrogens such as Tamoxifen and Toremifene: line, Rizatriptan and Sumatriptan; 0309 Antineoplastic adjuncts including folic acid replen 0293 Antinauseant drugs such as Acetylleucine Monoet ishers such as Frolinic Acid; hanolamine, Alizapride, BenZquinamide, Bietanautine, Bro 0310 Antiparkinsonian drugs such as Amantadine, mopride, Buclizine, Chlorpromazine, Clebopride, Cyclizine, Benserazide, Bietanautine, Biperiden, Bromocriptine, Dimenhydrinate, Dipheniodol, Domperidone, Granisetron, Budipine, Cabergoline, Carbidopa, Deprenyl (a/k/a L-depre Meclizine, Methalltal, Metoclopramide, Metopimazine, nyl, L-deprenil, L-deprenaline and selegiline), Dexetimide, Nabilone, Ondansteron, Oxypendyl, Pipamazine, Piprinhy Diethazine, Diphenhydramine, Droxidopa, Ethopropazine, drinate, Prochlorperazine, Scopolamine, Tetrahydrocannab Ethylbenzhydramine, Levodopa, Naxagolide, Pergolide, inols. Thiethylperazine. Thioproperzaine and Trimethoben Piroheptine, Pramipexole, Pridinol, Prodipine, Quinpirole, Zamide; Remacemide, Ropinirole, Terguride, Tigloidine and Trihex 0294 Antineoplastic drugs, including: Alkylating agents, yphenidyl Hydrochloride; Such as Alkyl Sulfonates such as BuSulfan, ImproSulfan and 0311 Antipheochromocytoma drugs such as Metyrosine, Piposulfan; Phenoxybenzamine and Phentolamine; 0295 AZiridines such as Benzodepa, Carboquone, 0312 Antipneumocystis drugs such as Efformithine, Pen Meturedepa and Uredepa; tamidine and Sulfamethoxazole; 0296 Ethylenimines and methylmelamines such as Altre 0313 Antiprostatic hypertrophydrugs such as Gestonor tamine, Triethylenemelamine, Triethylenephosphoramide, one Caproate, Mepartricin, Oxendolone and Proscar7; Triethylenethiophosphoramide and Trimethylolomelamine; 0314 Antiprotozoal drugs (Leshmania) Such as Antimony 0297 Nitrogen mustards such as Chlorambucil, Chlor Sodium Gluconate, Ethylstibamine, Hydroxy stilbamidine, naphazine, Chclophosphamide, Estramustine, Ifosfamide, N-Methylglucamine, Pentamidine, Stilbamidine and Urea Mechlorethamine, Mechlorethamine Oxide Hydrochloride, Stibamine; Melphalan, Novembichin, Phenesterine, Prednimustine, Tro 0315 Antiprotozoal drugs (Trichomonas) such as Acetar fosfamide and Uracil Mustard; Sone, Aminitrozole, Anisomycin, AZanidazole, Forminitra 0298 Nitrosoureas such as Carmustine, Chlorozotocin, Zole, Furazolidone, Hachimycin, Lauroguadine, Mepartricin, Fotemustine, Lomustine, Nimustine and Ranimustine; and Metronidazole, Nifuratel, Nifuroxime, Nimorazole, Secnida others such as Camptothecin, Dacarbazine, Mannomustine, Zole, Silver Picrate, Tenonitrozole and Timidazole; Mitobronitol, Mitolactol and Pipobroman; 0316 Antiprotozoal drugs (Trypanosma) such as Ben 0299 Antibiotics such as Aclacinomycins, Actinomycin Znidazole, Eflornithine, Melarsoprol, Nifurtimox, Oxophe F, Anthramycin, AZaserine, Bleomycins, Cactinomycin, narsine, Hydrochloride, Pentamidine, Propamidine, Puromy Carubicin, Carzinophilin, Chromomycins, Dactinomycin, cin, Quinapyramine, Stilbamidine, Suramin Sodium, Trypan Daunorubicin, 6-Diazo-5-oxo-L-norleucine, Doxorubicin, Red and Tryparasmide: Epirubicin, Mitomycins, Mycophenolic Acid, Nogalamycin, 0317 Antipuritics such as Camphor, Cyproheptadine, Olivomycins, Peplomycin, Plicamycin, Porfiromycin, Puro Dichlorisone, Glycine, Halometasone, 3-Hydroxycamphor, mycin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Menthol, Mesulphen, Methdilazine, Phenol, Polidocanol, Zinostatin and Zorubicin; Risocaine, Spirit of Camphor. Thenaldine, Tolpropamine and 0300 Antimetabolites, including: Folic acid analogs such Trimeprazine; as Denopterin, Methotrexate, Pteropterin and Trimetrexate: 0318 Antipsoriatic drugs such as Acitretin, Ammonium 0301 Purine analogs such as Fludarabine, 6-Mercaptopu Salicylate, Anthralin, 6-AZauridine, Bergapten(e), Chrysar rine. Thiamiprine and Thioguanaine; and obin, Etretinate and Pyrogallol; US 2012/O 141590 A1 Jun. 7, 2012 24

0319 Antipsychotic drugs, including: Butyrophenones 0331 Nitrofurans such as Furazolidone, 2-(Methoxym such as Benperidol, Bromperidol, properidol, Fluanisone, ethyl)-5-nitrofuran, Nidroxy Zone, Nifuroxime, Nifurzide and Haloperidol, Melperone, Moperone, Pipamperone, Sniper Nitrofurazone; one, Timiperone and Trifluperidol; 0332 Phenols such as Acetomeroctol, Bithionol, Cad 0320 Phenothiazines such as Acetophenazine, Butapera mium Salicylate, Carvacrol, Chloroxylenol, Clorophene, Zine, Carphenazine, Chlorproethazine, Chlorpromazine, Cresote, Cresol(s), p-Cresol, Fenticlor, Hexachlorophene, Clospirazine, Cyamemazine, Dixyrazine, Fluphenazine, Imi 1-Napthyl Salicylate, 2-Napthyl Salicylate, 2.4.6-Tribromo clopazine, Mepazine, Mesoridazine, Methoxypromazine, m-cresol, and 3',4',5'-Trichlorosalicylanilide; Metofenazate, Oxaflumazine, Perazine, Pericyazine, 0333 Quinolines such as Aminoquinuride, BenZOxiduine, Perimethazine, Perphenazine, Piperacetazine, Pipotiazine, Broxyquinoline, Chloroxine, Chlorquinaldol, Cloxyquin, Prochlorperazine, Promazine, Sulforidazine. Thiopropazate, Ethylhydrocupreine, Euprocin, Halquinol, Hydrastine, 8-Hy Thioridazine, Trifluoperazine and Triflupromazine: droxquinoline, 8-Hydroxquinoline Sulfate and Iodochlorhy 0321. Thioxanthenes such as Chlorprothixene, Clo droxyquin; and penthixol, Flupentixol and Thiothixene; 0334 others such as Aluminum Acetate Solution, Alumi 0322 other tricyclics such as Benzquinamide, num Subacetate Solution, Aluminum Sulfate, 3-Amino-4- Carpipramine, Clocapramine, Clomacran, Clothiapine, hydroxybutyric Acid, Boric Acid, Chlorhexidine, Chloroazo Clozapine, Opipramol, Prothipendyl, Tetrabenazine, and din, m-CresylAcetate, Cupric Sulfate, Dibromopropamidine, Zotepine; and Ichthammol, Negatol7, Noxytiolin, Ornidazole, Propiolac 0323 others such as Alizapride, Amisulpride, Buramate, tone, C-Terpineol; Fluspirilene, Molindone, Penfluridol, Pimozide, Spirilene 0335 Antispasmodic drugs such as Alibendol, Ambuceta and Sulpiride; mide, Aminopromazine, Apoatropine, Bevonium Methyl Sulfate, Bietamiverine. Butaverine, Butropium Bromide, 0324 Antipyretics Such as Acetaminophen, Acetaminos N-Butylscopolammonium Bromide, Caroverine, alol, Acetanilide, Cimetropium Bromide, Cinnamedrine, Clebopride, Coniine 0325 Aconine, Aconite, Aconitine, Alclofenac, Alumi Hydrobromide, Coniine Hydrochloride, Cyclonium Iodide, num Bis(acetylsalicylate), Aminochlorthenoxazin, Ami Difemerine, Diisopromine, Dioxaphetyl Butyrate, Diponium nopyrine, Aspirin, Benorylate, BenZydamine, Berberine, Bromide, Drofenine, Emepronium Bromide, Ethaverine, p-Bromoacetanilide, Bufexamac, Bumadizon, Calcium Feclemine, Fenalamide, Fenoverine, Fenpiprane, Fenpiver Acetysalicylate, Chlorthenoxazin(e), Choline Salicylate, inium Brcmide, Fentonium Bromide, Flavoxate, Flopropi Clidanac, Dihydroxyaluminum Acetylsalicylate, Dipyro one, Gluconic Acid, Guaiactamine, Hydramitrazine, Hyme cetyl, Dipyrone, Epirizole, Etersalate. Imidazole Salicylate, cromone, Leiopyrrole, Mebeverine, Moxaverine, Nafiverine, Indomethacin, IsofeZolac, p-Lactophenetide, Lysine Acetyl Octamylamine, Octaverine, Pentapiperide, Phenamacide salicylate, Magnesium Acetylsalicylate, Meclofenamic Acid, Hydrochloride, Phloroglucinol, Pinaverium Bromide, Piperi Morazone, Morpholine Salicylate, Naproxen, NifenaZone, late, Pipoxolan Hydrochloride, Pramiverin, Prifinium Bro 51-Nitro-2'-propoxyacetanilide, Phenacetin, Phenicarbazide, mide, Properidine, Propivane, Propyromazine, Prozapine, Phenocoll, Phenopyrazone, PhenylAcetylsalicylate, Phenyl Racefemine, Rociverine, Spasmolytol, Stilonium Iodide, Salicylate, PipebuZone, Propacetamol, Propyphenazone, Sultroponium, Tiemonium Iodide, Tiquizium Bromide, Tiro Ramifenazone, Salacetamide, Salicylamide O-Acetic Acid, pramide, Trepibutone, Tricromyl, Trifolium, Trimebutine, Sodium Salicylate, Sulfamipyrine, Tetrandrine and Tinori N,N-ITrimethyl-3,3-diphenyl-propylamine, Tropenzile, Tro dine; spium Chloride and Xenylropium Bromide: 0326 Antirickettsial drugs such as p-Aminobenzoic Acid, 0336 Antithrombotic drugs such as Anagrelide, Arga Chloramphenicol, Chloramphenicol Palmitate, Chloram troban, Cilostazol, Chrysoptin, Daltroban, Defibrotide, phenicol Pantothenate and Tetracycline; Enoxaparin, Fraxiparine-7, Indobufen, Lamoparan, OZagrel, 0327 Antiseborrheic drugs such as Chloroxine, 3-O-Lau Picotamide, Plafibride, Reviparin, Tedelparin, Ticlopidine, roylpyridoxol Diacetate, Piroctone, Pyrithione, Resorcinol, Triflusal and Warfarin; Selenium Sulfides and Tioxolone; 0337 Antitussive drugs such as Allocamide, Amicibone, 0328 Antiseptics, including: Guanidines such as Alexi Benproperine, BenZonatate, Bibenzonium Bromide, Bromo dine, Ambazone, Chlorhexidine and Picloxydine: form, Butamirate. Butethamate, Caramiphen Ethanedisul 0329 Halogens and halogen compounds such as Bismuth fonate, Carbetapentane, Chlophedianol, Clobutinol, Clop Iodide Oxide, Bismuth Iodosubgallate, Bismuth Tribro erastine, Codeine, Codeine Methyl Bromide, Codeine mophenate, Bornyl Chloride, Calcium Iodate, Chlorinated N-Oxide, Codeine Phosphate, Codeine Sulfate, Cyclex Lime, Cloflucarban, Fluorosalan, Iodic Acid, Iodine, Iodine anone, Dextromethorphan, Dibunate Sodium, Dihydroco Monochloride, Iodine Trichloride, Iodoform, Methenamine deine, Dihydrocodeinone Enol Acetate, Dimemorfan, Tetraiodine, Oxychlorosene, Povidone-Iodine, Sodium Dimethoxanate, C.C.-Diphenyl-2-piperidinepropanol, pro Hypochlorite, Sodium Iodate, Symclosene, Thymol Iodide, propizine, Drotebanol, Eprazinone, Ethyl Dibunate, Ethyl Triclocarban, Triclosan and Troclosene Potassium; morphine, Fominoben, Guiaiapate, Hydrocodone, Isoami 0330 Mercurial compounds such as Hydragaphen, Mera nile, Levopropoxyphene, Morciofone, Narceline, lein Sodium, Merbromin, Mercuric Chloride, Mercuric Chlo Normethadone, Noscapine, Oxeladin, Oxolamine, Pholcod ride, Ammoniated, Mercuric Sodium p-Phenolsulfonate, ine, Picoperine, Pipazethate, Piperidione, Prenoxdiazine Mercuric Succinimide, Mercuric Sulfide, Red, Mercurophen, Hydrochloride, Racemethorphan, Taziprinone Hydrochlo Mercurous Acetate, Mercurous Chloride, Mercurous Iodide, ride, Tipepidine and Zipeprol; Nitromersol, Potassium Tetraiodomercurate(II), Potassium 0338 Antiulcerative drugs such as Aceglutamide Alumi Triiodomercurate (II) Solution. Thimerfonate Sodium and num Complex, e-Acetamidocaproic Acid Zinc Salt, Acetox Thimerosal; olone, Arbaprostil, Benexate Hydrochloride, Bismuth Subci US 2012/O 141590 A1 Jun. 7, 2012

trate Sol (Dried), Carbenoxolone, Cetraxate, Cimetidine, 0352 Calcium channel blockers, including: Arylalky Enprostil, Esaprazole, Famotidine, Ftaxilide, Gefarnate, lamines such as Bepridil, Ditiazem, Fendiline, Gallopanil, GuaiaZulene, Irsogladine, Misoprostol, Nizatidine, Omepra Prenylamine, Terodiline and Verapamil: Zole, Ornoprostil, Y-Oryzanol, Pifamine, Pirenzepine, 0353. Dihydropyridine derivatives such as Felodipine, Plaunotol, Ranitidine, Rioprostil, Rosaprostol, Rotraxate, Isradipine, Nicardipine, Nifedipine, Nilvadipine, Nimo Roxatidine Acetate, Sofalcone, Spizofurone. Sucralfate, dipine, Nisoldipine and Nitrendipine; Teprenone, Trimoprostil, Thrithiozine, Troxipide and Zoli 0354 Piperazine derivatives such as Cinnarizine, Flunar midine; isine and Lidoflazine; and 0339 Antiurolithic drugs such as Acetohydroxamic Acid, 0355 others such as Bencyclane, Etafenone and Perhexy Allopurinol, Potassium Citrate and Succinimide: line; 0340 Antivenin drugs such as Lyovac7 Antivenin, 0356 Calcium regulators such as Calcifediol, Calcitonin, 0341 Antiviral drugs, including: Purines and pyrimidino Calcitriol, Clodronic Acid, Dihydrotachysterol, Elcatonin, nes such as Acyclovir, Cytarabine, Dideoxyadenosine, Etidronic Acid, Ipriflavone, Pamidronic Acid, Parathyroid Hormone and Teriparatide Acetate; Dideoxycytidine, Dideoxyinosine, Edoxudine, Floxuridine, 0357 Cardiotonics such as Acefylline, Acetyldigititoxins, Ganciclovir, Idoxuridine, Inosine Pranobex, MADU, Penci 2-Amino-4-picoline, Amrinone, Benfurodil Hemisuccinate, clovir, Trifluridine, Vidrarbine and Zidovudine; and Buclasdesine, Cerberoside, Camphotamide, Convallatoxin, 0342 others such as Acetylleucine Monoethanolamine, Cymarin, Denopamine, Deslanoside, Ditalin, Digitalis, Digi Amantadine, Amidinomycin, Cosalane, Cuminaldehyde Thi toxin, Digoxin, Dobutamine, Dopamine, Dopexamine, osemicarbZone, Foscarnet Sodium, Imiquimod, Interferon-C. Enoximone, Erythrophleine, Fenalcomine, Gitalin, Gitoxin, Interferon-B, Interferon-Y, Kethoxal, Lysozyme, Methisa Glycocyamine, Heptaminol, Hydrastinine, lbopamine, Lano Zone, Moroxydine, Podophyllotoxin, Ribavirin, Rimanta todises, Metamivam, Millrinone, Neriifolin, Oleandrin, Oua dine, Stallimycin, Statolon, Tromantadine, Xenazoic Acid, bain, Oxyfedrine, Prenalterol, Proscillaridin, Resilbufogenin, and the anti-influenza drugs Zanamivir and Oseltamivir phos Scillaren, Scillarenin, Strophanthin, Sulmazole. Theobro phate; mine and Xamoterol; 0343 Anxiolytic drugs, including: Arylpiperazines Such 0358 Chelating agents such as Deferozmine, Ditiocarb as Buspirone, Gepirone, Ipsapirone and TondoSpirone; Sodium, Edetate Calcium Disodium, Edetate Disodium, 0344 Benzodiazepine derivatives such as Alprazolam, Edeate Sodium, Edetate Trisodium, Penicillamine, Pentetate Bromazepam, Camazepam, Chlordiazepoxide, Clobazam, Calcium Trisodium, Pentectic Acid, Succimer and Trientine; CloraZepate, Chotiazepam, Cloxazolam, Diazepam, Ethyl 0359 Cholecystokinin antagonists such as Proglumide: Loflazepate, Etizolam, Fluidazepam, Flutazolam, Fluto 0360 Cholelitholytic agents such as Chenodiol, Methyl prazepam, Halazepam, Ketazolam, Lorazepam, Loxapine, tert-Butyl Ether, Monooctanoin and Ursodiol; Medazepam, Metaclazepam, Mexazolam, Nordazepam, 0361 Choleretics such as Alibendol, Anethole Trithion, Oxazepam, Oxazolam, Pinazepam, Prazepam and Tofiso AZintamide, Cholic Acid, Cicrotoic Acid, Clanobutin, pam, Cyclobutyrol, Cyclovalone, Cynarin(e), Dehydrocholic 0345 Carbamates such as Cyclarbamate, Emylcamate, Acid, Deoxycholic Acid, Dimecrotic Acid, O.-Ethylbenzyl Hydroxyphenamate, Meprobamate, Phenprobamate and Alcohol, Exiproben, Feguprol, Fencibutirol, Fenipentol, Flo Tybamate; and rantyrone, Hymecromone, Menbutone, 3-(o-Methoxyphe 0346 others such as Alpidem, Benzoctamine, Captodi nyl)-2-phenylacrylic Acid, Metochalcone, Moquizone, amine, ChlormeZanone, Etifoxine, Flesinoxan, Fluoresone, Osalmid, Ox Bile Extract, 4.4'-Oxydi-2-butanol, Piprozolin, Glutamic Acid, Hydroxyzine, Lesopitron, Mecloralurea, Prozapine, 4-Salicyloylmorpholine, Sincalide, Taurocholic Mephenoxalone, Mirtazepine, Oxanamide, Phenaglycodol, Acid, Timonacic, Tocamphyl, Trepibutone and Vanitiolide; Suriclone and Zatosetron; 0362 Cholinergic agents such as Aceclidine, Acetylcho 0347 Benzodiazepine antagonists such as Flumazenil; line Bromide, Acetylcholide Chloride, Aclatonium Napadisi late, BenZpyrinium Bromide, Bethanechol chloride, Carba 0348 Bronchodilators, including: Ephedrine derivatives chol, Carpronium chloride, Demecarium Bromide, such as Albuterol, Bambuterol, Bitolterol, Carbuterol, Clen Dexpanthenol, Diisopropyl Paraoxon, Echothiophate Iodide, buterol, Clorprenaline, Dioxethedrine, Ephedrine, Epiniph Edrophomium chloride, Eseridine. Furtrethonium, Isofluo rine, Eprozinol, Etafedrine, Ethylnorepinephrine, Fenoterol, rophate, Methacholine chloride, Muscarine, Neostigmine, Hexoprenaline, Isoetharine, Isoproterenol, Mabuterol, Oxapropanium Iodide, Physostigmine and Pyridostigmine Metaproterenol, N-Methylephedrine, Pirbuterol, Procaterol, Bromide; Protokylol, Reproterol, Rimiterol, Salmeterol, Soterenol, 0363 Cholinesterase inhibitors such as Ambenonium Terbutaline and Tulobuterol; Chloride, Distigmine Bromide and Galanthamine: 0349 Quaternary ammonium compounds such as Bevo 0364 Cholinesterase reactivators such as Obidoximine nium Methyl Sulfate, Clutropium Bromide, Ipratropium Bro Chloride and Pralidoxime Chloride; mide and Oxitropium Bromide: 0365 Central nervous system stimulants and agents such 0350 Xanthine derivatives such as Acefylline, Acefylline as Amineptine, Amphetimine, Amphetaminil, Bemegride, piperazine, Ambuphylline, Aminophylline, Bamifylline, Benzphetamine, Brucine, Caffeine, Chlorphentermine, choline Theophyllinate, Doxofylline, Dyphylline, Enprofyl Clofenciclan, Clortermine, Coca, Demanyl Phosphate, Dex line, Etamiphyllin, Etofylline, Guaithylline, Proxyphylline, oxadrol, Dextroamphetamine Sulfate, Diethlpropion, Theobromine, 1-Theobromineacetic Acid and Theophylline: N-Ethylamphetamine, Ethamivan, Etifelmin, Etryptamine, and Fencamfamine, Fenethylline, Fenosolone, Fluorothyl, Gal 0351 others such as Fenspiride, Medibazine, Monteku anthamine, Hexacyclonate Sodium, Homocamfin, Mazindol, last, Methoxyphenanime, Tretoquinol and Zafirkulast; Megexamide, Methamphetamine, Methylphenidate, US 2012/O 141590 A1 Jun. 7, 2012 26

Nikethamide, Pemoline, Pentylenetetrazole, Phenidimetra diol Benzoate, Estradio 17? 3-Cypionate, Estriol, Estrone, zine, Phenmetrazine, Phentermine, Picrotoxin, Pipradrol, Ethinyl Estradiol, Mestranol, Moxestrol, Mytatrienediol, Prolintane and Pyrovalerone; Quinestradiol and Quinestrol; 0366 Decongestants such as Amidephrine, Cafaminol, 0386 Gastric secretion inhibitors such as Enterogastrone Cyclopentamine, Ephedrine, Epinephrine, Fenoxazoline, and Octreotide; Indanazoline, Metizoline, Naphazoline, Nordefrin Hydro 0387 Glucocorticoids such as 21-Acetoxyprefnenolone, chloride, Octodrine, oxymetazoline, Phenylephrine Hydro AalclometaSone, Algestone, Amicinonide, Beclomethasone, chloride, Phenylpropanolamine Hydrochloride, Phenylpro Betamethasone, Budesonide, Chloroprednisone, Clobetasol, pylmethylamine, Propylhexedrine, Pseudoephedrine, Blovetasone, Clocortolone, Cloprednol, Corticosterone, Cor Tetrahydrozoline, Tymazoline and Xylometazoline: tisone, CortivaZol, Deflazacort, Desonide, DeSoximetaSone, 0367 Dental agents, including: Bisphosphonates (anti Dexamethasone. Diflorasone. Diflucortolone. Difluprednate, periodontal disease and bone resorption) Such as Alendr Enoxolone, Fluazacort, Flucloronide, Flumehtasone, onate, Clodronate, Etidronate, Pamidronate and Tiludronate; Flunisolide, Fluocinolone Acetonide, Fluocinonide, Fluocor Carries Prophylactics such as Arginine and Sodium Fluoride: tin Butyl, Fluocortolone, Fluorometholone, Fluperolone 0368. Desensitizing Agents such as Potassium Nitrate and Acetate, Fluprednidene Acetate, Fluprednisolone, Flurandre Citrate Oxalate; nolide. Formocortal, Halcinonide, Halometasone, Halopre 0369 Depigmentors such as Hydroquinine, Hydro done Acetate, Hydrocortamate, Hydrocortisone, Hydrocorti quinone and Monobenzone; Sone Acetate, ydrocortisone Phosphate, Hydrocortisone 0370 Diuretics, including: Organomercurials such as 21-Sodium Succinate, Hydrocortisone Tebutate, Chlormerodrin, Meralluride, Mercamphamide, Mercaptom Mazipredone, Medrysone, Meprednisone, Methyolpredniso erin Sodium, Mercumallylic Acid, Mercumatilin Sodium, lone, Mometasone Furoate, Paramethasone, Prednicarbate, Mercurous Chloride and Mersalyl: Prednisolone, Prednisolone 21-Diethylaminoacetate, Pred 0371 Pteridines such as Furterene and Triamterene: nisone Sodium Phosphate, Prednisolone Sodium Succinate, 0372 Purines such as Acefylline, 7-Morpholinomethylth Prednisolone Sodium 21-m-Sulfobenzoate, Prednisolone eophylline, Pamabrom, Protheobromine and Theobromine; 21-Stearoylglycolate, Prednisolone Tebutate, Prednisolone 0373 Steroids such as Canrenone, Oleandrin and 21-Trimethylacetate, Prednisone, Prednival, Prednylidene, Spironolactone; Prednylidene 21-Diethylaminoacetate, Tixocortal, Triamci 0374 Sulfonamide derivatives such as Acetazolamide, nolone, Triamcinolone Acetonide, Triamcinolone Bene Ambuside, AZosemide, Bumetanide, Butazolamide, tonide and Triamcinolone Hexacetonide; Chloraminophenamide, Clofenamide, Clopamide, Clorex 0388 Gonad-Stimulating principles such as Buserelin, olene, Diphenylmethane-4.4'-disulfonamide, Disulfamide, Clomiphene, Cyclofenil, Epimestrol, FSH, HCG and LH EthbXZolamide. Furosemide, Indapamide, Mefruside, Meth RH; aZolamide, Piretanide, QuinethaZone, Torasemide, Tripam (0389. Gonadotropic hormones such as LH and PMSG: ide and Xipamide; 0390 Growth hormone inhibitors such as Octreotide and 0375 Uracils such as Aminometradine and Amisometra Somatostatin; dine; 0391 Growth hormone releasing factors such as Semore 0376 others such as Amanozine, Amiloride, Arbutin, lin; Chlorazanil, Ethacrynic Acid, Etozolin, Hydracarbazine, 0392 Growth stimulants such as Somatotropin: Isosorbide, Mannitol, Metochalcone, Muzolimine, Perhexy 0393 Hemolytic agents such as Phenylhydrazine and Phe line, Ticrynafen and Urea; nylhydrazine Hydrochloride; 0377 Dopamine receptoragonists such as Bromocriptine, 0394 Heparin antagonists such as Hexadimethrine Bro Dopexamine, Fenoldopam, lbopamine, Lisuride, Naxagolide mide and Protamines; and Pergolide; 0395 Hepatoprotectants such as S-Adenosylmethionine, 0378 Ectoparasiticides such as Amitraz, Benzyl Ben Betaine, Catechin, Citolone, Malotilate, Orazamide, Phos Zoate, Carbaryl, Crotamiton, DDT. Dixanthogen, Isobornyl phorylcholine, Protoporphyrin IX, Silymarin-Group. Thiotic Thiocyanoacetate Technical, Lime Sulfurated Solution, Acid and Tiopronin; Lindane, Malathion, Mercuric Oleate, Mesulphen and Sul 0396. Immunomodulators such as Amiprilose, Bucil phur Pharmaceutical; lamine, Ditiocarb Sodium, Inosine Pranobex, Interferon-y, 0379 Enzymes, including: Digestive enzymes such as Interleukin-2, Lentinan, Muroctasin, Platonin, Procodazole, C.-Amylase (Swine Pancreas), Lipase, Pancrelipase, Pepsin Tetramisole, Thymomodulin, Thymopentin and Ubenimex: and Rennin; 0397 Immunosuppressants such as Azathioprine, 0380. Mucolytic enzymes such as Lysozyme; Cyclosporins and Mizoribine; 0381 Penicillin inactivating enzymes such as Penicilli 0398 Ion exchange resins such as Carbacrylic Resins, nase; and Cholestyramine Resin, Colestipol, Polidexide, Resodec and 0382 Proteolytic enzymes such as Collagenase, Chymo Sodium Polystyrene Sulfonate; papain, Chymotrypsins, Papain and Trypsin; 0399 Lactation stimulating hormone such as Prolactin; 0383 Enzyme inducers (hepatic) such as Flumecinol: 04.00 LH-RHagonists such as Buserelin, Goserelin, Leu 0384 Estrogens, including: Nonsteroidal estrogens such prolide, Nafarelin, and Triptorelin; as Benzestrol, Broparoestrol, Chlorotrianisene, Dienestrol, 0401 Lipotropic agents such as N-Acetylmethionine, Diethylstilbestrol, Diethylstilbestrol Diproprionate, Choline Chloride, Choline Dehydrocholate, Choline Dihy Dimestrol, Fosfestrol, Hexestrol, Methallenestril and Meth drogen Citrate, Inositol, Lecithin and Methionine: estrol; and 0402 Lupus erythematosus Suppressants such as Bismuth 0385 Steroidal estrogens such as Colpormon, Conjugated Sodium Triglycollamate, Bismuth Subsalicylate, Chloro Estrogenic Hormones, Equilenin, Equilin, Estradiol, Estra quine and Hydroxychloroquine; US 2012/O 141590 A1 Jun. 7, 2012 27

0403 Mineralcorticoids such as Aldosterone, Deoxycor Prostaglandin E, Prostagland in F. Rioprostil, Rosapros ticosterone, Deoxycorticosterone Acetate and Fludrocorti tol, Sulprostone and Trimoprostil; Sone; 0420 Protease inhibitors such as Aprotinin, Camostat, 0404 Miotic drugs such as Carbachol, Physostigmine, Gabexate and Nafamo.stat; Pilocarpine and Pilocarpus; 0421 Respiratory stimulants such as Almitrine, Beme 04.05 Monoamine oxidase inhibitors such as Deprenyl, gride, Carbon Dioxide, Cropropamide, Crotethamide, Dime Iproclozide, Iproniazid, Isocarboxazid, Moclobemide, Octo fline, Dimorpholamine, Doxapram, Ethamivan, Fominoben, moxin, Pargyline, Phenelzine, Phenoxypropazine, Pivalyl Lobeline, Mepixanox, Metamivam, Nikethamide, Picro benzhydrazine, Prodipine, Toloxatone and Tranylcypromine; toxin, Pimeclone, Pyridofylline, Sodium Succinate and 04.06 Mucolytic agents such as Acetylcysteine, Bromhex Tacrine; ine, Carbocysteine, Domiodol, Letosteine, Lysozyme, 0422 Sclerosing agents such as Ethanolamine, Ethy Mecysteine Hydrochloride, Mesna, Sobrerol, Stepronin, Tio lamine, 2-Hexyldecanoic Acid, Polidocanol, Quinine Bisul pronin and Tyloxapol; fate, Quinine Urea Hydrochloride, Sodium Ricinoleate, 04.07 Muscle relaxants (skeletal) such as Afloqualone, Sodium Tetradecyl Sulfate and Tribenoside; Alcuronium, Atracurium Besylate, Baclofen, BenZoctamine, 0423 Sedatives and hypnotics, including: Acyclic ureides Benzoquinonium Chloride, C-Calebassine, Carisoprodol, Such as Acecarbromal, Apronalide, BomisoValum, Capuride, Chlormezanone, Chlorphenesin Carbamate, Chlorproethaz Carbromal and Ectylurea; ine, ChloZoxazone, Curare, Cyclarbamate, Cyclobenzaprine, 0424 Alcohols such as Chlorhexadol, Ethchlorvynol, Dantrolene, Decamethonium Bromide, Diazepam, Meparfynol, 4-Methyl-5-thiazoleethanol, tert-Pentyl Alco Eperisone, Fazadinium Bromide, Flumetramide, Gallamine hol and 2.2.2-Trichloroethanol: Triethiodide, Hexacarbacholine Bromide, Hexafluorenium 0425 Amides such as Butoctamide, Diethylbromoaceta Bromide, Idrocilamide, Lauexium Methyl Sulfate, Leptodac mide, Ibrotamide, Isovaleryl Diethylamide, Niaprazine, Tric tyline, Memantine, Mephenesin, Mephenoxalone, Metaxa etamide, Trimetozine, Zolpidem and Zopiclone; lone, Methocarbamol, Metocurine Iodide, Nimetazepam, 0426 Barbituric acid derivatives such as Allobarbital, Orphenadrine, Pancuronium Bromide, Phenprobamate, Amobarbital, Aprobarbital, Barbital, Brallabarbital, Butabar Phenyramidol, Pipecurium Bromide, Promoxolane, Quinine bital Sodium, Butalbital, Butallylonal, Butethal, Carbubarb, Sulfate, Styramate. Succinylcholine Bromide, Succinylcho Cyclobarbital, Cyclopentobarbital, Enallyipropymal, line Chloride, Succinylcholine Iodine, Suxethonium Bro 5-Ethyl-5-(1-piperidyl) barbituric Acid, 5-Furfuryl-5-isopro mide, Tetrazepam, Thiocolchicoside, Tizanidine, Tolp pylbarbituric Acid, Heptabarbital, Hexethal Sodium, Hex erisone, Tubocurarine Chloride, Vecuronium Bromide and obarbital, Mephobarbital, Methitural, Narcobarbital, Neal ZoXolamine; barbital, Pentobarbital Sodium, Phenallymal, Phenobarbital, 0408 Narcotic antagonists such as Amiphenazole, Cycla Phenobarbital Sodium, Phenylmethylbarbituric Acid, Zocine, Levallorphan, Nadide, Nalmfene, Nalorphine, Nalor Probarbital, Propallylonal, Proxibarbal, Reposal, Secobar phine Dinicotinate, Naloxone and Naltrexone; bital Sodium, Talbutal, Tetrabarbital, Vinbarbital Sodium and 04.09 Neuroprotective agents such as Dizocilpine; Vinylbital; 0410 Nootropic agents such as Aceglutamide, Acetylcar 0427 Benzodiazepine derivatives such as Brotizolam, nitine, Aniracetam, Bifematlane, Exifone, Fipexide, Ide Doxefazepam, Estazolam, Flunitrazepam, Flurazepam, benone, Indeloxazune Hydrochloride, Nizofenone, Oxirac Haloxazolam, Loprazolam, Lorimetazepam, Nitrazepam, etam, Piracetam, Propentofylline, Pyritinol and Tacrine; Quazepam, Temazepam and Triazolam; 0411 Ophthalmic agents such as 15-ketoprostaglandins; 0428 Bromides such as Ammonium Bromide, Calcium Bromide, Calcium Bromolactobionate, Lithium Bromide, 0412 Ovarian hormone such as Relaxin; Magnesium Bromide, Potassium Bromide and Sodium Bro 0413. Oxytocic drugs such as Carboprost, Cargutocin, mide; Deaminooxytocin, Ergonovine, Gemeprost, Methylergono 0429 Carbamates such as Amyl Carbamate Tertiary, vine, Oxytocin, Pituitary (Posterior), Prostaglandin E. Pros Ethinamate, Hexaprpymate, Meparfynol Carbamate, taglandin F, and Sparteine; Novonal and Tricholorourethan; 0414 Pepsin inhibitors such as Sodium Amylosulfate; 0430. Chloral derivatives such as Carbocloral, Chloral 0415 Peristaltic stimulants such as Cisapride; Betaine, Chloral Formamide, Chloral Hydrate, Chloralan 0416 Progestogens Such as Allylestrenol, Anagestone, tipyrine, Dichloralphenazone, Pentaerythritol Chloral and Chlormadinone Triclofos; 0417 Acetate, Delmadinone Acetate, Demegestone, 0431 Piperidinediones such as Glutehimide, Methypry Desogestrel, Dimethisterone, Dydrogesterone, Ethisterone, lon, Piperidione, Pyrithyldione, Taglutimide and Thalido Ethynodiol, Fluorogestone Acetate, Gestodene, Gestonorone mide; Caproate, Haloprogesterone, 17-Hydroxy-16-methylene— 0432 Quinazolone derivatives such as Etaqualone, progesterone, 17C.-Hydroxyprogesterone, 17C-Hydroxyges Mecloqualone and Methaqualone; and terone Caproate, Lynestrenol, Medrogestone, MedroX 0433 others such as Acetal, Acetophenone, Aldol, Ammo yprogesterone, Megestrol Acetate, Melengestrol, nium Valerate, Amphenidone, d-Bornyl C-Bromoisovalerate, Norethindrone, Norethynodrel, Norgesterone, Norgestimate, d-Bornyl Isovalerate, Bromoform, Calcium 2-Ethylbu Norgestrel, Norgestrienone, Norvinisterone, Pentagestrone, tanoate, Carfinate, C.-Chlorolose, Clomethiazole, Cypripe Progesterone, Promegestone, Quingestrone and Trengestone; dium, Doxylamine, Etodroxizine, Etomidate, Fenadiazole, 0418 Prolactin inhibitors such as Metergoline; Homofenazine, Hydrobromic Acid, Mecloxamine, Menthyl 0419 Prostaglandins and prostaglandin analogs such as Valerate, Opium, Paraldehyde, Perlapine, Propiomazine, Ril Arbaprostil, Carboprost, Enprostil, Bemeprost, Limaprost, mazafone, Sodium Oxybate, Sulfonethylmethane and Sul Misoprostol, Ornoprostil, Prostacyclin, Prostaglandin E, fonmethane; US 2012/O 141590 A1 Jun. 7, 2012 28

0434 Thrombolytic agents such as APSAC, Plasmin, Pro such as Carboplatin, Cisplatin and Oxaliplatin: Retinoids Urokinase, Streptokinase, Tissue Plasminogen Activator and Such as All-trans retinoic acid; and Vinca alkaloids and Urokinase; derivatives such as Vinblastine, Vincristine, Vindesine and 0435 Thyrotropic hormones such as TRH and TSH: Vinorelbine. 0436 Uricosurics such as Benzbromarone, Ethebenecid, 0448. In certain embodiments, the agent to be delivered is Orotic Acid, Oxycinchophen, Probenecid, Sulfinpyrazone, one or more proteins, hormones, vitamins or minerals. In Ticrynafen and Zoxazolamine; certain embodiments, the agent to be delivered is selected 0437. Vasodilators (cerebral) such as Bencyclane, Cin from insulin, IGF-1, testosterone, Vinpocetin, hexarelin, narizine, Citicoline, Cyclandelate, Ciclonicate, Diisopropy GHRP-6 or calcium. In certain embodiments, the composi lamine Dichloractetate, Eburnamorine, Fenoxedil, Flunariz tions contain two or more agents. ine, Ibudilast, Ifenprodil, Nafronyl, Nicametate, Nicergoline, 0449 The above list of active agents is based upon those Nimodipine, Papaverine, Pentifylline, Tinofedrine, Vincam categories and species of drugs set forth on pages THER-1 to ine, Vinpocetine and Viduidil; THER-28 of The Merck Index, 12th Edition, Merck & Co. 0438 Vasodilators (coronary) such as Amotriphene, Rahway, N.J. (1996). This reference is incorporated by ref Bendazol, Benfurodil Hemisuccinate, BenZiodarone, Chloa erence herein in its entirety. cizine, Chromonar, Clobenfurol, Clonitrate, Dilazep, Dipy 0450. The macromolecules and small molecules can be ridamole, proprenilamine, Efloxate, Erythritol, Erythrityl characterized by their ability to interact with the counterion Tetranitrate, Etafenone, Fendiline, Floredil, Ganglefene, and antisolvent. Such as citrate (counterion) and isopropanol Hexestrol Bis(B-diethylaminoethyl ether), Hexobendine, (solvent), to form intact, discrete microspheres containing a Itramin Tosylate, Khellin, Lidoflazine, Mannitol Hexanitrate, high content of the macromolecule or small molecule. The Medibazine, Nicorandil, Nitroglycerin, Pentaerythritol Tet content of the macromolecule or Small molecule in the micro ranitrate, Pentrinitrol, Perhexyline, Pimethylline, Preny spheres can vary from about or at 5%, 10%, 15%, 20%, 25%, lamine, Propatyl Nitrate, Pyridofylline, Trapidil, Tricromyl, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, Trimetazidine, TroInitrate Phosphate and Visnadine: 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 0439 Vasodilators (peripheral) such as Aluminum Nico 99% or greater weight/weight (w/w) of the microspheres. In tinate, Bamethan, Bencyclane, Betahistine, Bradykinin, Some embodiments, the macromolecule or Small molecule Brovincamine, Bufoniode, Buflomedil, Butalamine, Cetiedil, content of the microsphere is substantially the same as the Ciclonicate, Cinepazide, Cinnarizine, Cyclandelate, Diiso amount of macromolecule or Small molecule initially in solu propylamine Dichloracetate, Eledoisin, Fenoxidil, Flunar tion, prior to forming the microspheres. isine, Heronicate, Ifenprodil, Inositol Niacinate, 1soXSuprine, 0451. The macromolecules used to prepare microspheres Kallidin, Kallikrein, Moxisylyte, Nafronyl, Nicametate, by the methods provided herein can include peptides, such as Nicergoline, Nicofuranose, Nicotinyl Alcohol, Nylidrin, Pen Somatostatins and leuprolides, polypeptides and proteins, tifylline, Pentoxifylline, Piribedil, Protaglandin E, Sulocti glycopeptides such as Vancomycin, carbohydrates, including dil and Xanthinal Niacinate; lipids, fatty acids, polysaccharides and nucleic acids (DNA, 0440 Vasoprotectants such as Benzarone, Bioflavonoids, RNA or PNA, siRNA, tRNA), viruses, such as tobacco Chromocarb, Clobeoside, Diosmin, Dobesilate Calcium, mosaic virus, virus particles, viroids and prions. In some Escin, Rolescutol, Leucocyanidin, Metescufylline, Querce embodiments, the macromolecules are proteins, including tin, Rutin and Troxerutin; therapeutic proteins such as DAS181 (the sialidase fusion 0441 Vitamins, vitamin Sources, and vitamin extracts protein having the sequence of amino acid residues set forth such as Vitamins A, B, C, D, E, and Kand derivatives thereof, in SEQID NO:17), alpha1-antitrypsin, PI8, eglin c, Ecotin, Calciferols, Glycyrrhiza and Mecobalamin; aprotinin, recombinant human DNase, insulin, interferons, 0442 Vulnerary agents such as Acetylcysteine, Allantoin, recombinant human DNAse (rhDNAse, useful, for example, Asiaticoside, Cadexomer Iodine, Chitin, Dextranomer and in the treatment of cystic fibrosis as an inhalation therapeutic Oxaceprol; (Genentech); see also Shaket al., Proc. Natl. Acad. Sci. USA, 0443 Anticoagulants such as heparin; 87:9188-9192 (1990)), human serum albumin, human growth 0444 Miscellaneous such as Erythropoietin (Hematinic), hormone, parathyroid hormone and calcitonin. In some Filgrastim, Finasterlde (Benign Prostate Hypertrophy) and embodiments, the protein is DAS181, the counterion is Interferon B1-C. (Multiple Sclerosis). Sodium sulfate or sodium citrate, and the antisolvent is iso 0445 Nucleic acid based-therapeutics, such as antisense propanol. In other embodiments, the macromolecule is a nucleic acids and siRNA, or genes for gene therapy. nucleic acid, e.g., siRNA, the counterion is polyethylene 0446 Gene delivery vehicles for gene therapy, such as imine (PEI) and the antisolvent is isopropanol. In yet other viruses, virus particles and viroids. embodiments, the macromolecule is a virus, e.g., tobacco 0447 Chemotherapeutic agents, including Alkylating mosaic virus, the counterion is Na-sulfate/Na-acetate, and the agents such as Cyclophosphamide, Mechlorethamine, antisolvent is isopropanol. In further embodiments, the mac Chlorambucil and Melphalan; Anthracyclines such as romolecule is a peptide, e.g., leuprolide or somatostatin, the Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitox counterion is sodium glutamate, and the antisolvent is isopro antrone, Valrubicin; Cytoskeletal disruptors such as Pacli panol. taxel and Docetaxel, and other taxanes; Epothilones; Inhibi 0452. The small molecules used to prepare microspheres tors of topoisomerase II Such as Etoposide, Teniposide and by the methods provided herein can include antibiotics, such Tafluposide; Nucleotide analogs and precursor analogs such as the aminoglycosides tobramycin and kanamycin, penicil as AZacitidine, AZathioprine, Capecitabine, Cytarabine, lins and tetracyclines, sterols, steroid hormones, prostaglan Doxifluridine, Fluorouracil, Gemcitabine, Mercaptopurine, dins, chemotherapeutic agents, such as paclitaxel, or any Methotrexate and Tioguanine (formerly Thioguanine); Pep other small molecule of interest. For example, in one embodi tide antibiotics Such as Bleomycin; Platinum-based agents ment, the Small molecule is tetracycline, the counterion is US 2012/O 141590 A1 Jun. 7, 2012 29 arginine, and the antisolvent is isopropanol. In another oxaloacetate, malate, maleate, oxalate, piruvate, citrate. Suc embodiment, the Small molecule is kanamycin or tobramy cinate, fumarate, ketoglutarate, butanetricarboxylic acid, cin, the counterion is itaconic acid, and the antisolvent is hydromuconic acid, cyclobutanedicarboxylic acid, dimethyl isopropanol. In yet another embodiment, the Small molecule maleate, deoxyribonucleic acid, polyglutamic acid, folic is paclitaxel, the solvent is t-butanol, the antisolvent is water acid, lactic acid, ascorbic acid, carminic acid, Sorbic acid, (in which sodium citrate is dissolved to form a citrate buffer), malonic acid, EDTA, MOPS, TES, MES, PIPES, pyridine, and the counterion is sodium citrate. tricine, betaine, Sulfuric acid, thiosulfuric acid, phosphoric 0453 The methods provided herein can avoid the use of acid, adenosine triphosphate, nitric acid, itaconic acid, pivalic conditions, such as heat, that can compromise the activity of acid, dimethylmalonic acid, and perchloric acid. In some the compound, e.g., melting of a small molecule compound or embodiments, itaconic, pivalic, dimethylmalonic, and Suc denaturation of a protein, and reduce its activity. The micro cinic acids are used as counterions in the methods provided spheres provided according to the methods provided herein herein. therefore can be used to prepare vaccines or other therapeutic 0458 Cationic counterions can be inorganic (ammonium, medications that require compounds to retain their activity, phosphonium, Sulfonium, cesium, rubidium, etc.) or organic e.g., proteins or peptides to be present in their native confor compounds that carry groups known as amine, amide, imine, mation. imide, guanidine, imidazole, dioxane, aniline. Organic com 0454. The concentration of the compound in solution, pounds carrying other cationic groups or have positive charge used during precipitation of the microspheres, can be between polarizability due to other molecular characteristics also can about or at 0.1 mg/ml to about or at 0.2,05, 0.8, 1.0, 2.0, 5.0, be used. Compounds that can be used as cationic counterions 10.0, 12.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, also include, but are not limited to, the following: Tris, Bis 70.0, 80.0, 90.0, 100, or 200 mg/ml. In some embodiments, Tris, Bis-Tris propane, diaminopropane, piperazine, pipera the concentration is between about or at 1 mg/ml and about or dine, pentylamine, diaminobutane, propylamine, trimethy at 20 mg/ml. Depending on the characteristics of the molecule lamine, triethylamine, spermine, spermidine, putrescine, (pl. hydrophobicity, solubility, stability, etc.) and other pro cadaverine, ethanolamine, diethanolamine, triethanolamine, cess parameters, the concentration of molecule can empiri imidazole, tetramethylammonium, trimethylammonium, cally be determined to achieve formation of microspheres of ammonium, cesium, rubidium, imidazole, polyethileneimine a desired size. In general, molecules with lower solubility in (PEI), DEAE, TEAE, QAE. the solvent prior to adding counterion and organic solvent can 0459 Zwitterionic counterions possessing any charged be used at lower concentrations (0.1-5 mg/ml) to form micro groups in any combination can also be used. Compounds that spheres according to the methods herein, while molecules can be used as Zwitterionic counterions include, but are not with higher solubility can be used at 1-20 mg/ml or higher. If limited to, the following: HEPES, BICINE, glycine, glycylg the formation of amorphous aggregates or aggregated micro lycine, 6-aminohexanoic acid, piperidic acid, natural and spheres is observed, the concentration of the molecule gen non-natural amino acids (e.g., histidine, glutamine, arginine, erally should be decreased to reduce or prevent Such aggre lysine). gation. 0460. The counterions can be used as acids (e.g. sulfuric 0455 Nature and Concentration of Counterion acid) or bases (e.g. imidazole) or their salts (e.g. sodium 0456. The counterion can be any compound capable of sulfate orimidazole-HCl). Counterions that can be used in the neutralizing one or more oppositely charged groups on the methods provided herein include those listed by the National molecule at the pH at which the method is performed. Formulary, United States Pharmacopeia, Japanese Pharma Depending on the characteristics of the molecule (pK, pl. copeia, or European Pharmacopeia, the clinical safety of nature and quantity of charged groups, distribution of charge which has been demonstrated (citric acid, malic acid, amino groups on the Surface, Solubility and structural stability under acids, Sulfate, etc.). In some embodiments, counterions used different pH conditions), the pH can empirically be deter in the methods provided herein include ones for which safety mined for microsphere formation. In general, for a macro has been established or as falling into the GRAS (generally molecule Such as a protein, if precipitation is performed at a regarded as safe) category. The counterions (or their salts) can pH below the pK of the macromolecule, anionic counterions be solidat room temperature (about 25°C.), or at the intended can be used. In general, if precipitation is performed at a pH temperature of use and storage). Combinations of two or above the pK of the macromolecule, cationic counterions can more counterions also can be used. Volatile and liquid coun be used. The counterion can empirically be selected based on terions also can be used in the methods provided herein. its Suitability to initiate microsphere formation. In some 0461 The concentration of counterion generally is main embodiments, the counterion can have a molecular weight of tained between about or at 0 mMandabout or at 0.1, 0.2,0.5, 60 Daltons or greater, or about 75 Daltons or greater. The 0.8, 1.0, 2.0, 3.0, 5.0, 7.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, counterion can be a polymer, Such as polyethylene glycol 60.0, 70.0, 80.0, 90.0 and 100.0 mM. In some embodiments, (PEG) or polyethyleneimine (PEI). the concentration of the counterion is between about or at 0.5 0457. The counterions can be anionic, cationic or Zwitte mMandabout or at 20 mM. Depending on the characteristics rionic. Anionic counterions can be inorganic (phosphate, Sul of the macromolecule or Small molecule (pl (e.g., for proteins phate, thiocyanate, thiosulfate, hypochlorate, nitrate, bro or peptides), hydrophobicity, solubility, stability, etc.) and mine, iodine, etc.) or organic compounds that carry charge other process parameters, the concentration of the counterion polarizable groups including enol, hydroxy, —SH, can empirically be determined using, for example, a high carboxylic, carboxymethyl, Sulfopropyl. Sulfonic, and phos throughput format as provided herein. In general, the forma phoric. Organic compounds carrying otheranionic groups or tion of oversized microspheres, amorphous aggregates or having negative charge due to other molecular characteristics aggregated microspheres indicates that the concentration of also can be used. Compounds that can be used as anionic counterion should be decreased, while failure to form micro counterions also include, but are not limited to, the following: spheres (broken glass-like crystals or flakes) or formation of US 2012/O 141590 A1 Jun. 7, 2012 30 microspheres below the desired size indicates that the con nation of an aqueous liquid and a non-aqueous and/or organic centration of counterion should be increased. liquid, or both can be non-aqueous and/or organic liquids. 0462 Counterions that Produce Microspheres in the 0468 Nature and Concentration of Solvent Absence of Added Compound 0469 A number of macromolecules and small molecules, among the microparticle-forming compounds of interest, are 0463. In the course of screening conditions for micro soluble in water and aqueous solutions; hence, the solvent for sphere formation, including empirical variation of the type Such molecules generally is aqueous. For compounds that are and nature of solvent, antisolvent, solvent/antisolvent system not soluble in aqueous solvents, the solvent used in the meth and counterions (sometimes in a buffer, in other embodiments ods provided herein generally can be water miscible and is present without a buffer) for each compound of interest, it was selected from among alcohols (methanol, ethanol. 1-pro found that several control reactions containing no added com panol, isopropanol, butanol, tert-butyl alcohol), chloroform, pound produced microspheres of counterion/buffer. For dimethyl chloride, polyhydric Sugar alcohols (glycerin, example, a solution of 15 mMunbuffered arginine with 25% erythritol, arabitol. Xylitol, Sorbitol, mannitol), aromatic isopropanol produced microspheres with a rating of 7, with hydrocarbons, aldehydes, ketones, esters, ethers (di-ethyl Some crystallinity present. A solution containing 2 mM Na ether), alkanes (hexane, cyclohexane, petroleum ether), alk sulfate with 0.2 mMNa-Acetate buffer at either pH4 or pH 6 enes, conjugated dienes, toluene, dichloromethane, acetoni in 15% isopropanol, resulted in microspheres with a rating of trile, ethyl acetate, polyols, polyimids, polyesters, polyalde 7, at both conditions. Although some clumping was present, hydes, dimethyl formamide (DMF), dimethyl sulfoxide many Small, well-separated, discrete microspheres also were (DMSO), carbon tetrachloride, and mixtures thereof. In some observed. Itaconic acid also showed a propensity to form embodiments, the solvent can be volatile. In other embodi microspheres independently, with no added compound. ments, when incorporation of the solvent into the micro When a 2 mM solution of itaconic acid was buffered with spheres is desired, non-volatile solvents can be used that sodium hydroxide at pH 4 in the presence of 15% isopro provide, for example, novel characteristics to the micro panol, microspheres were formed. A similar cocktail contain spheres (e.g., Sustained release or added mechanical ing 2 mMitaconic acid buffered to pH 7 with 5% isopropanol, strength). The concentration of the solvent generally can be produced hygroscopic microspheres. Similarly, pivalic acid maintained between about or at 0.1%, to about or at 0.5%, also was found to make microspheres independent of an 1%, 2%. 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, additional compound. For example, when a 2 mM solution of Volume/volume (v/v). In some embodiments, the concentra pivalic acid was titrated to pH 5 with sodium hydroxide in the tion of the solvent is between about or at 1% to about or at presence of 15% isopropanol, microspheres of pivalic acid 30%, V/v. Organic compounds that are partially miscible or were produced that had a rating of 6. completely immiscible with water also can be used as Sol 0464. The above types of counterions can be useful as a vents for water-insoluble compounds. tool for catalyzing microsphere formation in molecules that 0470 Organic solvents that can be used in the methods otherwise might not form micro particles. provided herein include alcohols and others listed as Class 3 0465 Solvent/Antisolvent System and 2 solvents in International Conference on Harmonisation 0466. A solvent/antisolvent system suitable for use in the (ICH) Harmonised Tripartite Guideline (Impurities: Guide methods of microsphere formation provided herein can be line for Residual Solvents), safe handling of which has been based on the relative solubilities of the compound of interest established in pharmaceutical and food industries. in the solvent and in the antisolvent, as known and available to 0471 Depending on the characteristics of the molecule those of skill in the art. Alternately, the solubilities of the (hydrophobicity, solubility, stability, etc.) and other process compound of interest in the solvent and/or the antisolvent can parameters, the choice and concentration of the solvent can be be determined empirically, by varying the types and concen optimized, for example, using high-throughput screening on trations of various solvents, antisolvents and counterions in a microtiter plates or similar chips or other device. In general, high-throughput format, as provided herein, or by other meth uncontrolled precipitation before the initiation of cooling, the ods known to those of skill in the art including, but not limited formation of oversized microspheres, amorphous aggregates, to, dissolution Saturation testing. aggregated microspheres or Sticky aggregates indicates that 0467. In general, the compound of interest that is used to solvent that affords higher solubility of the drug should be form the microspheres is soluble in the selected solvent (from used, while failure to form microspheres (broken glass-like about or at 1 mg/ml to about or at 100 mg/ml). The antisolvent crystals or flakes) or formation of microspheres below the can be selected from among a group of solvents in which the desired size indicates that use of solvent with lower drug compound of interest has limited or no solubility. The solvent solubility may be beneficial. and antisolvent generally are selected Such that they are mis 0472 Nature and Concentration of Antisolvent cible, or partially miscible, at the temperatures used for dis 0473. In general, if the compound of interest is water Solution to prepare the cocktail solution. In some embodi soluble and in an aqueous solution, the antisolvent is an ments it is possible, however, that the solvent and antisolvent organic solvent. On the other hand, if the compound of inter can have different freezing points; therefore, lowering the est is water-insoluble, the antisolvent is an aqueous solvent. temperature can cause one of the components to freeze, The solvent and the antisolvent can, however, both be organic thereby increasing the concentration of the antisolvent, Solvents. Under conditions of mixing of the cocktail reagents thereby inducing precipitation (e.g., in some preparations of and/or precipitation by chilling to initiate microsphere for microspheres of DAS181, using 5% isopropanol as the anti mation, the antisolvent generally is miscible or partially mis Solvent). In general, it is desirable to select a solvent/antisol cible with the solvent in which the compound forming the vent system that does not facilitate precipitation of compo microparticle is dissolved. Such solvents include, for nents other than the compound of interest (e.g., counterion example, water and other aqueous solutions, such as buffers, and excipients). The solvent and antisolvent can be a combi alcohols (methanol, ethanol. 1-propanol, isopropanol, US 2012/O 141590 A1 Jun. 7, 2012 butanol, tert-butyl alcohol), chloroform, polyhydric Sugar pH of the cocktail, using a different counterion or decreasing alcohols (glycerin, erythritol, arabitol. Xylitol, Sorbitol, man the concentration of the compound in the cocktail, can con nitol), aromatic hydrocarbons, aldehydes, ketones, esters, Veniently and rapidly be performed in semi high-throughput ethers (di-ethyl ether), alkanes (hexane, cyclohexane, petro or high throughput format. In general, for forming protein or leum ether), alkenes, conjugated dienes, toluene, dichlo polypeptide-based microspheres, a pH value that is below the romethane, carbon tetrachloride, dimethylformamide pl of the protein provides optimal microsphere formation. (DMF), dimethyl sulf oxide (DMSO), acetonitrile, ethyl Such empirical optimization methods are applicable to other acetate, polyols, polyimides, polyesters, polyaldehydes, and macromolecules and Small molecules as provided and exem mixtures thereof. plified herein. 0474. In some embodiments, the organic solvent can be 0480 Ionic Strength volatile. In other embodiments, when incorporation of the 0481. The ionic strength of the cocktail solution can be organic solvent into the microspheres is desired, non-volatile modulated by adjusting the concentration of the counterion or organic solvents can be used that provide, for example, novel other salts, such as chlorides or acetates. In some embodi characteristics to the microspheres (e.g., Sustained release or ments, no additional salt is required to produce microspheres. added mechanical strength). The concentration of the organic In certain embodiments, the ionic strength can be adjusted to Solvent generally can be maintained between about or at preserve the structural integrity and activity of the molecule. 0.1%, to about or at 0.5%, 1%, 2%. 5%, 10%, 15%, 20%, Examples of other applications where the presence of specific 25%, 30%, 40% or 50%, volume/volume (v/v). In some salts can be beneficial include formulations of parenteral and embodiments, the concentration of the organic solvent is other drugs, or foods where specific tonicity or buffering between about or at 1% to about or at 30%, V/v. Organic capacity may be required upon reconstitution of micro compounds that are partially miscible or completely immis spheres. cible with water also can be used. 0482 Cooling Ramp 0475 Organic solvents that can be used in the methods 0483 The cocktail containing a molecule, a counterion provided herein include alcohols and others listed as Class 3 and a suitable solvent/antisolvent system initially is prepared, and 2 solvents in International Conference on Harmonisation prior to cooling, at a temperature at which the molecule is (ICH) Harmonised Tripartite Guideline (Impurities: Guide soluble, generally about -15° C. to about 30° C. In some line for Residual Solvents), safe handling of which has been embodiments, the initial temperature, prior to cooling is at established in pharmaceutical and food industries. ambient temperature (18°C. to 25-30°C.). In other embodi 0476 Depending on the characteristics of the molecule ments, for example, with small molecules, the compound can (hydrophobicity, solubility, stability, etc.) and other process be dissolved in the solvent and/or antisolvent system at much parameters, the choice and concentration of the anti-solvent higher temperatures, for example, about or at 50° C., 60°C., can be optimized, for example, using high-throughput screen 65° C., 70° C., 75° C., 80° C., 85°C., 90° C., 95°C., 100° C., ing on microtiter plates or similar chips or other device. In 125° C., 150° C., 175°C., 200° C. or greater, then cooled to a general, uncontrolled precipitation before the initiation of temperature of, for example, about or at 190° C. 170° C. cooling, the formation of oversized microspheres, amorphous 150° C. 125° C., 100° C., 80° C., 75° C., 60° C., 50° C., 400 aggregates, aggregated microspheres or Sticky aggregates C., 30° C., 20°C., 15° C. or lower, at which the microspheres indicates that the concentration of anti-solvent should be are formed. The microspheres are formed by a process Such as decreased, while failure to form microspheres (broken glass precipitation, phase separation or colloid formation upon like crystals or flakes) or formation of microspheres below the gradual cooling to a temperature below the temperature at desired size indicates that the concentration of the anti-sol which the macromolecule is dissolved and in solution. The vent should be increased. rate at which cooling is performed can control the formation 0477 pH and other characteristics such as size of the microspheres. In 0478. In addition to initiating microsphere formation, the general, when the molecule is a protein, flash-freezing in counterion also can serve as a buffer. Alternately, in some liquid nitrogen does not generate microspheres. embodiments, a buffering compound can be used to obtain the 0484 The rate at which cooling and freezing of the cock desired pH. In some embodiments, the buffering compound is tail (cooling ramp) is performed can determine the final size 60 Da or larger. Depending on the characteristics of the mol of the microspheres. In general, a faster cooling ramp yields ecule (pl. hydrophobicity, solubility and stability at a specific Smaller microspheres whereas a slower cooling ramp yields pH, etc.) and other process parameters, the optimal pH can larger microspheres. Without being bound by any theory, the empirically be adjusted to achieve formation of microspheres cooling rate can determine the rate of: (1) nucleation that of desired dimensions and preserve the activity of the mol produces initial Smaller microspheres and (2) a fusion process ecule. In general, failure to form microspheres (broken glass in which the initial microspheres coalesce (aggregate) and like crystals or flakes) indicates that the molecule may be too anneal into larger microspheres. Fusion of the Smaller par soluble under the conditions used. Formation of amorphous ticles into larger ones is a time dependent process that can be aggregates can indicate that precipitation is not well con determined, for example, by the duration for which liquid trolled and the molecule. Such as a protein, may not be stable Suspension of microspheres exists prior to freezing. Due to or soluble at the pH used. the reversible nature of the bonds between molecules, in the 0479. It has been observed that certain compound/counte microsphere compositions provided herein, Smaller micro rion combinations can cause immediate and uncontrolled spheres annealing into larger particles can generate micro precipitation at certain pH values. The high-throughout spheres with Smooth Surfaces. Depending on the size of screening methods provided herein can be used to empirically microparticles desired, the cooling rate can be from about determine the appropriate combination of protein, pH and 0.01° C.Amin or 0.01° C./min to about 20° C./min or 20° counterion to form microspheres of desired dimensions. For C./min: from about or at 0.05° C./min or about or at 0.1° example, empirical determinations including changing the C./min to about or at 10° C./min or about or at 15° C./min, US 2012/O 141590 A1 Jun. 7, 2012 32 from about or at 0.2° C./min to about or at 5° C./min, from conjunction with cationic, anionic or Zwitterionic counteri about or at 0.5°C/minto about or at 2°C./min, or about or at ons and antisolvents at various concentrations can be 1° C./min. In some embodiments, the cooling ramp can be screened. For example, the screening can be performed using between 0.1° C. per minute and about 40° C. per minute. In several identical microtiter plates, to each of which the mol other embodiments, a cooling ramp can be between about ecule of interest is added at various concentrations. Each set 0.5°C. per minute and 15° C. per minute. of test conditions can be screened in duplicate. In some 0485 Depending on the specific needs, in some embodi embodiments, microplates with flat-bottom wells can be used ments it can be desirable to adapt the production process to with the skirt of the microtiter plate broken off to permit good the specific equipment. In some embodiments, a lyophilizer heat transfer between the lyophilizer shelf and the bottoms of with temperature-controlled shelves can be used for the cool the wells. The microplates can be placed on the shelves of the ing. In other embodiments, endothermic reactions can be lyophilizer and cooled to form microspheres and to Subse used for the cooling. If the microspheres produced are larger quently solidify the Suspensions. Upon freezing of the con than desired, other parameters of the process including con tents of the wells, a vacuum can be applied. At the end of centration of the molecule, antisolvent, counterion, ionic lyophilization, one of the duplicate plates can be reconstituted strength and/or pH can be modified to achieve the desired with water or a buffer of choice to observe if certain condi reduction in size of the microspheres. tions rendered the molecule insoluble or reduced its activity. 0486 For a faster cooling ramp (smaller particle size), the Conditions that resulted in material that can readily be res cocktail solution can be passed through a heat exchanger, olubilized or provide microspheres with desirable character Such as that used in a continuous mode. If the size of micro istics can be subjected to further analysis by spectroscopic, spheres needs to be increased, increased concentrations of chromatographic, enzymatic or other assays to confirm that one of the cocktail ingredients (molecule, antisolvent, coun native structure and activity are preserved. Lyophilized mate terion) can provide the desired increase in the size of micro rial in a duplicate plate can be used for microscopy to deter spheres. mine whether microspheres are formed. Conditions that pro 0487. In general, the cooling should be performed uni duced microspheres can further be modified and fine-tuned to formly and at a steady rate to prevent the formation of aggre produce microspheres of desirable size and characteristics. gates and crystals or glass-like particulates. Depending on the 0491 Kits for performing high-throughput screens can be concentration of the antisolvent, the precipitation of the mol provided and can contain all the ingredients used in the meth ecule into microspheres can occur in several ways. At higher ods provided herein including one or more of a molecule, concentrations of antisolvent (about 5%-40%, dependent on buffers, pre-dispensed cocktail of known composition (anti the actual components used) the microspheres generally can Solvent, counterion) and/or salts. Kits can contain 3, 4, 5, 10. form when the cocktail solution is still in liquid form. At 15, 20, 30, 40, 50, 100 or more (in some embodiments, 96 or lower concentrations of antisolvent (2-25%, dependent on the more) buffers with predetermined pH, counterion, ionic actual components used) ice crystals can form first, following strength and antisolvent in each microtiter plate. The micro which the expelled molecules and antisolvent reach can reach titer plate supplied with the kit can be modified so that the a critical local concentration and precipitate. A further bottoms of the wells are in direct contact with the shelf of the decrease of temperature in the near-bottom layer of the lyo lyophilizer. philizertray can lead to complete solidification of the liquid Suspension and further expulsion of the antisolvent into the C. Large-Scale Manufacture of Microparticles top layer. An excess of antisolvent in the top layer can cause 0492. The methods provided herein can be scaled for the uncontrolled precipitation of the molecule and aggregation of manufacture of large quantities of microspheres. For microspheres. This effect usually can be alleviated by select example, the Batch Process described herein is suitable for ing appropriate ratios of the components—molecule, counte the manufacture of high quality dry powder microspheres in rion, antisolvent, salts, etc. in the cocktail. In addition, main an amount ranging from, for example, milligrams of to about taining a thin layer of cocktail in the lyophilization tray or a kilogram, based on the capacity of the mixing tank and/or mixing of the cocktail while being chilled can prevent forma lyophilizer shelf space. An alternative “continuous” process tion of aggregates and crystals and yield uniform micro described herein can be used to manufacture amounts ranging spheres. For example, if a relatively low concentration of from, for example, hundreds of grams to hundred or more Isopropanol (e.g. 2-6%) is used, and a thin layer of cocktail kilograms (100 grams to 100 kg and above). An additional (10-20 mm) is filled into the tray, and the tray is placed on a advantage of the continuous process is better control over the pre-chilled shelf (generally, -30° C. to -75° C.), uniform chilling of the cocktail. microspheres can be obtained. 0493. The large scale manufacture by a batch process or by 0488. The methods provided hereincan, under some con a continuous process can follow, for example, one or more of ditions, lead to substantially all or all the molecule being the steps described below in any combination: incorporated from the Solution into the microspheres 0494 Precipitation of the molecule into microspheres. 0489 High-Throughout Screening of Microparticle For This step can be performed in a batch mode by placing mation Conditions and Optimization of Particle Formation the cocktail solution containing the desired concentra 0490 Depending on the characteristics of the molecule, tion of molecule, organic solvent and counterion in lyo the composition of the cocktail solution used to prepare the philization tray(s) and placing the tray(s) onto lyo microspheres according to the methods provided herein can philizer shelves. Alternatively, trays can be chilled and be optimized. The optimization can rapidly be performed in a frozen on a chilled platform or other type of equipment medium or high throughput format using, for example micro (e.g., a freezer) and stored for a period of time frozen and titer plate(s) or chips where tens to hundreds to thousands to lyophilized later. Alternatively, the microspheres can be tens of thousands of cocktails can be screened simulta formed by precipitationina vessel with stirring, wherein neously. In some embodiments, a number of pH values in the vessel is placed onto a cold Surface or a cooling coil US 2012/O 141590 A1 Jun. 7, 2012 33

is immersed into liquid or while the cocktail is being ski S.J. Biopharmaceutical powders: particle formation and recirculated through a heat exchanger using a peristaltic formulation considerations. Curr Pharm Biotechnol 2000; pump. Alternatively, the microspheres can be formed by 1(3):283-302; MaaY F. Nguyen PA, Hsu SW. Spray-drying precipitation in a continuous mode, by passing the cock of air-liquid interface sensitive recombinant human growth tail solution through a heat exchanger(s) once using a hormone. J Pharm Sci 1998: 87(2): 152-159; Vanbever R, peristaltic pump. Mintzes J. D. Wang Jet al. Formulation and physical charac 0495 Removal of bulk liquid. The suspension of the terization of large porous particles for inhalation. Pharm Res microspheres can be concentrated using standard cen 1999; 16(11):1735-1742: Bot AI, Tarara TE, Smith DJ, Bot trifugation, continuous flow centrifugation (e.g., CARR SR, Woods CM, Weers J.G. Novellipid-based hollow-porous ViaFuge Pilot), or filtration (e.g., on glass fiber, sintered microparticles as a platform for immunoglobulin delivery to glass, polymer filters, hollow fiber cartridges (e.g., those the respiratory tract. Pharm Res 2000; 17(3):275-283; MaaY manufactured by GE Healthcare) or tangential flow fil F. Nguyen PA, Sweeney T. Shire SJ, Hsu C C. Protein tration cassettes (TFF cassettes. Such as those manufac inhalation powders: Spray drying VS spray freeze drying. tured by Millipore or Sartorius)). The removal of bulk Pharm Res 1999; 16(2):249-254: Sellers S P Clark G. S. liquid (50% or greater) can result in a faster drying cycle Sievers R. E. Carpenter J. F. Dry powders of stable protein and higher efficiency and throughput. formulations from aqueous solutions prepared using Super 0496 Drying the microspheres. The recovered micro critical CO(2)-assisted aerosolization. Pharm Sci 2001; spheres formed by any mode, can be dried by conven 90(6):785-797: Garcia-Contreras L. Morcol T. Bell SJ, tional lyophilization. Alternatively, the microspheres Hickey A.J. Evaluation of novel particles as pulmonary deliv can be dried under ambient temperature and atmo ery systems for insulin in rats. AAPS PharmSci 2003; 5(2): spheric pressure, eliminating the use of lyophilizer. E9; Pfutzner A, Flacke F. Pohl R et al. Pilot study with technosphere/PTH(1-34)—a new approach for effective pull D. Microparticle Compositions monary delivery of parathyroid hormone (1-34). Horm Metab 0497. The molecules contained in the microparticle com Res 2003: 35(5):319-323; Alcock R, Blair JA, O’Mahony D positions obtained by the methods provided herein are sub J. Raoof A, Quirk A. V. Modifying the release of leuprolide stantially structurally and chemically unchanged by the meth from spray dried OED microparticles. J Control Release ods. For example, when the molecule is a macromolecule 2002; 8202-3):429-440; Grenha A, Seijo B, Remunan-Lopez such as Green Fluorescent Protein or Red Fluorescent Pro C. Microencapsulated chitosan nanoparticles for lung protein tein, their fluorescence and native conformation and activity delivery. EurJ Pharm Sci 2005; 25(4-5):427-437; Edwards D of the proteins are retained in the microparticles. The dry A, Hanes J. Caponetti G. et al. Large porous particles for microspheres, obtained by volatilizing substantially all of the pulmonary drug delivery. Science 1997: 276(5320): 1868 solvents and/or moisture except for the solvent and other 1871; McKenna BJ, Birkedal H, Bartl M H, Deming T J, components associated with the microspheres, can be stored Stucky G. D. Micrometer-sized spherical assemblies of and their activity can Substantially be recovered upon recon polypeptides and Small molecules by acid-base chemistry. stitution. The relatively low moisture content of the micro Angew Chem Int Ed Engl 2004; 43(42):5652-5655; Oh M, particles provided herein, for example, between about or at Mirkin CA. Chemically tailorable colloidal particles from 0.01% to about or at 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 1.0%, infinite coordination polymers. Nature 2005:438(7068):651 2.0%. 3.0%. 4.0%, 5.0%, 5.5%, 6.0%, 6.5%. 7.0%. 7.5%, 654; U.S. Pat. No. 5,981,719, U.S. Pat. No. 5,849,884 and 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, U.S. Pat. No. 6,090,925; U.S. Patent application No. 12.0%, 12.5%, 14%, 15%, 16%, 17%, 18%. 19%, or 20%, can 20050234.114: U.S. Pat. No. 6,051,256). provide improved stability. The microspheres obtained by the 0498. The microparticles obtained by the methods pro methods provided herein also are homogeneous in size and vided herein can be of any shape—a regular geometric shape shape, and can be obtained reproducibly with the desired including, but not limited to, spherical, elliptical, square, characteristics. Other techniques traditionally used for prepa triangular and polyhedral, or an irregular shape. The micro ration of dry formulations (salt precipitation, alcohol or particles can have sizes (mean width or diameters) in the acetone precipitation, lyophilization, e.g.) can result in com range of from about or at 0.001 micron to about or at 0.002, plete or partial inactivation of the molecule, e.g., denaturation 0.005, 0.01, 0.02, 0.03, 0.05, 0.1, 0.02, 0.03,0.5, 1.0, 2.0, 2.5, of a protein. In addition, the microspheres prepared by the 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, methods provided herein avoid the need for complex or spe 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, or 50.0 or greater cialized spray drying, spray freeze-drying, Supercritical fluid microns. For pulmonary administration to the alveoli, anti-solvent based processes or milling processes (See, for depending on the application, the size can be from about 0.1 example, Laube BL. The expanding role of aerosols in sys micron or less to about or at 0.5 micron or greater, up to about temic drug delivery, gene therapy, and vaccination. Respir or at 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2.0 or 5.0 microns or greater. Care 2005:50(9): 1161-1176: Taylor G, Gumbleton M. Aero For administration by inhalation to the throat, trachea and sols for Macromolecule Delivery: Design Challenges and bronchi, the size can be from about or at 0.5 microns to about Solutions. American Journal of Drug Delivery 2004; 2(3): or at 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 143-155; Smyth HDC, Hickey A.J. Carriers in Drug Powder 7.5, 8.0, 8.5, 9.0, 9.5, 9.5, 10.0 10.0, 15.0 or 20.0 microns or Delivery. Implications for Inhalation System Design. Ameri greater, or in Some embodiments from about or at 1.0 micron can Journal of Drug Delivery 2005:3(2):117-132: Cryan SA. to about or at 2.0 microns. In some embodiments, the micro Carrier-based strategies for targeting protein and peptide particles are substantially spherical in shape. drugs to the lungs. AAPSJ 2005: 7(1):E20-E41; LiCalsi C, 0499. The molecules that can be used to form micropar Maniaci M.J, Christensen T. Phillips E, Ward G. H. Witham C. ticles according to the methods provided herein can include A powder formulation of measles vaccine for aerosol deliv preventative agents, prophylactic agents, therapeutic and ery. Vaccine 2001; 19(17-19):2629-2636; MaaY F. Prestrel diagnostic agents, processed foods, dietary Supplements, US 2012/O 141590 A1 Jun. 7, 2012 34 nutritional Supplements, cosmetic compounds and polymers. teins, peptides, nucleic acids, polymers or combinations In some embodiments, cross-linking agents, salts, or other thereof, or certain Small molecules such as amino acids, compounds can be included in the formulation cocktail to Sodium acetate, sodium Sulfate, Sodium citrate or combina modify solubility of the microspheres and/or enhance their tions thereof. Therapeutic agents or other active agents can be mechanical strength. In some embodiments, microspheres added at the time of microsphere formation or added to the that are insoluble in most aqueous or organic solvents can be Suspension of formed microspheres. Alternatively, therapeu used to manufacture particles Such as chromatographic resins tic agents can be blended with the dry microsphere composi and dispersible abrasives. In other embodiments, micro tions by mixing, tumbling or other techniques practiced in spheres with partial solubility in solvents such as pharmaceu pharmaceutical and food industries. tical vehicles for delivery can be useful in the manufacture of 0504 Polymers that can serve as micro-carriers for other Sustained release active agent or therapeutic formulations. therapeutic agent(s) in the microspheres provided herein can 0500. In some embodiments, the microparticles provided be any of those defined herein including, but not limited to, herein can be used in combination with an inhalation device nucleic acids such as deoxyribonucleic acid (DNA), ribo to deliver a therapeutic dose of microspheres to the respira nucleic acid (RNA), and mixed DNA or RNA derivatives, or tory airways and lungs of a Subject. For example, when the peptide nucleic acids (PNA), polyacrylamides, polystyrenes, molecule is the DAS181 protein (sequence set forth in SEQ polyalkyl-substituted Styrenes, polyacrylates, polymethacry ID NO: 17), microspheres of about 0.5 micron to about 8 lates, polyacrylic acid, polymethacrylic acid, polyvinyl chlo microns, or about 1 micron to about 5 micron can be obtained ride, polyvinyl acetate, polybutadiene, polyisoprene, poly by the methods provided herein, using sodium sulfate as the ethylene glycol and polyethyleneimine. Other exemplary counterion and isopropanol as the organic solvent. For organic or inorganic polymers, natural and synthetic poly DAS181 microspheres, which are administered to prevent or mers, include, but are not limited to, agarose, cellulose, nitro treat viral infections that initiate in the respiratory tract, such cellulose, cellulose acetate, other cellulose derivatives, dext as influenza, it can be desirable to deposit the microspheres in ran, dextran-derivatives and dextran co-polymers, other the throat, trachea or bronchi. The DAS181 fusion protein polysaccharides, glass, silica gels, proteins such as gelatin, formulated as microspheres can act by degrading the receptor polyethylene glycols, polyethyleneimines, polyethyleneim sialic acids in the throat/trachea/bronchi, thus preventing ides, polyvinyl pyrrolidone, rayon, nylon, polyethylene, viral binding and infection at these sites. For optimal delivery polypropylene, polybutylene, polycarbonate, polyesters, of the DAS181 microspheres to sites where respiratory viral polyamides, vinyl polymers, polyvinylalcohols, polystyrene infection can be initiated, i.e., in the throat, trachea or bronchi, and polystyrene copolymers, polystyrene cross-linked with the microspheres must not be (a) So big that they are trapped divinylbenzene or the like, acrylic resins, acrylates and at the front end in the mouth (i.e., microspheres are too big, acrylic acids, acrylamides, polyacrylamides, polyacrylamide about 8 microns or greater); or (b) so Small that they are blends, co-polymers of vinyl and acrylamide, methacrylates, absorbed deep in the lungs and absorbed systemically into the methacrylate derivatives and the like. blood stream through the alveoli where they are not active 0505. In some embodiments, the micro-carriers can be and/or can be toxic (i.e., 0.5 micron or smaller). For delivery materials that are capable of forming hydrogels. Hydrogels of the DAS181 microspheres to the throat, trachea and bron are water-swellable polymeric matrices that can absorb water chi, a size range of about 1 micron to about 5.5-6 microns to form elastic gels. Hydrogels and hydrogel microspheres generally can be suitable. Similar behavior is observed with have been tested as drug delivery systems for topical and microparticles of a much smaller exemplary molecule, van systemic delivery to a variety of target tissues, including eye comycin, prepared by the methods provided herein. and bone. The manufacture of hydrogel microspheres has 0501. The inhaler can be used to treat any medical condi previously been accomplished using complex methods. Such tion in which the protein or other molecule can be adminis as the oil/water emulsion method. The methods provided tered by inhalation therapy. Typical inhalation devices can herein facilitate the simple manufacture of hydrogel micro include dry powder inhalers, metered dose inhalers, and elec spheres. trostatic delivery devices. Typical applications of inhalation 0506 Examples of materials capable of forming hydro delivery devices include the deep lung delivery of insulin and gels include but are not limited to various natural, genetically other therapeutic proteins, and Vancomycin. engineered, derivatized, and synthetic polymers such as pro 0502. In some embodiments, the microspheres obtained teins (collagen, gelatin, silk) and polysaccharides (chitosan, by the methods provided herein also can be delivered by oral dextran, gellan gum, agarose). Examples 22 and 23 demon ingestion, intranasally, intravenously, intramuscularly, Sub strate that materials capable of forming hydrogels (gelatin, cutaneously, transdermally, topically and by other delivery dextran) can be incorporated into microsphere formulations methods suitable for the delivery of therapeutic, diagnostic, prepared by the methods provided herein, resulting in micro nutritional or cosmetic molecules. The microsphereformula spheres capable of forming hydrogels. The therapeutic agent tions for pulmonary delivery generally can be in a size range or active agent of interest can be added to the cocktail formu of about 0.5 micron to about 5-6 microns, while those lation containing the hydrogel-forming material at any time designed for other types of delivery, Such as Subcutaneous and in any sequence during the steps leading to the formation delivery, parenteral delivery or intramuscular delivery can be of microspheres according to the methods provided herein. in a range of from about or at 10 micron to about or at 30, 40 Alternatively, the therapeutic agent or active agent can be or 50 microns. added to the solution used to hydrate/swell the microspheres 0503. In some embodiments, the microspheres provided or can be added to the Suspension of Swollen microspheres herein have no direct therapeutic effect but can serve as and allowed to diffuse into the particles. micro-carriers for other therapeutic agent(s). Examples of 0507. The hydrogel microspheres can be crosslinked to molecules useful for preparation of Such microspheres decrease their solubility/erosion and to provide a more Sus include but are not limited to polysaccharides, glycans, pro tained release. Cross-linking can be performed using a variety US 2012/O 141590 A1 Jun. 7, 2012

of cross-linking functionalities known to those of skill in the mg, 80 mg. 85 mg. 90 mg, 95 mg, 100 mg, 125 mg, 150 mg. art including, but not limited to, carboxyl, amino, hydroxyl, 175 mg, 200 mg, 250 mg. 300 mg. 350 mg. 400 mg, 500 mg. phosphate, and/or sulfhydryl groups using natural condensa 600 mg, 700 mg. 800 mg,900 mg or 1000 mg compound per tion or agents that mediate cross-linking, Such as EDC dose. (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro 0513. The molecule component of the microsphere can be chloride) or other compounds employing carbodiimide and any molecule capable of forming microspheres according to non-carbodiimide chemistries. the methods provided herein. For example, small molecule 0508. In some embodiments, cross-linking agents, poly compounds Such as those understood by those of skill in the mers, lipophilic Substances, salts such as those with poor art and provided herein, including therapeutics, diagnostic solubility in aqueous solvents, or combinations thereof or molecules, nutritional Supplements and cosmetics, are con other compounds can be included in the formulation cocktail templated for the preparation of microparticles according to solution to modify the solubility of the microspheres and/or the methods provided herein. Exemplified herein are small enhance their mechanical strength. Slow dissolution of the molecules belonging to a variety of classes of small molecule microspheres can be useful in Sustained release of therapeu compounds, including the aminoglycosides tobramycin and tics delivered by oral ingestion, inhalation, intranasally, intra kanamycin, the penicillin compound amplicillin, and tetracy venously, intramuscularly, transdermally, topically, Subcuta cline. Other Small molecule compounds can include, but are neously, and by other delivery methods suitable for the not limited to, sterols such as cholesterol and lanosterol, delivery or application of therapeutic, diagnostic, nutritional steroids such as estrogen, testosterone, canrenone, oleandrin or cosmetic molecules. In some embodiments, the micro and spironolactone, Sulfonamide derivatives such as Aceta spheres can be delivered by oral ingestion in a form of a pill Zolamide, Ambuside, AZosemide, Bumetanide, ButaZola or capsule with an enteric coating, endocytosed from the mide, Diphenylmethane-4.4'-disulfonamide, Disulfamide, duodenum, and the molecule released into the blood stream or Furosemide, uracils such as Aminometradine and Amisome other site of action. tradine, and the like, and prostaglandins. An organic or inor 0509. In some embodiments, for example when the mol ganic natural or synthetic pharmaceutical compound or drug ecule is a protein or other macromolecule, the microspheres can be incorporated into the microspheres by attaching the can be rendered insoluble by partial denaturation of the mac drug to the Small molecule, and then forming the micro romolecule, which upon delivery becomes renatured and bio spheres from the molecule-drug complex or conjugate. available. 0514. In other embodiments, the molecule is a macromol 0510. In other embodiments, the microspheres are sub ecule including a protein, including enzymes and recombi stantially spherical in shape, and can have mean diameters nant proteins, peptides Such as Somatostatins and leuprolides, within the range of from about 0.1 microns to 30.0 microns. In glycopeptides such as Vancomycin, carbohydrates, lipids, yet other embodiments, the mean diameter of the micro fatty acids, polysaccharides, carbohydrate- or polysaccha spheres can be within the range of from about 0.5 microns to ride-protein conjugates, nucleic acids such as DNA, PNA, 5.0 microns, or from about 1.0 microns to 2.0 microns. RNA, siRNA, tRNA, virus, virus particles, viroids, prions, 0511. In yet another aspect, provided herein are devices conjugates of Small molecules (such as a hapten) and pro and methods for delivering the microspheres to a Subject, teins, or mixtures thereof. In some embodiments, an organic Such as an animal or human patient in need of medical treat or inorganic natural or synthetic pharmaceutical compound ment. Suitable delivery routes can include parenteral, such as or drug can be incorporated into the microspheres by attach i.m., i.v. and s.c., and non-parenteral. Such as oral, buccal, ing the drug to a macromolecule. Such as a protein, and then intrathecal, nasal, pulmonary, transdermal, transmucosal, and forming the microspheres from the macromolecule-drug the like delivery routes. Delivery devices can include complex or conjugate. It will be understood by those of skill Syringes, both needleless and needle containing, and inhalers. in the art that the macromolecule can be a portion of a mol 0512. The delivery devices can contain a single dose of the ecule Such as, for example, a peptide, a single-stranded seg microspheres for treating a condition that is treatable by rapid ment of a double-stranded nucleic acid molecule, or a virus or Sustained release of the macromolecule in Vivo, or they can particle, or other macromolecule having a tertiary and/or contain multiple doses of microspheres, or can be multi quaternary structure. chambered and deliver more than one type of compound 0515. In some embodiments, the macromolecule is a formulated as microspheres. The number of microspheres therapeutic protein including, but not limited to, a sialidase, a present in the single dose is dependent on the type and activity sialidase fusion protein, a fusion protein containing a siali of the molecule. The single dose can be selected to achieve dase catalytic domain fused to a GAG-binding domain, a Sustained release over a period of time that has been opti protease, a protease inhibitor, insulin, interferons, human mized for treating the particular medical condition. For growth hormone, calcitonin, rhDNase or parathyroid hor example, when the molecule is a macromolecule. Such as, for mone, and the protein content of the microspheres can be example, the DAS181 fusion protein (SEQ ID NO:17), the from about or at 50% to about or at 60%, 65%, 70%, 75%, delivery dosage of microsphere compositions containing 80%, 85%, 90%. 95%, 96%, 97%, 98%, 99% or greater. For DAS181 can be from between about or at 0.5 mg protein per pulmonary administration, the microspheres can have an dose to about or at 100 mg protein per dose, or about or at 0.75 average size in the range of from about or at 0.5 microns to mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg. about or at 5.0 microns, and in some embodiments, between 30 mg, 40 mg, 45 mg, 50 mg, 55 mg or 60mg protein perdose. about or at 1 micron and about or at 2 microns. When the molecule is a small molecule, the delivery dosage 0516. Other proteins and peptides that can be used to form can be from between about or at 0.1 mg compound perdose to microspheres by the methods provided herein can include, about or at 1000 mg compound perdose, or about or at 0.2 mg, but are not limited to, therapeutic proteins including DAS181 0.5 mg, 1 mg, 1.5 mg, 2 mg.3 mg, 5 mg, 10 mg, 15 mg, 20 mg. (DAS181; SEQ ID NO:17), al-antitrypsin, Ecotin, eglin c. 30 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65mg, 70 mg, 75 serpin, Pulmozyme (rhDNase), BetaxololTM, DiclofenacTM, US 2012/O 141590 A1 Jun. 7, 2012 36 doxorubicin, acetyl cysteine, leuprolide acetate, luteinizing 0521. When preparing microspheres containing a protein, hormone releasing hormone (LHRH), (D-Tryp6)-LHRH, a proteinstabilizer Such as glycerol, fatty acids, Sugars such as nafarelin acetate, insulin, Sodium insulin, Zinc insulin, prota Sucrose, ions such as Zinc, Sodium chloride, or any other mine, lysozyme, alpha-lactalbumin, basic fibroblast growth protein stabilizers known to those skilled in the art can be factor (bFGF), beta-lactoglobulin, Trypsin, calcitonin, par added prior to cooling the cocktail during microsphere for athyroid hormone, carbonic anhydrase, ovalbumin, bovine mation, to minimize protein denaturation. Such stabilizers serum albumin (BSA), human serum albumin (HSA), phos also can be added to microspheres formulated from other phorylase b, alkaline phosphatase, beta-galactosidase, IgG, macromolecules or Small molecules. fibrinogen, poly-L-lysine, IgM, DNA, desmopressin acetate, 0522. In some embodiments the microspheres can further growth hormone releasing factor (GHRF), Somatostatin, leu be coated on the surface with suitable molecules and/or coat prolide, antide, Factor VIII, G-CSF/GM-CSF, human growth ing agents. Such as those that lend resistance to acids, such as hormone (hGH), beta interferon, antithrombin III, alpha digestive acids, or proteases. In other embodiments, the interferon, alpha interferon 2b. microspheres can be non-covalently coated with compounds 0517. The term “macromolecule' or “small molecule' Such as fatty acids or lipids. The coating can be applied to the also can include a plurality of different macromolecules and/ microspheres by immersion in the solubilized coating Sub or Small molecules and includes combinations such as a com stance, then spraying the microspheres with the Substance, or bination of a pharmaceutical compound and an affinity mol by using other methods known to those of skill in the art. In ecule for targeting the pharmaceutical compound to a tissue, some embodiments, the fatty acids or lipids are added directly organ or tumor requiring treatment. An affinity molecule can to the microsphere-forming cocktail solution. be, for example, a ligand or a receptor. Examples of ligands 0523 Formation of the microspheres by decreasing tem can include viruses, bacteria, polysaccharides, or toxins that perature can be performed by a multitude of conventional can act as antigens to generate an immune response when methods in batch or continuous modes. Microsphere forma administered to an animal and cause the production of anti tion can further be triggered by other methods including, but bodies. The microspheres provided herein also can be pre not limited to, modulating atmospheric pressure, g-force or pared from combinations or mixtures of macromolecules and Surface expansion, including seeding. Microsphereformation Small molecules can occur immediately upon exposure to these conditions or 0518. An inhaler device can be used to deliver a therapeu can require an extended period of time as provided herein. tic compound or diagnostic compound, such as those listed above, to the respiratory airways and lungs of a subject. For D. Exemplary Compounds example, protein microspheres, or antibiotic microspheres, Such as Vancomycin microspheres, can be prepared, for 0524 A. Peptides example by contacting an aqueous solution of the protein or 0525 Exemplary peptides that can be used to form micro Vancomycin with a carboxylic acid Such as citrate, or Sulfate particles by the methods provided herein are described below or other counterion and an organic solvent Such as isopro 0526. Somatostatins panol, and cooling the Solution to form the microspheres. The 0527. Somatostatin (also known as growth hormone protein can be a therapeutic protein, Such as a sialidase, a inhibiting hormone (GHIH) or somatotropin release-inhibit protease inhibitor, insulin, human growth hormone, calcito ing hormone (SRIF)) is a peptide hormone that regulates the nin, rhDNase or parathyroid hormone, and the protein or endocrine system and affects neurotransmission and cell pro Vancomycin content of the microspheres can be about or at liferation via interaction with G-protein-coupled somatosta 70% to about or at 90% or more, 95% or more, or at least tin receptors and inhibition of the release of numerous sec about 99% or more. For pulmonary administration, the ondary hormones. Somatostatin has two active forms microspheres, for example DAS181 microspheres or Vanco produced by alternative cleavage of a single preproprotein: mycin microspheres, can be sized to have a mean diameter in one of 14 amino acids, the other of 28 amino acids. Exem the range of from about 0.5 microns to 5.0 microns, or plary sequences corresponding to preprosomatostatin (con between about 1 micron to about 2 microns. tains signal sequence and propeptide), presomatostatin (con 0519 Incubation conditions for forming the microspheres tains propeptide), somatostatin 28 (SS-28, the 28 amino acid can be optimized to incorporate at least about 70%, 75%, peptide) and Somatostatin 14 (SS-14, the 14 amino acid pep 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, tide) are set forth in SEQID NOS: 18-21, respectively. 98%, or 99% or greater of the total amount of the molecule 0528. Somatostatin is primarily produced by neuroendo present in the Solution prior to formation of the microspheres, crine neurons of the periventricular nucleus of the hypothala by adjusting parameters including pH, temperature, concen mus, and is secreted in several locations in the digestive tration of molecule, or duration of reaction or incubation. system, including the stomach, intestine and delta cells of the 0520. In some embodiments, a molecule or compound that pancreas. does not produce microspheres of desirable characteristics, 0529. Somastatin is of therapeutic significance, for can be incorporated into microspheres having desirable char example, in the treatment of neuroendocrine disorders and acteristics, e.g., of size, delivery profile, mechanical strength, tumors, due to its various biological actions, including inhib by incorporation or coupling of the compound with a carrier iting the release of growth hormone (GH), thus opposing the molecule that can form microspheres with desirable charac effects of Growth Hormone-Releasing Hormone (GHRH): teristics. In some embodiments, the carrier macromolecule is inhibiting the release of thyroid-stimulating hormone (TSH): a protein, and the molecule or compound is bound inside and Suppressing the release of gastrointestinal hormones Such and/or on the surface of the microsphere. In some embodi as Gastrin, Cholecystokinin (CCK), Secretin Motilin, Vaso ments, the molecule or compound also can serve as the coun active intestinal peptide (VIP), Gastric inhibitory polypeptide terion and initiate and/or facilitate the formation of micro (GIP), Enteroglucagon (GIP); and pancreatic hormones, glu spheres. cagon and insulin. US 2012/O 141590 A1 Jun. 7, 2012 37

0530 Leuprolide for preparation in the form of microspheres according to the 0531 Leuprorelin (INN) or leuprolide acetate (USAN) is methods provided herein. An exemplary chemotherapeutic a gonadotropin-releasing hormone agonist (GnRH agonist). agent is paclitaxel. By causing constant stimulation of the pituitary GnRH recep 0543 Paclitaxel tors, it initially causes stimulation (flare), but thereafter 0544 Paclitaxel is a mitotic inhibitor drug used in the decreases pituitary secretion (downregulation) of gonadotro treatment of cancer. Paclitaxel is an effective drug for the pins luteinizing hormone (LH) and follicle-stimulating hor treatment of a variety of cancers, including lung, ovarian, mone (FSH). Like other GnRH agonists, leuprolide may be breast cancer, and advanced forms of Kaposi's sarcoma. used in the treatment of hormone-responsive cancers such as Together with docetaxel, it forms the drug category of the prostate cancer or breast cancer, estrogen-dependent condi taxanes. Paclitaxel also is used for the prevention of resteno tions (such as endometriosis or uterine fibroids), to treat pre sis (recurrent narrowing) of coronary stents; locally delivered cocious puberty, and to control ovarian stimulation in IVF. It to the wall of the coronary artery, a paclitaxel coating limits also is considered a possible treatment for paraphilias. An the growth of neointima (scar tissue) within stents. exemplary sequence of leuprolide is set forth in SEQID NO: 0545 D. Nucleic Acids 22. 0546 Nucleic acids, including those of therapeutic sig 0532. B. Antibiotics nificance, are contemplated for the preparation of micro 0533. An antibiotic includes any compound that inhibits spheres as provided herein. An exemplary therapeutic nucleic or abolishes the growth of microorganisms. Such as bacteria, acid is siRNA. fungi, or protozoans. Exemplary antibiotics that can be used (0547 siRNA to form microparticles by the methods provided herein are 0548 Small interfering RNA (siRNA), sometimes known described below. as short interfering RNA or silencing RNA, are a class of 0534 Aminoglycosides 20-25 nucleotide-long double-stranded RNA molecules that 0535 Aminoglycosides are a group of antibiotics that are play a variety of roles in biological systems. siRNA is effective against certain types of bacteria. They include ami involved in the RNA interference (RNAi) pathway, where the kacin, gentamicin, kanamycin, neomycin, netilmicin, paro siRNA interferes with the expression of a specific gene. In momycin, streptomycin, tobramycin and apramycin. Ami addition to their role in the RNAi pathway, siRNAs also can noglycosides are believed to work by binding to the bacterial act in RNAi-related pathways, e.g. as an antiviral mechanism 30S ribosomal subunit (some work by binding to the 50s or in shaping the chromatin structure of agenome. Given their subunit), inhibiting the translocation of the peptidyl-tRNA potential ability to knock down essentially any gene of inter from the A-site to the P-site and also causing misreading of est, RNAi, via siRNAs has generated a great deal of interest in mRNA, leaving the bacterium unable to synthesize proteins possible therapeutic applications, such as influenza treat vital to its growth. ment. There are an increasing number of large-scale RNAi 0536 Glycopeptides screens that are designed to identify the important genes in 0537 Glycopeptide antibiotics are a class of antibiotic various biological pathways. Because disease processes also drugs. They contain a glycosylated cyclic or polycyclic non depend on the activity of multiple genes, it is expected that in ribosomal peptide. Exemplary glycopeptide antibiotics some situations turning off the activity of a gene with a siRNA include Vancomycin, teicoplanin, ramoplanin, and decapla could produce a therapeutic benefit. For example, as nin. This class of drugs inhibit the synthesis of cell walls in described in Qing et al., (2003) Proc. Nat. Acad. ScL USA, Susceptible microbes by inhibiting peptidoglycan synthesis, 100:2718-2723, some siRNAs have been shown to inhibit thus inhibiting microbial growth. PR8 and WSN influenza production in MDCK cells. The 0538 Penicillins sequences of these siRNAs (sense and antisense Strands) are 0539 Penicillins are a class of B-lactam antibiotics that set forth in SEQID NOS: 23-26. include compounds such as Ampicillin, AZlocillin, Carbeni (0549. E. Prostaglandins cillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, 0550 A prostaglandin is any member of a group of lipid Nafcillin, Penicillin, Piperacillin and Ticarcillin. B-lactaman compounds that are derived enzymatically from fatty acids, tibiotics work by inhibiting the formation of peptidoglycan are hormone or hormone-like, and have important and pleio cross links in the bacterial cell wall. The B-lactam moiety of tropic functions and effects in the animal body. Every pros penicillin binds to the enzyme (transpeptidase) that links the taglandin contains 20 carbon atoms, including a 5-carbon peptidoglycan molecules in bacteria, and this weakens the ring. They are mediators and have a variety of physiological cell wall of the bacterium (in other words, the antibiotic effects. The prostaglandins together with the thromboxanes causes cytolysis or death). and prostacyclins form the prostanoid class of fatty acid (0540 Tetracyclines derivatives; the prostanoid class is a Subclass of eicosanoids. 0541 Tetracyclines are a class of natural and synthetic 0551. Due to their pleiotropic effects, prostaglandins have broad-spectrum antibiotics whose members include, for a variety of clinical applications, including: example, Tetracycline, Chlortetracycline, Oxytetracycline, 0552. To induce childbirth, parturition or abortion (PGE Demeclocycline, Semi-synthetic Doxycycline, Lymecycline, or PGF2, with or without mifepristone, a progesterone Meclocycline, Methacycline, Minocycline and Rolitetracy antagonist); cline. Tetracyclines inhibit cell growth by inhibiting transla 0553 To prevent closure of patent ductus arteriosus in tion. The tetracyclines bind to the 16S part of the 30S ribo newborns with particular cyanotic heart defects (PGE): somal subunit and prevent the amino-acyl tRNA from binding 0554) To prevent and treat peptic ulcers (PGE): to the A site of the ribosome. 0555 As a vasodilator in severe Raynaud's phenomenon 0542. C. Chemotherapeutic Agents Pharmacologic agents or ischemia of a limb; that are useful in the treatment of cancer fall under the general 0556. In pulmonary hypertension; umbrella of chemotherapeutic agents, and are contemplated 0557. In treatment of glaucoma; and US 2012/O 141590 A1 Jun. 7, 2012

0558 To treat erectile dysfunction or in penile rehabilita C.(2.3)-Gal (Ito et al. (2000) Microobiol Immunol 44:423 tion following Surgery. 730). The human respiratory epithelium expresses both forms 0559 F. Viruses of sialic acids, but C.(2,6)-linked sialic acid is more abundant 0560 Viruses have a variety of applications as carriers or than O.(2,3)-linked sialic acid. The low abundance of O.(2.3)- vectors, including in gene therapy and as inactivated viruses linked sialic acid is most likely the basis for the species barrier in vaccines. Microparticles of viruses, including derivative for avian viruses, and indicates that reducing the level of a forms of the viruses such as virus particles or inactivated receptorsialic acid expressed on the airway epithelium would viruses, including, but not limited to, animal viruses, plant likely reduce the infectivity of an influenza virus. Thus, siali viruses, phages, influenza virus, parainfluenza virus, aden dases, which remove terminal sialic acid residues from Sialo oviruses, retroviruses, respiratory syncytial virus, DNA glycoconjugates, present themselves as potential influenza based viruses, coronavirus and rotavirus are contemplated for virus therapeutic agents that function to reduce the levels of preparation according to the methods provided herein. Exem receptor Sialic acids. Sialidases also can act as therapeutic plified herein is the tobacco mosaic virus (TMV) agents for any other pathogen that utilizes sialic acids in the 0561 Tobacco Mosaic Virus (TMV) infection process including, but not limited to, M. pneumo 0562 Tobacco mosaic virus (TMV) is an RNA virus that niae, M. catarrhalis, H. pylori, H. influenzae, S. pneumonia, infects plants, especially tobacco and other members of the P. aeruginosa, parainfluenza viruses and some coronaviruses family Solanaceae. The virus has a rod-like appearance. Its and rotaviruses. capsid is made from 2130 molecules of coat protein and one 0568 Sialidases tend to be highly substrate specific. They molecule of genomic RNA, 6390 bases long. The coat protein can target particular types of complex molecules. Such as self assembles into the rod like helical structure around the glycoproteins or glycolipids; specific Sugarlinkages (e.g. 2-3, RNA, which forms a hairpin loop structure. The protein 2-6, or 2-8); or can be sensitive to the nature of the linkage monomer constains 158 amino acids that are assembled into Sugar itself (e.g. D-galactose, N-acetyl-D-galactosamine). four main alpha-helices, which are joined by a prominent Substrate molecules include, but are not limited to, oligosac loop proximal to the axis of the virion. charides, polysaccharides, glycoproteins, gangliosides, and 0563. In addition to its impact on crop losses, the highly synthetic molecules. For example, a sialidase can cleave detailed knowledge regarding the structure of TMV, and the bonds having C.(2.3)-Gal, C.(2,6)-Gal, or C.(2.8)-Gal linkages fact that it does not infect animals, makes it a valuable tool for between a sialic acid residue and the remainder of a substrate investigations in areas including structural molecular biol molecule. A sialidase also can cleave any or all of the linkages ogy, X-ray diffraction, and Virus assembly and disassembly. between the sialic acid residue and the remainder of the 0564 G. Proteins Substrate molecule. Many sialidase proteins have been puri 0565 Exemplary proteins that can be used to form micro fied from microbes and higher eukaryotes and of these, sev particles by the methods provided herein are described below eral have been shown to catalyze the removal of terminal 0566 Sialidases sialic acid residues than can serve as receptors for pathogenic 0567 Sialidases, also referred to as neuraminidases and microorganisms. For example, among the large bacterial N-acylneuraminosylglycohydrolases, are a family exogly sialidases are those that that can degrade the influenza recep cosidases that catalyze the removal of terminal sialic acid torsialic acids Neu5Ac C.(2.6)-Gal and Neu5Ac C.(2.3)-Gal, residues from Sialo-glycoconjugates. Sialic acids are a family including Sialidases from Clostridium perfingens, Actino of a keto acids with 9-carbon backbones that are usually myces viscosus, Arthrobacter ureafaciens, and Micromono found at the outermost positions of the oligosaccharide chains spora viridifaciens. Other sialidases that can serve as thera attached to glycoproteins and glycolipids. These molecules peutic agents include the human sialidases, such as those are involved in a variety of biological functions and pro encoded by the genes NEU2 and NEU4. cesses. Such as the regulation of innate immunity, cell adhe 0569. Sialidase-GAG. Fusion Proteins Sion, and the interaction between inflammatory cells and 0570 Sialidase-GAG fusion proteins are proteins that are target cells, possibly mediated through the binding of various made up of a sialidase protein, or catalytically active portion lectins (Varki et al. (1992) Curr Opin Cell Biol. 4:257-266). thereof, fused to a glycosaminoglycan (GAG)-binding Sialic acids also are excellent Sources of carbon, nitrogen, sequence. As such, these proteins effectively contain an energy, and precursors of cell wall biosynthesis. Further still, anchoring domain (the GAG-binding sequence) and a thera sialic acids on eukaryotic cells can be used as receptors or peutic domain (the sialidase protein, or catalytically active coreceptors for pathogenic microorganisms, including, but portion thereof). The sialidase-GAG fusion proteins are not limited to, influenza virus, parainfluenza virus, some designed to bind to the epithelium and remove the surround coronavirus and rotavirus Haemophilus influenzae, Strepto ing Sialic acids, and can therefore be used as a therapeutic coccus pneumonia, Mycoplasma pneumoniae, Moaxella agent against pathogens that utilize sialic acids in the infec catarrhalis, Helicobacter pylori and Pseudomonas aerugi tion process. The ability of the fusion protein to bind to the nosa. The most prominent member of the Sialic acid family is epithelium increases its retention when the fusion protein is N-acetylneuraminic acid (Neu5Ac), which is the biosynthetic administered, for example, as an inhalant to treat influenza precursor for most of the other types. Two major linkages infection. The GAG-binding sequence acts as an epithelium between Neu5Ac and the penultimate galactose residues of anchoring domain that tethers the Sialidase to the respiratory carbohydrate side chains are found in nature, Neu5Ac C.(2. epithelium and increases its retention and potency. 3)-Gal and Neu5Ac C.(2.6)-Gal. Both Neu5Ac C.(2.3)-Gal 0571 Heparan sulfate, closely related to heparin, is a type and Neu5Ac C.(2.6)-Gal molecules can be recognized by of glycosaminoglycan (GAG) that is ubiquitously present on influenza viruses and used as the receptor through which the cell membranes, including the Surface of respiratory epithe virus binds and initiates infection. Human influenza viruses, lium. Many proteins specifically bind to heparin/heparan Sul however, seem to prefer Neu5Ac C.(2,6)-Gal, while avian and fate, and the GAG-binding sequences in these proteins have equine influenza viruses predominantly recognize Neu5Ac been identified. For example, the GAG-binding sequences of US 2012/O 141590 A1 Jun. 7, 2012 39 human platelet factor 4 (PF4) (SEQID NO:3), human inter platelets and appears to be involved in the regulation of furin leukin 8 (IL8) (SEQ ID NO:4), human antithrombin III (AT activity and, therefore, platelet aggregation (LeBlond et al. III) (SEQ ID NO:5), human apoprotein E (ApoE) (SEQ ID (2006) Thromb Haemost 95:243-252). NO:6), human angio-associated migratory cell protein 0576. In addition to their role in the regulation of endog (AAMP) (SEQ ID NO:7), or human amphiregulin (SEQ ID enous biological processes, such as coagulation, serine pro NO:8) have been shown to exhibit high affinity for heparin tease inhibitors also can function to inhibit the biological (Lee et al. (1991) PNAS 88:2768-2772: Goger et al. (2002) activities of exogenous microorganisms. For example, a num Biochem. 41: 1640-1646; Witt et al. (1994) Curr Bio 4:394 400; Weisgraber et al. (1986) J Bio Chem 261:2068-2076). ber of serine protease inhibitors have been shown to reduce The GAG-binding sequences of these proteins are distinct influenza virus activation in cultured cells, chicken embryos from their receptor-binding sequences, so they do not induce and in the lungs of infected mice. The serpins bind to hemag the biological activities associated with the full-length pro glutinin (HA) molecules on the surface of the influenza virus teins or the receptor-binding domains. These sequences, or and inhibit its activity, thus reducing the infectivity of the other sequences that can bind heparin/heparan Sulfate, can be virus. For example trypsin inhibitors, such as: aprotinin (Zhi used as epithelium-anchoring-domains in sialidase-GAG mov et al. (2002) JVirol 76:8682-8689), leupeptin (Zhimov et fusion proteins. al. (2002) J Virol 76:8682-8689: Tashiro et al. (1987) J Gen Virol 68:2039–2043), soybean protease inhibitor (Barbey 0572. In the context of a sialidase-GAG fusion protein, the Morel et al. (1987) J Infect Dis 155:667-672), e-aminocap sialidase can include the entire Sialidase protein, or a catalyti roic acid (Zhimov et al. 1982. Arch Virol 73:263-272) and cally active portion thereof. For example, sialidase-GAG n-p-tosyl-L-lysine chloromethylketone (TLCK) (Barbey fusion protein can contain the 901 amino acid Sialidase pro tein from A. viscosus set forth in SEQID NO:1. In another Morel et al. (1987) J Infect Dis 155:667-672) have all been example, the sialidase-GAG fusion protein can contain the shown to inhibit influenza virus infection, and are candidate 394 amino acid catalytically active portion of a sialidase therapeutic agents for use in the treatment of influenza virus protein from A. viscosus set forth in SEQ ID NO:2. The infection. Thus, as a related trypsin inhibitor, PI8 also can be GAG-binding sequence can be linked to the sialidase by used as a therapeutic agent in the treatment of influenza virus recombinant methods. In some examples, the fusion protein infection. can include an amino acid linker, Such as four glycine resi 0577 Surface Active Agents dues. Furthermore, linkage can be via the N- or C-terminus of 0578. The compositions provided herein can contain one the GAG-binding sequence, or the N- or C-terminus of the or more surface active agents that are added in an amount sialidase. Exemplary examples of Sialidase-GAG fusion pro sufficient to stabilize the cocktail solutions and/or the micro teins include those polypeptides set forth in SEQ ID NOS: spheres. The selection of an appropriate amount of Surface 9-13, and 17. In a further example, the sialidase and GAG active agent is a function of the nature of the compound, binding sequence components can be linked using chemical Solvent and antisolvent. or peptide linkers, by any method known in the art. 0579. In certain embodiments, the surface active agent can 0573. Proteinase Inhibitor 8 be selected from sodium lauryl sulfate; sorbitan laurate, sor 0574) Proteinase inhibitor 8 (PI8), also known as Serpin bitan palmitate, sorbitan stearate (available under the trade B8, is a serine protease inhibitor (Serpin) Serpins are a large name SpanR 20-40-60 etc.); polysorbates such as polyoxy Superfamily of structurally related proteins that are expressed ethylene (20) sorbitan monolaurate, polyoxyethylene (20) in viruses, insects, plants and higher organisms, but not in Sorbitan monopalmitate, polyoxyethylene (20) Sorbitan bacteria or yeast. Serpins regulate the activity of proteases monostearate (available under the tradename TWEENS(R) involved in many biological process, including coagulation, 20-40-60 etc.); benzalkonium chloride, mixed chain phos fibrinolysis, inflammation, cell migration, and tumorigenesis. pholipids, cationic lipids, oligolipids, phospholipids, car They contain a surface-exposed reactive site loop (RSL), nitines, sphingosines, sphingomyelins, ceramides, glycolip which acts as a "bait for proteases by mimicking a protease ids, lipoproteins, apoproteins, amphiphilic proteins, Substrate sequence. On binding of the target protease to the amphiphilic peptides, amphiphilic synthetic polymers, and serpin, the RSL is cleaved, after which the protease is combinations thereof. Other exemplary Surface active agents covalently linked to the serpin. The protease in the newly for use herein include, but are not limited to formed serpin-protease complex is inactive (Huntington et al. 0580 i) Natural lipids, i.e. Cholesterol, Sphingosine and (2000) Nature 407:923-926). Derivatives, Gangliosides, Sphingosine derivatives (Soy (0575 PI8 is a member of a subfamily of serpins of which Bean), Phytosphingosine and derivatives (Yeast), Choline chicken ovalbumin is the archtype. Like other serpins that (Phosphatidylcholine), Ethanolamine (Phosphatidylethano belong to this family, PI8 lacks a typical cleavable N-terminal lamine), Glycerol (Phosphatidyl-DL-glycerol), Inositol signal sequence, resulting in a 374 amino acid protein (SEQ (Phosphatidylinositol), Serine (Phosphatidylserine (Sodium IDNO:14) that resides mainly intracellularly. Other members Salt)), Cardiolipin, Phosphatidic Acid, Egg Derived, Lyso of this human ovalbumin-like Subfamily include plasminogen (Mono Acyl) Derivatives (Lysophosphatides), Hydrogenated activator inhibitor type 2 (PAI-2), monocyte neutrophil Phospholipids, Lipid Tissue Extracts, elastase inhibitor (MNEI), squamous cell carcinoma antigen 0581 ii) Synthetic lipids, i.e. Asymmetric Fatty Acid, (SCCA)-1, leupin (SCCA-2) maspin (PI5), protease inhibitor Symmetric Fatty Acid Saturated Series, Symmetric Fatty 6 (PI6), protease inhibitor (PI9) and bomapin (PI10). Within Acid Unsaturated Series, Acyl Coenzyme A (Acetoyl this family the serpins P16, PI8, and PI9 show the highest Coenzyme A. Butanoyl Coenzyme A, Crotanoyl Coenzyme structural homology (up to 68% amino acid identity) (Spre A, Hexanoyl Coenzyme A, Octanoyl Coenzyme A, Decanoyl cher et al. (1995) J Biol Chem 270:29854-29861). PI-8 has Coenzyme A. Lauroyl Coenzyme A, Myristoyl Coenzyme A, been shown to inhibit trypsin, thrombin, factor Xa, subtilisin Palmitoyl Coenzyme A, Stearoyl Coenzyme A, Oleoyl Coen A furin, and also chymotrypsin in vitro. It is released by Zyme A, Arachidoyl Coenzyme A, Arachidonoyl Coenzyme US 2012/O 141590 A1 Jun. 7, 2012 40

A, Behenoyl Coenzyme A, Tricosanoyl Coenzyme A. Ligno 0586 vii) Fluorescent lipids, i.e.: Glycerol Based (Phos ceroyl Coenzyme A, Nervonoyl Coenzyme A, Hexacosanoyl phatidylcholine (NBD), Phosphatidic Acid (NBD), Phos Coenzyme A, phatidylethanolamine (NBD), Phosphatidylglycerol (NBD), 0582 iii) Sphingolipids, i.e. D-erythro (C-18) Derivatives Phosphatidylserine (NBD)), Sphingosine Based (Ceramide (Sphingosine, Such as: D-erythro Sphingosine (synthetic), (NBD), Sphingomyelin (NBD), Phytosphingosine (NBD), Galactosyl Cerebroside (NBD)), Headgroup Labeled Lipids Sphingosine-1-Phosphate, N.N. Dimethylsphingosine, N.N. (Glycerol Based) (Phosphatidylethanolamine (NBD), Phos N-Trimethylsphingosine, Sphingosylphosphorylcholine, phatidylethanolamine (Lissamine Rhodamine B), Dioleoyl Sphingomyelin and Glycosylated Sphingosine), Ceramide Phosphatidylethanolamine (Dansyl, Pyrene, Fluorescein), Derivatives (Ceramides, D-erythro Ceramide-1-Phosphate, Phosphatidylserine (NBD), Phosphatidylserine (Dansyl)), Glycosulated Ceramides), Sphinganine (Dihydrosphin 25-NBD-Cholesterol, gosine) (Sphinganine-1-Phosphate, Sphinganine (C20), 0587 viii) Other lipids including, but not limited to Leci D-erythro Sphinganine, N-Acyl-Sphinganine C2, N-Acyl thin, Ultralec-P (ADM), Soy powder, Sphinganine C8, N-acyl-Sphinganine C16, N-Acyl-Sphinga 0588 ix) Surfactants including, but not limited to polyeth nine C18, N-Acyl-Sphinganine C24, N-Acyl-Sphinganine ylene glycol 400; sodium lauryl sulfate; sorbitan laurate, sor C24:1), Glycosylated (C18) Sphingosine and Phospholipid bitan palmitate, sorbitan stearate (available under the trade Derivatives (Glycosylated-Sphingosine) (Sphingosine, BD name SpanR 20-40-60 etc.); polysorbates such as Glucosyl, Sphingosine, BD-Galactosyl, Sphingosine, polyoxyethylene (20) Sorbitan monolaurate, polyoxyethyl BD-Lactosyl), Glycosylated-Ceramide (D-Glucosyl-31-1' ene (20) sorbitan monopalmitate, polyoxyethylene (20) sor Ceramide (C8), D-Galactosyl-B1-1" Ceramide (C8), D-Lac bitan monostearate (available under the tradename tosyl-B1-1" Ceramide (C8), D-Glucosyl-B1-1" Ceramide TWEENSR 20-40-60 etc.); benzalkonium chloride. (C12), D-Galactosyl-B1-1'Ceramide (C12), D-Lactosyl-(1- 0589. In certain embodiments, the phospholipids for use 1' Ceramide (C12)), Glycosylated-Phosphatidylethanola are phosphatidylcholines, phosphatidylethanolamines, phos mine (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N- phatidylserines, phosphatidylglycerols, phosphatidylinosi Lactose), D-erythro (C17) Derivatives (D-erythro tols, phosphatidic acids, mixed chain phospholipids, lyso Sphingosine, D-erythro Sphingosine-1-phosphate), phospholipids, hydrogenated phospholipids, partially D-erythro (C20) Derivatives (D-erythro Sphingosine), hydrogenated phospholipids, and mixtures thereof. L-threo (C18) Derivatives (L-threo Sphingosine, Safingol 0590. In certain embodiments, the surface active agent is (L-threo Dihydrosphingosine)), Sphingosine Derivatives selected from polysorbate-80, lecithin and phosphatidylcho (Egg, Brain & Milk) (D-erythro-Sphingosine, Sphingomy line. The Surface active agents are present in an amount Suf elin, Ceramides, Cerebrosides, Brain Sulfatides), Ganglio ficient to stabilize the cocktail solution and/or the resulting sides (Gangliosides Structures, Gangliosides—Ovine Brain, microspheres. Gangliosides Porcine Brain), Sphingosine Derivatives (Soy Bean) (Glucosylceramide), Phytosphingosine Deriva 0591. The amount of surface active agent can be empiri tives (Yeast) (Phytosphingosine, D-ribo-Phytosphingosine cally determined and is a function of the agent selected, and 1-Phosphate, N-Acyl Phytosphingosine C2, N-Acyl the desired form of the resulting microsphere composition. Phytosphingosine C8, N-Acyl Phytosphingosine C18, The amount included can be from less than 0.1% by weight up to 35% or more. In certain embodiments, the surface active 0583 iv) Acyl coenzyme A, i.e. Acetoyl Coenzyme A agent is present at a concentration of about 1%, 2%. 3%, 4%, (Ammonium Salt). Butanoyl Coenzyme A (Ammonium 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% by weight up to Salt), Crotanoyl Coenzyme A (Ammonium Salt), Hexanoyl about 30% by weight of the total weight of the composition. Coenzyme A (Ammonium Salt), Octanoyl Coenzyme A In certain embodiments, the Surface active agent is present at (Ammonium Salt), Decanoyl Coenzyme A (Ammonium a concentration of about 1 weight% up to about 20 weight% Salt), Lauroyl Coenzyme A (Ammonium Salt), Myristoyl of the total weight of the composition. In certain embodi Coenzyme A (Ammonium Salt), Palmitoyl Coenzyme A ments, the Surface active agent is presentata concentration of (Ammonium Salt), Stearoyl Coenzyme A (Ammonium Salt), about 1 weight% up to about 15 weight% of the total weight Oleoyl Coenzyme A (Ammonium Salt), Arachidoyl Coen of the composition. In other embodiments, the Surface active Zyme A (Ammonium Salt), Arachidonoyl Coenzyme A (Am agent is present at a concentration of about 1 weight% up to monium Salt), Behenoyl Coenzyme A (Ammonium Salt), about 10 weight% of the total weight of the composition. In Tricosanoyl Coenzyme A (Ammonium Salt), Lignoceroyl other embodiments, the Surface active agent is present at a Coenzyme A (Ammonium Salt), Nervonoyl Coenzyme A concentration of about 1 weight% up to about 8 weight% of (Ammonium Salt), Hexacosanoyl Coenzyme A (Ammonium the total weight of the composition. In other embodiments, Salt), Docosahexaenoyl Coenzyme A (Ammonium Salt), the Surface active agent is presentata concentration of about 0584 V) Oxidized lipids, i.e. 1-Palmitoyl-2-AZelaoyl-sn 1 weight% up to about 6 weight% of the total weight of the Glycero-3-Phosphocholine, 1-O-Hexadecyl-2-AZelaoyl-sn composition. In other embodiments, the Surface active agent Glycero-3-Phosphocholine, 1-Palmitoyl-2-Glutaroyl-sn is present at a concentration of about 1 weight% up to about Glycero-3-Phosphocholine (PGPC), 1-Palmitoyl-2-(9'-oxo 4 weight % of the total weight of the composition. In other Nonanoyl)-sn-Glycero-3-Phosphocholine, 1-Palmitoyl-2- embodiments, the Surface active agent is present at a concen (5'-oxo-Valeroyl)-sn-Glycero-3-Phosphocholine, tration of about 20 weight % of the total weight of the com 0585 vi) Ether lipids, i.e.: Diether Lipids (Dialkyl Phos position. In other embodiments, the Surface active agent is phatidylcholine, Diphytanyl Ether Lipids), Alkyl Phospho present at a concentration of about 15 weight % of the total choline (Dodedylphosphocholine), O-Alkyl diacylphos weight of the composition. In other embodiments, the Surface phatidylcholinium (1,2-Diacyl-sn-Glycero-3- active agent is presentata concentration of about 13 weight% Ethylphosphocholine), Synthetic PAF & Derivatives of the total weight of the composition. In other embodiments, (1-Alkyl-2-Acyl-Glycero-3-Phosphocholine & Derivatives), the Surface active agent is presentata concentration of about US 2012/O 141590 A1 Jun. 7, 2012

11 weight% of the total weight of the composition. In other nts, muscle relaxants, neoplastics, glycoproteins, nucleopro embodiments, the Surface active agent is present at a concen teins, lipoproteins, ophthalmics, psychic energizers, tration of about 8 weight% of the total weight of the compo sedatives, steroids, sympathomimetics, parasympathomimet sition. In other embodiments, the Surface active agent is ics, tranquilizers, urinary tract drugs, vaccines, vaginal drugs, present at a concentration of about 6 weight % of the total Vitamins, minerals, nonsteroidal anti-inflammatory drugs, weight of the composition. In other embodiments, the Surface angiotensin converting enzymes, polynucleotides, polypep active agent is presentata concentration of about 4 weight% tides, polysaccharides, and nutritional Supplements including of the total weight of the composition. In other embodiments, herbal Supplements. the Surface active agent is presentata concentration of about 0596) The level of agent to be delivered is from about 2 weight % of the total weight of the composition. In other 0.01% up to about 50%, from about 0.1% up to about 40%, embodiments, the Surface active agent is present at a concen from about 0.1% up to about 30%, from about 0.1% up to tration of about 1 weight% of the total weight of the compo about 20%, from about 0.1% up to about 10%, from about sition. 0.1% up to about 9%, from about 0.1% up to about 8%, from 0592 Optional additional Agents about 0.1% up to about 7%, from about 0.1% up to about 6%, 0593. The compositions provided herein can optionally, in from about 0.1% up to about 5%, from about 0.1% up to about addition to the compound of interest, contain one or more 4%, from about 0.1% up to about 3%, from about 0.1% up to pharmaceutical or nutraceutical or diagnostic or cosmetic or about 2%, from about 0.1% up to about 1% by weight of the other Such active agent for administering to a subject. Gen composition. The agent to be delivered can be water soluble, erally the agents are those that have a function in a host, e.g., slightly water soluble, or soluble in an organic solvent or an immune regulation, regulation of biochemical processes, or oil. In certain embodiments, the agent to be delivered is enzymatic activity. Any agent that can be formulated as selected from among antibiotics, chemotherapeutics, antivi described herein can be administered in the compositions rals, anticonvulsants, analgesics, antiparkinsons, anti-inflam provided herein. Where the agent is a therapeutic, the com matories, calcium antagonists, anesthetics, antimicrobials, positions contain a therapeutically effective amount of an antimalarials, antiparasitics, antihypertensives, antihista agent to be delivered. The particular amount of active agent in mines, antipyretics, alpha-adrenergic agonists, alpha-block a dosage will vary widely according to the nature of the active ers, biocides, bactericides, bronchial dilators, beta-adrenergic agent, the nature of the condition being treated, the age and blocking drugs, contraceptives, cardiovascular drugs, cal size of the Subject, and other parameters. In addition, the cium channel inhibitors, depressants, diagnostics, diuretics, compound forming the microsphere can itself also be an electrolytes, enzymes, hypnotics, hormones, hypoglycemics, active agent. hyperglycemics, muscle contractants, muscle relaxants, neo 0594 Generally, the amount of additional active agent or plastics, glycoproteins, nucleoproteins, lipoproteins, non nutrient besides the compound in the composition will vary denatured whey protein, ophthalmics, psychic energizers, from less than about 0.01% by weight to about 20% by weight sedatives, steroids, sympathomimetics, parasympathomimet of the composition, or more and typically are formulated for ics, tranquilizers, urinary tract drugs, vaccines, vaginal drugs, single dosage administration. A single dosage can vary from Vitamins, minerals, nonsteroidal anti-inflammatory drugs, about 0.01 g to 10 mg of an agent per kilogram of body angiotensin converting enzymes, polynucleotides, polypep weight of the host, with dosages from about 0.1 g to 1 mg/kg tides, polysaccharides, and nutritional Supplements including being commonly employed. These concentrations, however, herbal Supplements. are general guidelines only and particular amounts and dos 0597 Exemplary active agents are the same as the classes ages may be selected based on the active agent being admin of compounds listed as being Suitable compounds for prepar istered, the condition being treated, and the treatment regi ing microparticles, and they are set forth in the “Macromol men being employed means an amount of a drug or an active ecules and Small Molecules' section herein as “Exemplary agent that is sufficient to provide the desired local or systemic Active Agent Categories for Macromolecules and Small Mol effect and performance at a reasonable benefit/risk ratio to a ecules.” Subject attending any medical treatment. E. Uses of the Compositions 0595 Agents can be selected from inorganic and organic drugs including, but not limited to drugs that act on the 0598. Therapeutic and diagnostic applications of the peripheral nerves, adrenergic receptors, cholinergic recep microspheres include drug delivery, vaccination, gene tors, nervous system, skeletal muscles, cardiovascular sys therapy, and in vivo tissue or tumor imaging. Routes of tem, Smooth muscles, blood circulatory system, synaptic administration include oral or parenteral administration; sites, neuro-effector junctional sites, endocrine system, hor mucosal administration; ophthalmic administration; intrave mone systems, immunological system, reproductive system, nous, Subcutaneous, intra articular, or intramuscular injec skeletal system, autocoid systems, alimentary and excretory tion; inhalation administration; and topical administration. systems, histamine systems, and the like. The active agents 0599. The diseases and disorders can include, but are not that can be delivered using the compositions provided herein limited to neural disorders, respiratory disorders, immune include, but are not limited to, anticonvulsants, analgesics, system disorders, muscular disorders, reproductive disorders, antiparkinsons, anti-inflammatories, calcium antagonists, gastrointestinal disorders, pulmonary disorders, digestive anesthetics, antimicrobials, antimalarials, antiparasitics, anti disorders, metabolic disorders, cardiovascular disorders, hypertensives, antihistamines, antipyretics, alpha-adrenergic renal disorders, proliferative disorders, cancerous diseases agonists, alpha-blockers, biocides, bactericides, bronchial and inflammation. dilators, beta-adrenergic blocking drugs, contraceptives, car 0600 The microparticles provided herein can be used to diovascular drugs, calcium channel inhibitors, depressants, treat Infectious diseases, such as arboviral infections, botu diagnostics, diuretics, electrolytes, enzymes, hypnotics, hor lism, brucellosis, candidiasis, campylobacteriosis, chicken mones, hypoglycemics, hyperglycemics, muscle contracta pox, chlamydia, cholera, coronovirus infections, staphyllo US 2012/O 141590 A1 Jun. 7, 2012 42 coccus infections, coxsackie virus infections, Creutzfeldt of skill in the art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052, Jakob disease, cryptosporidiosis, cyclospora infection, 558 and 5,033,252. Examples of pharmaceutical packaging cytomegalovirus infections, Epstein-Barr virus infection, materials include, but are not limited to, blister packs, bottles, dengue fever, diphtheria, ear infections, encephalitis, influ tubes, inhalers, pumps, bags, vials, containers, bottles, and enza virus infections, parainfluenza virus infections giardia any packaging material Suitable for a selected formulation sis, gonorrhea, Haemophilus influenzae infections, hantavi and intended mode of administration and treatment. rus infections, viral hepatitis, herpes simplex virus infections, 0606. The following examples are included for illustrative HIV/AIDS, helicobacter infection, human papillomavirus purposes only and are not intended to limit the scope of the (HPV) infections, infectious mononucleosis, legionellosis, invention. leprosy, leptospirosis, listeriosis, lyme disease, lymphocytic choriomeningitis, malaria, measles, marburg hemorrhagic Example 1 fever, meningitis, monkeypox, mumps, mycobacteria infec tion, mycoplasma infection, norwalk virus infection, pertus Preparation of Microspheres of the Sialidase Fusion sis, pinworm infection, pneumococcal disease, Streptococcus Protein, DAS181 pneumonia infection, Mycoplasma pneumoniae infection, Moraxella catarrhalis infection, Pseudomonas aeruginosa 0607 A. Purification of DAS181 infection, rotavirus infection, psittacosis, rabies, respiratory 0608 DAS181 is a fusion protein containing the heparin syncytial virus infection, (RSV), ringworm, rocky mountain (glycosaminoglycan, or GAG) binding domain from human spotted fever, rubella, salmonellosis, SARS, scabies, sexually amphiregulin fused via its N-terminus to the C-terminus of a transmitted diseases, shigellosis, shingles, sporotrichosis, catalytic domain of Actinomyces Viscosus (sequence of streptococcal infections, syphilis, tetanus, trichinosis, tuber amino acids set forth in SEQIDNO:17). The DAS181 protein culosis, tularemia, typhoid fever, viral meningitis, bacterial was purified as described in Malakhov et al., Antimicrob. meningitis, west nile virus infection, yellow fever, adenovi Agents Chemother, 1470–1479, 2006, which is incorporated rus-mediated infections and diseases, retrovirus-mediated in its entirety by reference herein. Briefly, the DNA fragment infectious diseases, yersiniosis Zoonoses, and any otherinfec coding for DAS181 was cloned into the plasmid vector tious respiratory, pulmonary, dermatological, gastrointestinal pTrc99a (Pharmacia: SEQID NO:16) under the control of a and urinary tract diseases. IPTG (isopropyl-B-D-thiogalactopyranoside)-inducible pro 0601. Other diseases and conditions, including arthritis, moter. The resulting construct was expressed in the BL21 asthma, allergic conditions, Alzheimer's disease, cancers, strain of Escherichia Coli (E. Coli). cardiovascular disease, multiple sclerosis (MS), Parkinson's 0609. The E. Coli cells containing the expressed construct disease, cystic fibrosis (CF), diabetes, non-viral hepatitis, were lysed by sonication in 50 mM phosphate buffer, pH 8.0: hemophilia, bleeding disorders, blood disorders, genetic dis 0.3 MNaCl and 10% glycerol. The clarified lysate was passed orders, hormonal disorders, kidney disease, liver disease, through an SP-Sepharose column. Proteins were eluted from neurological disorders, metabolic diseases, skin conditions, the column with lysis buffer that contained 0.8 MNaCl. The thyroid disease, osteoporosis, obesity, stroke, anemia, fraction eluted from SP-Sepharose was adjusted to 1.9 M inflammatory diseases and autoimmune diseases. ammonium sulfate ((NH4)2SO4), clarified by centrifugation, and loaded onto abutyl-Sepharose column. The column was F. Combinations, Kits, Articles of Manufacture washed with two volumes of 1.3 M (NH)SO, and the 0602 Combinations and kits containing the combinations DAS181 fusion protein was eluted with 0.65 M (NHA)SO. provided herein, including microparticles or ingredients for 0610 For the final step, size exclusion chromatography forming the microparticles Such as a small molecule or a was performed on Sephacryl S-200 equilibrated with phos macromolecule of interest, counterions, solvents, buffers, or phate-buffered saline (PBS). The protein purity was deter salts and optionally including instructions for administration mined to be greater than 98% as assessed by sodium dodecyl are provided. The combinations include, for example, the Sulfate-polyacrylamide gel electrophoresis, reversed-phase compositions as provided herein and reagents or Solutions for high-pressure liquid chromatography, and enzyme-linked diluting the compositions to a desired concentration for immunosorbent assay with antibodies generated against E. administration to a host Subject, including human beings. The Coli cell proteins. The purified DAS181, molecular weight combinations also can include the compositions as provided 44,800 Da, was dialyzed against 2 mM sodium acetate buffer, herein and additional nutritional and/or therapeutic agents, pH 5.0. including drugs, as provided herein. 0611 B. Activity of DAS181 0603 Additionally provided herein are kits containing the 0612. The sialidase activity of DAS181 was measured above-described combinations and optionally instructions for using the fluorogenic substrate 4-methylumbelliferyl-N- administration by oral, Subcutaneous, transdermal, intrave acetyl-C-D-neuraminic acid (4-MU-NANA; Sigma). One nous, intramuscular, ophthalmic or other routes, depending unit of Sialidase is defined as the amount of enzyme that on the protein and optional additional agent(s) to be delivered. releases 10 nmol of MU from 4-MU-NANA in 10 minutes at 0604. The compositions provided herein can be packaged 37° C. (50 mM CHCOOH-NaOH buffer, pH 5.5) in a reac as articles of manufacture containing packaging material, a tion that contains 20 nmol of 4-MU-NANA in a 0.2 ml Vol composition provided herein, and a label that indicates that ume (Potier et al., Anal. Biochem., 94:287-296, 1979). The the composition, e.g., a DAS181 formulation or a Vancomy specific activity of DAS181 was determined to be 1,300 U/mg cin formulation, is formulated for oral, pulmonary or other protein (0.77 ug DAS181 protein per unit of activity). delivery. 0613 C. Preparation of Microspheres Using Purified 0605. The articles of manufacture provided herein can DAS181 contain packaging materials. Packaging materials for use in 0614 DAS181 (10 mg/ml), purified and prepared as packaging pharmaceutical products are well known to those described under Section A above, was used to form 200 ul US 2012/O 141590 A1 Jun. 7, 2012

cocktails as shown below. The cocktails contained either gly were cooled from ambient temperature (about 25°C.) to 4° cine or citrate as counterions, and isopropanol as organic C., followed by cooling to -20°C., followed by freezing to solvent, as follows: -80°C., as described in Example 1. Upon freezing to 80°C., 0615. 1) DAS181+5 mM glycine, pH 5.0; the tubes are placed in a lyophilizer and the volatiles (water 0616. 2) DAS181+5 mM glycine, pH 5.0+10% isopro and isopropanol) were removed by Sublimation, leaving the panol: dry powder containing microspheres. 0617 3) DAS181+5 mM sodium citrate, pH 5.0; 0628 Results: Microsphere formation was observed with 0618. 4) DAS181+5 mM sodium citrate, pH 5.0+10% iso all three concentrations: 10%, 20%, or 30%, of the organic propanol: Solvent isopropanol. The dimensions of the microspheres 0619 Plastic microcentrifuge tubes containing the cock however varied, depending on the concentration of the tails with ingredients as described in 1)-4) above were gradu organic solvent. The sizes of the microspheres as determined ally cooled from: by comparing the particles to a grid on a hemocytometer were 0620 (a) ambient temperature (about 25°C.) to 4°C. by estimated to be 2 microns using 10% isopropanol, 4 microns placing the cocktails in a refrigerator, followed by: using 20% isopropanol, and 5-6 microns using 30% isopro 0621 (b) cooling to -20°C. by placing the resulting cock panol. These results demonstrate that the size of the micro tail from (a) in a freezer, followed by: particles can be engineered as desired using an appropriate 0622 (c) freezing to -80° C. by placing the resulting cock concentration of organic solvent. tail from b) in a freezer. 0623. Under optimal conditions, microspheres would be Example 3 expected to form between about 4°C. to about -20°C. (gen erally in the range of about -2°C. to about -15°C.). Freezing Size of DAS181 Microspheres as a Function of Pro to -80° C. is carried out to remove ingredients from the tein Concentration cocktail other than the microspheres (e.g., solvent, etc.) by 0629 DAS181 was purified and used to prepare micro freeze-drying. Cocktail 4) was prepared in triplicate, two spheres as described above in Example 1 (see cocktail 4)), aliquots in plastic tubes and one in a glass tube. One aliquot using a combination of DAS181 protein (5 mg/ml or 10 (in a plastic tube) was cooled as described above, while the mg/ml), citrate counterion (sodium citrate, 5 mM) and iso two other aliquots (one in a plastic tube and one in a glass propanol (5% or 20%). The resulting cocktail solutions were tube) were subjected to Snap cooling/freezing by dipping the cooled from ambient temperature (about 25°C.) to 4°C., tubes into liquid nitrogen. followed by cooling to -20°C., followed by freezing to -80° 0624 Upon freezing, all tubes were placed into the lyo C., as described in Example 1. Upon freezing to -80°C., the philizer and the volatiles (water and isopropanol) were tubes were placed in a lyophilizer and the volatiles (water and removed by sublimation, leaving the dry pellets. isopropanol) were removed by Sublimation, leaving the dry 0625 Results: The dry pellets recovered from the cock powder containing microspheres. tails treated as described above, were tested for the presence Results: Microsphere formation was observed with both con of microspheres. Of the above samples, microspheres with centrations of protein (5 mg/ml and 10 mg/ml), and both good dispersivity characteristics, about 2 microns (Lm) in concentrations of organic solvent (5% or 20%). The dimen size, were observed only with cocktail 4) containing citrate sions of the microspheres however varied. Cocktails contain counterion and isopropanol and Subjected to gradual cooling. ing 5 mg/ml or 10 mg/ml protein and 5% isopropanol pro The counterion glycine did not prove to be optimal for the duced microspheres estimated to be about 1.5 micron in size. DAS181 protein (cocktail2)), showing a mixture of glass-like The cocktail containing 5 mg/ml protein and 20% isopro crystals and agglomerates with only a few microspheres. panol produced microspheres of an estimated size of about 3 When no organic solvent was present, a glass-like mass of microns, while the cocktail containing 10 mg/ml protein and lyophilized DAS181 protein was obtained and no micro 20% isopropanol produced microspheres of an estimated size spheres were observed (cocktails 1) and 3)). Snap-freezing of of about 4 microns. These results demonstrate that the size of cocktail 4) in a glass tube produced glass-like crystals and no the microparticles can be engineered as desired using an microspheres, while Snap-freezing of cocktail 4) in a plastic appropriate concentration of protein, or an appropriate com tube (cooling rate is slightly slower due to slower diffusion of bination of concentration of organic solvent and concentra heat through plastic than through glass) produced agglomer tion of protein. ated microspheres. 0626. This example demonstrates that microspheres with Example 4 narrow size distribution and good dispersivity (minimal agglomeration) can be produced by a combination of appro Size of DAS181 Microspheres as a Function of priate protein, counterion, organic solvent and gradual cool Counterion Concentration ing, using the methods provided herein. 0630 DAS181 was purified and used to prepare micro spheres as described above in Example 1 (see cocktail 4)), Example 2 using a combination of DAS181 protein (10 mg/ml), citrate Size of DAS181 Microspheres as a Function of counterion (sodium citrate; 2 mM, 3 mM or 6 mM) and Organic Solvent Concentration isopropanol (20%). The cocktail solutions were mixed in glass vials and cooled from +20° C. to -40° C. at a freeze 0627 DAS181 was purified and used to prepare micro ramp of 1° C. per minute in a Millrock Lab Series lyophilizer. spheres as described above in Example 1 (see cocktail 4)), Volatiles (water and isopropanol) were removed by sublima using a combination of DAS181 protein (10 mg/ml), citrate tion at 100 mTorr with primary drying at -30°C. for 12 hours counterion (sodium citrate, 5 mM) and isopropanol organic and secondary drying at 30° C. for 3 hours, leaving the dry solvent (10%, 20% or 30%). The resulting cocktail solutions powder containing microspheres. US 2012/O 141590 A1 Jun. 7, 2012 44

0631 Results: Microsphere formation was observed at all Millrock Lab Series lyophilizer. Volatiles were removed by three tested concentrations of citrate counterion. The size of sublimation at 100 mTorr, with a primary drying at -30°C. the microspheres increased from 1 micron at 2 mM citrate, to for 12 hours and secondary drying at 30° C. for 3 hours. 3 microns at 3 mM citrate, to 5 microns at 6 mM citrate. 0637 Results: The results are shown in Table 1 below. For Addition of 1 mM sodium acetate or 1 mM sodium chloride to the BSA protein, combinations (of counterion and organic the cocktail containing 2 mM citrate did not affect formation Solvent, respectively) that produced the most uniform micro of the microspherestriggered by the citrate counterion. These spheres with minimal crystallization or aggregation include: results demonstrate that the size of the microparticles can be (1) citrate--isopropanol engineered as desired using an appropriate concentration of (2) citrate+acetone counterion. (3) itaconic acid--1-propanol Example 5 (4) glycine--dioxane (5) glycine-1-propanol DAS181 Microspheres Formed in the Presence of (6) rubidium+1-propanol Surfactants (7) perchlorate+1-propanol 0632. The addition of surfactants to macromolecular (e.g., protein) microspheres often can improve characteristics of TABLE 1 the microspheres that render them suitable for administration to a subject, Such as flowability, dispersivity and disposition High-throughput screening of BSA microspheres for a particular route of administration, Such as intranasal or formed under different conditions oral inhalation. To test whether Surfactants can be incorpo Counterion pH Organic Solvent Product description rated into the methods of manufacturing microspheres as 5 mM pivalic 4.05% Cyclohexanol 0.5-1 micron microspheres provided herein, the production of DAS181 microspheres acid with occasional crystals was undertaken as described in Example 1 above, except that 5 mM pivalic 4.05% 1-propanol 0.5-1 micron microspheres in addition, a Surfactant was added to the Solution. acid with some aggregates 5 mM pivalic 4.05% butyl alcohol Aggregated microspheres 0633) To a cocktail solution containing 5 mg/ml DAS181, acid 5 mM sodium citrate, and 20% isopropanol, was added a 5 mM pivalic 4.05% p-Dioxane Aggregated microspheres surfactant (3.5% w/w lecithin, 0.7% w/w Span-85.R. (sorbitan acid trioleate), or 3.5% w/w oleic acid). The microspheres were 5 mM rubidium 9.05% Cyclohexanol 0.5-1 micron microspheres. chloride Aggregates and occasional formed by cooling the solutions to 4°C., followed by cooling crystals to -20°C., followed by freezing to -80°C. for lyophilization 5 mM rubidium 9.05% 1-propanol 0.5-1 micron microspheres as described above in Example 1. Upon freezing, the tubes chloride were placed into a lyophilizer and the volatiles (water and 5 mM rubidium 9.05% butyl alcohol Few microspheres (0.5-1 chloride micron). Mostly aggregates isopropanol) were removed by Sublimation, leaving the dry and crystals powder containing microspheres. 5 mM rubidium 9.05% p-Dioxane 1-2 microns microspheres 0634. Results: The microspheres resulting from treatment chloride with some aggregates of each of the above cocktails as described above were spread 5 mM sodium 4.05% Cyclohexanol 1-2 microns microspheres bromide with some aggregates on glass slides using cover slips rubbed in a circular motion. 5 mM sodium 4.05% 1-propanol Few microspheres (0.5-2 Efficient microsphere formation was observed in all cases. bromide micron). Mostly aggregates When the samples containing Surfactant were compared to and crystals the sample containing all the remaining ingredients but no 5 mM sodium 4.05% butyl alcohol Few microspheres (0.5-1 bromide micron). Mostly aggregates added surfactant, it was noted that the microspheresformed in and crystals the presence of Surfactant had improved dispersivity (lesser 5 mM sodium 4.05% p-Dioxane 1-2 microns microspheres agglomeration or aggregation). bromide with some aggregates 5 mM sodium 4.05% Cyclohexanol 0.5-2 microns microspheres Example 6 perchlorate with some crystals and aggregates 5 mM sodium 4.05% 1-propanol 0.5-1 micron microspheres Preparation of Microspheres of Bovine Serum Albu perchlorate min (BSA) by Selection of Suitable Types and Con 5 mM sodium 4.05% butyl alcohol Few 1-2 microns microspheres. centrations of Organic Solvents and Counterions perchlorate Mostly crystals and aggregates 5 mM sodium 4.05% p-Dioxane Aggregated microspheres 0635. As described herein, the methods provided herein perchlorate can empirically be optimized in high-throughput format to 5 mM calcium 4.05% Cyclohexanol Few 1-2 microns microspheres, phosphate mostly aggregates obtain microspheres having desired characteristics including 5 mM calcium 4.05% 1-propanol 1-2 microns microspheres size, flowability and dispersivity. The purpose of this experi phosphate with some aggregates ment was to demonstrate that by varying types and concen 5 mM calcium 4.05% butyl alcohol Few 1-2 micron microspheres. trations of organic solvents and counterions, as well as pH of phosphate Mostly crystals and aggregates 5 mM calcium 4.05% p-Dioxane Aggregated microspheres the cocktail, size and quality of microspheres of a protein of phosphate interest, in this case bovine serum albumin (BSA), can be 5 mM trieth- 9.05% Cyclohexanol 0.5-1 micron microspheres adjusted. ylamine with some crystals and aggregates 0636 Cocktail solutions containing 5 mg/ml of BSA and 5 mM trieth- 9.05% 1-propanol 1-2 micron microspheres various organic solvents and counterions at indicated pH and ylamine with some aggregates concentrations (see Table 1) were placed in a microtiter plate 5 mM trieth- 9.05% butyl alcohol Few 1-2 micron microspheres. (final volume per well of 0.1 ml). Cocktails were cooled from ylamine Mostly crystals and aggregates +20° C. to -40°C. at a freeze ramp of 1° C. per minute in a US 2012/O 141590 A1 Jun. 7, 2012 45

were dialyzed against 2 mM sodium acetate buffer, pH 5.0, TABLE 1-continued and concentrated on a Centriprep (Amicon). High-throughput screening of BSA microspheres 0643 Purification of 6xHis tagged PI8: A construct formed under different conditions encoding PI8 with an N-terminal His tag was expressed in E. Coli as 6xHis-tagged PI8. Purification was performed as Counterion pH Organic Solvent Product description described for 6xHis RFP and 6xHis GFP above, with the 5 mM trieth- 9.05% p-Dioxane Aggregated microspheres exception that all buffers used in the various chromatographic ylamine purification steps contained 1 mM TCEP (Tris(2-carboxy 5 mM glycine 9.05% Cyclohexanol 0.5-1 micron microspheres ethyl)phosphine hydrochloride). with some crystals and aggregates 0644 Preparation of microspheres: Cocktail solutions 5 mM glycine 9.05% 1-propanol 0.5-2 micron microspheres containing 5 mg/ml of protein and various counterions, with occasional aggregates organic solvents and pH as listed below were prepared in a 5 mM glycine 9.05% butyl alcohol Few 1-2 micron microspheres. Mostly crystals and aggregates microtiter plate as described above in Example 6. 5 mM glycine 9.05% p-Dioxane 1-2 micron microspheres 5 mM sodium 4.0 15% isopropanol 1-2 micron microspheres TABLE 2 citrate 5 mM sodium 4.0 15% acetone 0.5-1 micron microspheres Combinations Used to Produce Microspheres of Different Proteins citrate 5 mMitaconic 4.0 15% 1-propanol 1-2 micron microspheres Microsphere acid Organic Size Protein Counterion pH Solvent (microns) 0638. These results demonstrate that, for each protein, Trypsin 5 mMarginine 8.05% isopropanol OS-1 Lysozyme 5 mM citrate 8.05% isopropanol 4-5 multiple formulations can readily be screened for the best PIN 168 (PI8) 5 mM citrate 5.0 7% isopropanol 2-5 microsphere formation (desired dimensions, uniformity, dis DNase I 5 mM citrate 4.05% isopropanol 0.4-1 persivity, minimal aggregation and crystal formation, etc.) in RNaseA 5 mM citrate 4.05% isopropanol 0.4-1 high-throughput format. The combinations of reagents and Hemoglobin 5 mM glycine 5.0 10% isopropanol O4-O-7 Ovalbumin 5 mM pivalic 4.0 10% isopropanol OS-1 conditions (counterion, organic solvent, pH, concentrations) acid selected from the initial screen can then further be fine-tuned Red fluores- 5 mM pivalic 7.0 10% 1-propanol 1-4 (occasional as desired. cent protein acid aggregates) Green fluores- 5 mM pivalic 7.0 10% 1-propanol O.S.-1.5 Example 7 cent protein acid Preparation of Microspheres Using a Variety of Pro 0645. The microtiter plate was cooled from +20° C. to teins -40°C. at a freeze ramp of 1° C. per minute in a Millrock Lab Series lyophilizer. Volatiles (water and isopropanol) were 0639. The methods provided herein can be used to prepare removed by sublimation at 100 mTorr with primary drying at microspheres using a variety of proteins. In addition to -30° C. for 12 hours and secondary drying at 30° C. for 3 DAS181 and BSA exemplified above, the methods were used hours, leaving the dry powder containing microspheres. to prepare microspheres from trypsin, hemoglobin, DNase I. 0646. The dry powders were spread on glass slides and lysozyme, ovalbumin, RNASe A, hexahistidine-tagged microphotography was performed through either 32x or 100x human proteinase inhibitor 8 (PI8, having the sequence of objective. All the combinations listed in Table 2 above pro amino acids set forth in SEQ ID NO:15), red fluorescent duced microspheres of good quality (uniform size distribu protein (RFP) and green fluorescent protein (GFP). tion, dispersivity, with few aggregates and/or crystals). The 0640 DNase I, trypsin and hemoglobin were purchased microspheres varied in size from about 0.4-1 micron (RNAse from Worthington. Lysozyme, ovalbumin, and RNAse A A, DNAse I) to about 2-5 microns (6xHis PI8, lysozyme), were purchased from Sigma. Purification of 6xHis tagged depending on the protein. This example demonstrates that the PI8, GFP and RFP: 6xHis tagged PI8, GFP and RFP were methods provided herein can be used to produce micro expressed and purified essentially as described for DAS181 in spheres from a wide variety of proteins. Example 1 above, with the following modifications: 0641 Purification of 6xHis tagged GFP and 6xHis tagged RFP: Constructs encoding Red Fluorescent protein and Example 8 Green Fluorescent protein with N-terminal His tags were expressed in E. Colias 6xHis-tagged proteins. Expression of (0647. Aerodynamic ParticleSize Distribution of DAS181 Red Fluorescent protein was allowed to proceed overnight in Microspheres for Inhalation: a Comparison of the Method LB medium with 1 mMIPTG. Green Fluorescent protein was Provided Herein with Spray-Drying induced for 3 hour in TB medium with 1 mM IPTG. Cell 0648. As described herein, the methods provided herein lysates from 4 liters of induced cultures were clarified by can be used to produce microspheres in any desired size centrifugation and the proteins were purified by metal chelate range, including a range of about 0.5 micron to about 6-8 affinity chromatography on Fast-Flow Chelating resin (GE microns for delivery via inhalation. Healthcare) charged with Nickel and packed into C-10 col 0649) A. Preparation of Microspheres umns (GE Healthcare). 0650. To test the aerodynamic particle size distribution of 0642. The proteins were further purified by Gel Filtration DAS181 dry powder (microspheres) formulated for delivery Chromatography on a 0.5 cmx70 cm Sephacryl 200 column by inhalation, DAS181 microspheres were prepared using equilibrated with phosphate buffered saline. The proteins two methods as follows: US 2012/O 141590 A1 Jun. 7, 2012 46

(a) A DAS181 aqueous solution containing 14 mg/ml ever, the aerodynamic particle size distribution of the two DAS181.5 mM sodium citrate, pH 5.0 was spray dried into an preparations differs significantly between the two methods. air stream at 55° C., to produce microspheres. For the microspheres produced according to a method as (b) Alternately, DAS181 microspheres were produced provided herein (Le., method (b) as set forth in section A above), less than 25% remained trapped in the mouth (throat/ according to the methods provided herein. To a DAS181 cone of the impactor assembly), while greater than 70% of the aqueous solution containing 14 mg/ml DAS181, 5 mM microspheres were delivered to the trachea and lungs (with Sodium citrate, pH 5.0, was added 5% isopropanol as organic greater than 40% in the terminal bronchi and alveoli). In solvent. The resulting solution was cooled from +20° C. to comparison, less than 50% of the DAS181 microspheres -40°C. at a freeze ramp of 1° C. per minute in a Milirock Lab formed by spray-drying (method (a) as set forth in section A Series lyophilizer. Volatiles (water and isopropanol) were above) was delivered to the trachea and lungs (less than 20% removed by sublimation at 100 mTorr with primary drying at in the terminal bronchi and alveoli). The results demonstrate -30° C. for 12 hours and secondary drying at 30° C. for 3 that methods provided herein can produce microspheres for hours, leaving the dry powder containing microspheres. delivery into deep lungs, and that the microspheres produced 0651 B. Aerodynamic Particle Size Distribution of by methods provided herein have Superior disagglomeration Microspheres and flowability properties (provide a higher delivered dose) 0652 The microspheres prepared as described in Example compared to microspheres produced by a spray-drying 8A were tested by Andersen Cascade Impaction. The depo method.

TABLE 3 Results of Cascade Impaction Analyses of DAS181 MicroSpheres Percent Deposition of DAS181 Component of the Corresponding Expected Deposition Microspheres Produced Microspheres Andersen Cascade Size Cut-Off in Respiratory by Method (a) (i.e., Produced by Impactor (microns) Airways Spray Drying) Method (b) Throat + Cone >10 oral cavity 42.9 16.6 -2 (S+ P) 8.0-10 oral cavity 3.7 4.9 -1 (S+ P) 6.5-8.0 oropharynx 5.9 5.5 -O S.2-6.5 pharynx 5.8 4.0 3.5-5.2 trachea bronchi 12.5 9.3 26-35 secondary bronchi 11.6 12.6 1.7-2.6 terminal bronchi 11.0 24.0 1.0-1.7 alveoli 4.5 19.2 O43-1.O Alveoli 1.4 3.5 sition of pharmaceuticals in the respiratory tract can be pre Example 9 dicted by the aerodynamic behavior of particles (micro spheres) on the stages/collection plates of the cascade Large Scale Manufacture of Microspheres impactor. 0655 This example demonstrates that the methods pro 0653. The cascade impaction experiment was performed vided herein can be scaled for the manufacture of large quan using DAS181 microspheres prepared by one of the two tities of DAS181. The Batch Process described herein is alternate methods described in section A above, i.e., either by suitable for the manufacture of high quality dry powder spray-drying or by the methods provided herein. The micro microspheres in an amount ranging from, for example, mil spheres (10 mg) were loaded into gelatin capsules. The gela ligrams to about a kilogram and is limited by the capacity of tin capsules were placed into a CycloHaler (PharmaChemie) the mixing tank and/or lyophilizer shelf space. An alternative dry powder inhaler and Subjected to cascade impaction. An “continuous” process described herein can be used to manu 8-stage, non-viable Andersen Cascade Impactor (Thermo facture amounts ranging from, for example, hundreds of Electron, Boston) modified for use at 90 liters per minute of grams to hundred or more kilograms (100 grams to 100 kg air flow and equipped with a USP throat, induction cone and and above). Additional advantage of continuous process is a no preseparator, was used. The collection plates of the impac better control over the chilling of the cocktail. tor representing various areas/stages of deposition post-inha 0656. The large scale manufacture by a batch process or by lation (trachea, primary and secondary bronchi, terminal a continuous process can follow, for example, one or more of bronchi, alveoli, etc.) were coated with silicon spray to pre the steps described below in any combination of steps or vent bouncing of the microspheres. The microspheres from specific alternative methods: the stages and collection plates were recovered into a phos 0657 Precipitation of protein into microspheres. This phate buffered saline containing 0.1% Tween, and the amount step can be performed in a batch mode by placing the of deposited DAS181 recovered from each stage and collec cocktail solution containing the desired concentration of tion plate was quantified by measuring absorbance at 280 nm. protein, organic solvent and counterion in lyophilization 0654 Results: The geometric size of microspheres pro tray(s) and placing the tray(s) onto lyophilizer shelves. duced by the two methods was assessed by light microscopy Alternatively, trays can be chilled and frozen on a chilled and found to be essentially identical (range of 1.5-3.0 platform or other type of equipment (e.g., a freezer) and microns) for both methods. As shown in Table 3 below, how stored for a period of time frozen and lyophilized later. US 2012/O 141590 A1 Jun. 7, 2012 47

Alternatively, the microspheres can be formed by pre particle size distribution to the microspheres produced by cipitation in a vessel with stirring, wherein the vessel is batch mode. When the formulated DAS181 cocktail solution placed onto a cold Surface or a cooling coil is immersed was not chilled (not passed through heat exchanger, thus no into liquid or while the cocktail is being recirculated precipitation of microspheres was induced) and poured through a heat exchanger using a peristaltic pump. Alter directly into liquid nitrogen, no microspheres were observed natively, the microspheres can be formed by precipita and, instead, glass-like crystals were observed after lyo tion in a continuous mode, by passing the cocktail solu philization. tion through a heat exchanger(s) once using a peristaltic Example 10 pump. 0661 Batch Mode Process and Formulation of DAS181 0658) Removal of bulk liquid. The suspension of the Microspheres for Delivery to Upper and Central Respiratory microspheres can be concentrated using standard cen Airways trifugation, continuous flow centrifugation (e.g., CARR 0662. This example describes formulation and a process ViaFuge Pilot), or filtration (e.g., on glass fiber, sintered for manufacture of DAS181 microspheres. The contents of glass, polymer filters, hollow fiber cartridges (e.g., those the DAS181 cocktail solution and their relative amounts are manufactured by GE Healthcare) or tangential flow fil shown in Table 4 below.

TABLE 4 Batch Manufacturing Formula for DAS181 Microspheres. Final Amount for one batch' concentration

Stock solution Amount in formulated Ingredient concentration added cocktail Function DAS181 protein 19.55 g/L 3.306 L, API 12 g/L Active ingredient Sodium acetate’ 1.12 mM Solution 0.688 mM pH buffer Acetic acid'’ O.63 mM 0.0387 mM pH buffer Sodium Sulfate SOO mM O.O21.5 L 2 mM Microparticle formation agent (counterion) Isopropanol 100% viv O-269 L 5% viv Microparticle formation agent Calcium chloride SOO mM O.OO28 L. 0.268 mM Stability enhancing agent Water for neat 1.79 L NA Diluent irrigation Batch size: final volume of formulated cocktail 5.38 L. Theoretical yield 74 g of bulkDAS181 Dry Powder. Components of the DAS181 protein (API) stock solution.

tration cassettes (TFF cassettes. Such as those manufac 0663 A. Production of Bulk Drug Substance tured by Millipore or Sartorius)). The removal of bulk 0664. The terms Drug Substance, Active Pharmaceutical liquid (50% or greater) can result in a faster drying cycle Ingredient, and API are used interchangeably in this example and higher efficiency and throughput. and refer to the DAS181 protein. Production of DAS181 0659 Drying the microspheres. The recovered micro protein in bulk was conducted as follows. First, bulk amounts spheres formed by any mode, can be dried by conven of DAS181 were expressed in E. coli (BL21 strain) essen tional lyophilization. Alternatively, the microspheres tially as described in Example 1. The E. coli cells expressing can be dried under ambient temperature and atmo the DAS181 protein were washed by diafiltration in a fermen spheric pressure, eliminating the use of lyophilizer. tation harvest wash step using Toyopearl buffer 1, UFP-500 0660 Results: DAS181 protein was successfully pro E55 hollow fiber cartridge (GE Healthcare) and a Watson cessed into dry powder (microspheres) by a continuous mode Marlow peristaltic pump. as described herein. Cocktail containing 10 mg/ml DAS181, 0665. The recombinant DAS181 protein was then purified 20% isopropanol. 2 mM sodium sulfate was passed through in bulk from the cells. The detailed specifications of the 35 SERIES heat exchanger (Exergy, Garden City, NY) components and buffers used in the bulk purification of coupled with a NESLAB circulating cryostatusing a peristal DAS181 are provided in Tables 5 and 6 below. The harvested tic pump so that during the passage the cocktail was cooled and washed cells were lysed in a homogenization step by from about 25°C. to about -12°C. The resulting suspension passing the cells twice through using Niro-Soave Panda cell of microspheres exiting the heat exchanger was pumped into disruptor. The homogenate thus obtained was clarified by a prechilled lyophilization tray (-40°C.), frozen and lyo microfiltration using the Toyopearl buffer 1, Hydrosart 0.2 philized or, alternatively, pumped directly into liquid nitrogen micron TFF cassette and a Watson Marlow pump. The clari and then lyophilized. The resulting microspheres, which were fied homogenate was then concentrated by allowing the lysate analyzed by microscopy and cascade impaction, showed uni to recirculate without fresh buffer feed. Next, DAS181 pro form microspheres with minimal aggregation and good dis tein was captured from the clarified homogenate on a Toyo persivity and were similar in dimensions and aerodynamic pearl SP-550C resin which was washed in a series of buffers US 2012/O 141590 A1 Jun. 7, 2012 48

(see Table 5) before the DAS181 protein was eluted from the resin. The sodium chloride concentration of the eluate was TABLE 5-continued adjusted to 1.0 M in a final buffer of 50 mM phosphate at pH 8.0. The DAS181-containing eluate was then passed through Purification of bulk DAS181 drug substance a Toyopearl Hexyl-650C resin for further purification using a 11 Purpose DAS181 polishing in flow-through mode Resin Amberchrome CG300M Toyopearl Buffer 4. The resin eluate containing DAS181 Activity Step Buffer Name protein was then buffer-exchanged into 5 mM sodium acetate Loading Sample Load Amberchrom Load 12 Purpose Concentration & Diafiltration in a diafiltration step (see step 8 in Table 5). The concentrated TFF Cartridge HydroSart 10K 0.6 m? Specifications protein was next passed through a Sartorius Q SingleSep Activity Buffer Name Recirc. Limin Filter in order to remove DNA in a flow-through mode. Iso Recirculation Formulation Buffer 15-16 Concentration Amberchrom Product Pool 15-16 propanol was added to the Q SingleSep filtrate to a final Diafiltration Formulation Buffer 15-16 concentration of 20% v/v. The DAS181 protein in the buffer *Volumes in liters, except 4x denotes multiples of the retentate volume was passed through an Amberchrome CG300M resin equili CW = Column Wolumes brated with an Amberchrom buffer (see step 11 in Table 5). NR=Not Recorded The purified bulk DAS181 protein was then buffer-ex NS = Not Specified changed into formulation buffer and concentrated by diafil tration (see step 12 of Table 5). TABLE 6 Buffers used during the DAS181 purification process TABLE 5 Buffer Name Buffer Composition Purification of bulk DAS181 drug substance Toyopearl Buffer 1 50 mM potassium phosphate, 0.3M NaCl, pH 8.0 1 Purpose Fermentation Harvest Wash Toyopearl Buffer 2 1.1 mM potassium phosphate, 2.9 mM Cartridge GEUFP-500-E55 Specifications sodium phosphate, 154 mM NaCl, pH 7.4 Activity Buffer Name Inlet PSI Toyopearl Buffer 3 1.1 mM potassium phosphate, 2.9 mM Diafiltration Toyopearl Buffer 1 25-35 sodium phosphate, 154 mM NaCl, 1% Triton 2 Purpose Homogenization X-100, 0.1% SDS, 0.5% sodium deoxycholate, Activity Step Buffer Name pH 7.4 Equilibration Equilibration Harvest Buffer Toyopearl Buffer 4 50 mM potassium phosphate, 1.OM NaCl, pH 8.0 Homogenization 1st Pass Sample Load Toyopearl Buffer 5 50 mM potassium phosphate, 0.5M NaCl, pH 8.0 Homogenization 2nd Pass Sample Load Toyopearl Buffer 6 5 mM sodium acetate, pH 5.0 3 Purpose Homogenate Toyopearl Buffer 7 5 mM sodium acetate, 60% isopropanol, pH 5.0 Clarification (Diafiltration) Formulation Buffer 1.75 mM sodium acetate, pH 5.0 TFF Cartridge HydroSart 10K 0.6 m? Specifications 3% Isoproyl Alcohol 3% isopropanol Activity Buffer Name Inlet PSI Amberchrom Buffer 5 mM sodium acetate, 20% isopropanol, Recirculation Sample Load 40 pH 5.0 adjusted with acetic acid Diafiltration Toyopearl Buffer 1 <50 1. ONNaOH.3% 1.ON NaOH, 3% isopropanol 4 Purpose Permeate Concentration Isopropanol TFF Cartridge HydroSart 10K 0.6 m? Specifications 1. ONNaOH 1.ONNaOH Activity Buffer Name Inlet PSI O.SN NaOH O.SN NaOH Recirculation Sample Load NS Concentration Sample Load <50 70% Isopropyl 70% isopropanol 5 Purpose DAS181 capture performed in bind and elute mode Alcohol Resin Toyopearl SP-550C 20% EtOH 20% ethyl alcohol Activity Step Buffer Name Loading Sample Load Clar. Homogenate Wash SP Wash 1 Toyopearl Buffer 1 0.666 B. Batch Manufacturing Process SP Wash2 Toyopearl Buffer 2 0667 The ingredients set forth in Table 4 above were SP Wash 3 Toyopearl Buffer 3 combined to form DAS181 microspheres in a large scale SP Wash4 Toyopearl Buffer 2 SP Wash 5 Toyopearl Buffer 1 batch process as described below. Elution Elution Toyopearl Buffer 4 Step I: Thawing of Bulk Drug Substance 6 Purpose Adjust NaCl Concentration Method Add NaCl to 1.OM 0668 Frozen 0.2 um-filtered bulk Drug Substance in plas Final Buffer 50 mM phosphate, 1.OM NaCl, pH 8.0 tic bottles was thawed overnight at ambient temperature 7 Purpose DAS181 purification in flow-through mode (25+3° C.). Resin Toyopearl Hexyl-650C Activity Step Buffer Name Step II: Weighing of the Excipients and Preparation of Solu Loading Sample Load Cond. Hexyl Load tions 8 Purpose Concentration & Diafiltration TFF Cartridge HydroSart 10K 0.6 m? Specifications 0669 35.51 g of Sodium Sulfate anhydrous powder was Activity Buffer Name Recirc. Limin weighed and Q.S. to 500 mL with Water For Irrigation, then Recirculation Toyopearl Buffer 6 15-16 stirred to obtain a clear solution. 18.38g of Calcium Chloride Concentration Hexyl Product Pool 15-16 dihydrate powder was weighed and Q.S. to 250 mL with Diafiltration Toyopearl Buffer 6 15-16 Water For Irrigation, then stirred to obtain a clear solution. Recirculation Toyopearl Buffer 6 NS 9 Purpose Remove DNA in flow-through mode Step III: Preparation of the DAS181 Cocktail Solution Resin Sartorius Q SingleSep Filter Activity Step Buffer Name 0670. To 3.3 L of concentrated Drug Substance (19.55 Loading Sample Load g/L), 1.79 L of Water For Irrigation was added slowly with 10 Purpose Buffer Adjustment stirring, followed by 0.0215 L of Sodium Sulfate solution, Method Add Isopropanol to 20% 0.0028 L of Calcium Chloride Solution and 0.269 L of iso Final Buffer 5 mM Acetate, 20% Isopropanol, pH 5.0 propanol. The solution was stirred to ensure complete mixing of components. US 2012/O 141590 A1 Jun. 7, 2012 49

Step IV: Filtration of Formulated Cocktail Solution Through 0682. The protein concentration in the final formulation 0.2 um Filter could be varied (10-14 mg/ml), as could the concentration of 0671 The formulated cocktail solution of Step III was counterion (1-5 mM) and isopropanol (2-30% V/v), without filtered through a 0.2 um filter into sterile media bags to Substantial impact on the physical properties of the micro control particulates and bioburden. spheres or the activity of the DAS181 protein in the micro Step V: Filling into Lyophilization Trays spheres. At higher concentrations of isopropanol (15-30%), 0672. The formulated filtered solution was dispensed into the microspheres formed while the cocktail was still fully autoclaved Lyoguard lyophilization trays. To ensure even liquid. At lower concentrations (2-15%), ice crystals began to cooling of the Solution and formation of high quality micro form first, followed by precipitation to form microspheres. spheres, 6 trays were each filled with 0.9L or less of cocktail 0683 C. Yield of DAS181 in the Microspheres Solution. 0684. The theoretical yield of DAS181 in the dry micro Step V 1: Freezing and Lyophilization spheres is calculated according to the following formula: Theoretical yield=DAS181 protein, g-protein fraction 0673. The trays were placed onto lyophilizer (Hull in Dry Powder (microspheres) 120FSX200) shelves pre-chilled to -45+5° C. and the solu tion was allowed to chill and freeze. Formation of micro 0685. The protein fraction value (0.866) was established spheres occurred while the solution was being frozen. The empirically by analysis of several manufactured batches of freezing is allowed to proceed for 1-2 h to ensure complete DAS181 microspheres. The theoretical yield for the amounts solidification. The product temperature was verified by read as set forth in Table 2 is 64.56 g--0.866–74.55g. The actual ing the thermocouples attached to two of the six trays. yield of DAS181 Dry Powder was found to be 64 g. 0674. The lyophilization cycle steps are as follows: 0686 Results: The suitability of the microspheres pre 0675 a) Set vacuum to 160 microns and allow to evacu ate to 100-200 microns; pared as described in section B above for administration by 0676 b) Ramp shelf temperature to +10° C. over 3 h; oral inhalation was tested by Andersen Cascade Impaction. 0677 c) Hold shelf temperature at +10° C. for 36 h The results are summarized in Table 7 below. The deposition (primary drying); of pharmaceuticals in the respiratory tract can be predicted by 0678 d) Thermocouple traces examined to verify that deposition of particles (microspheres) on the stages/collec primary drying phase is completed and the product tem tion plates of the cascade impactor. For a pharmaceutical, perature has stabilized at +10° C.t5° C. for 15-30 h. e.g., DAS181 microspheres, that is administered to prevent or 0679 e) Ramp shelf temperature to +30° C. over 1 hand treat viral infections that initiate in the respiratory tract, such hold for 3-5 h (secondary drying). as influenza, it is desirable to deposit the pharmaceutical in Step VII: Transfer of Bulk DAS181 Microspheres into Con the throat, trachea, bronchi (upper and central respiratory tainer and Mixing airways). The DAS181 fusion protein delivered to upper and 0680 A section on the bottom film of each Lyoguard lyo central respiratory airways cleaves off the receptor Sialic philization tray was cleaned using sanitizing wipes and a 3x3 acids from mucous membranes, thus preventing viral binding cm opening was made with a scalpel. The dry microspheres and infection at these sites. For optimal delivery of the were transferred into a plastic bottle. The bottle was capped DAS181 microspheres to sites where respiratory viral infec and tumbled forty times, changing directions with each inver tion can be initiated, i.e., in the throat, trachea or bronchi, the sion. The tumbling was to ensure uniformity of bottle content. microspheres must not be (a) So big that they are trapped at the Samples for analytical testing were taken and the bottle was front end in the mouth (i.e., microspheres are too big, about 8 recapped and sealed into plastic bags for storage. microns or greater); or (b) so Small that they are deposited in 0681. In the DAS181 microsphere bulk manufacturing deep lungs and absorbed systemically into the blood stream process as described above, Sulfate was demonstrated to be a (i.e., 0.5 microns or smaller). For delivery of the DAS181 safe Substance for use as a counterion, and reproducibly pro microspheres to the throat, trachea and bronchi, a size range duced microspheres with a narrow size distribution. Further, of about 1 micronto about 5.5-6 microns generally is suitable. the organic solvent isopropanol was a good solvent of choice 0687 DAS181 microspheres manufactured as described because (1) a class 3 solvent, (2) it can produce microspheres above were characterized by Andersen cascade impaction in a wide range (2-30%, V/v) of concentrations, and (3) it has and found to be suitable for delivery to upper and central a relatively high freezing point So its vapors can efficiently be respiratory airways with sufficiently low percentage (<5%) trapped during lyophilization. deposited in the alveoli.

TABLE 7 Aerodynamic Particle Size Distribution of DAS181 dry powder at 60 liters per minute. Percent of DAS181 total Component of protein DAS181 Andersen Cascade Corresponding size Expected deposition in deposited (in protein Impactor cut-off, microns respiratory airways mg) recovered Inhaler (Cyclohaler) 1.57 0.11 20.13% >10 Oral cavity O.93 + O.19 11.92% US 2012/O 141590 A1 Jun. 7, 2012 50

TABLE 7-continued Aerodynamic Particle Size Distribution of DAS181 dry powder at 60 liters per minute. Percent of DAS181 total Component of protein DAS181 Andersen Cascade Corresponding size Expected deposition in deposited (in protein Impactor cut-off, microns respiratory airways mg) recovered -1 (Stage + Plate) 8.6-10 Oral cavity OSO 0.10 6.41% -0 (Stage + Plate) 6.5-8.6 oropharynx O40 O.O3 S.13% 1 (Stage + Plate) 4.4-6.5 pharynx O58, O.O3 7.44% 2 (Stage + Plate) 3.3-4.4 trachea bronchi O.83 O.O7 10.64% 3 (Stage + Plate) 2.0-3.3 Secondary bronchi 18O, O.O9 23.08% 4 (Stage + Plate) 1.1-2.0 Terminal bronchi O.82 0.08 10.51% 5 (Stage + Plate) O54-11 alveoli O.23 O.O3 2.95% 6 (Stage + Plate) O.25-0.54 alveoli O.14 O.O3 1.79% XACI (Emitted) 6.24 O.1O 80.00% 10+ 1.0 mg of DAS181 Dry Powder (8.5 mg + 10% DAS181 protein) was filled into HPMC capsule XACI(Emitted) fraction is the sum of all material recovered from USP Throat, Induction Cone and stages -1 to 6.

DAS181 microspheres were further characterized by laser pulmonary administration could carry a risk of inducing air diffraction, which demonstrated, consistent with the cascade way hyperresponsiveness (Agrawal et al., Lung, 183:375-387 impaction results, that the majority of the microspheres pro (2005)). This example therefore tested alternate salts, such as duced by the method described in this Example are within a salts of other metals such as potassium, magnesium and cal size range of between 1 micron and 5 microns in size. Scan ning Electron Microscopy (FEI Quanta 200 Scanning Elec C1. tron Microscope. Everhart Thornley (ET) detector) of the 0689) DAS181 microspheres were manufactured as DAS181 microspheres prepared according to the method described above in Example 1. Cocktail solutions containing described in this Example revealed that the microspheres are 12 mg/mL DAS181 and 5% (v/v) isopropanol contained as present as agglomerates of hundreds and thousands of indi counterions the indicated Sulfates at 2 mM concentration, pH vidual particles approximately 0.5-3 micron in size. The 4.5-5.0. The microspheres were formed by cooling the solu agglomerates however are easily dissipated by air turbulence tions from +25° C. to -45° C. Upon freezing, the volatiles produced during the actuation through dry powder inhaler (as (water and isopropanol) were removed by Sublimation, leav demonstrated by Andersen Cascade Impaction or laser dif ing the dry powder containing microspheres. fraction). Light microscopy of microspheres dispersed in a liquid surfactant (e.g. TritonX-100 or Tween 20) or non-polar 0690. The aerodynamic particle size distribution of the dry Solvent (e.g., alcohol, acetone, or acetonitrile) that does not powder was assessed by Andersen Cascade Impaction, and dissolve the microspheres, confirmed that aggregates are eas the amount of DAS181 per stage was determined by UV ily dissipated into individual uniform microspheres. measurement at 226 nm (A). The results are shown below in Table8. The results demonstrate that sulfate salts other than Example 11 the sodium salt can be used as counterion to obtain DAS181 Preparation of DAS181 Microspheres Using Sulfates microspheres of a size range Such that the majority are deliv Other than the Sodium Salt ered to the throat, trachea and bronchi, in an amount that is 0688 Studies have shown that in certain instances, e.g., in comparable to the amount delivered when sodium sulfate is Some asthmatics, the presence of sodium in formulations for used as the counterion.

TABLE 8 Aerodynamic Particle Size Distribution of DAS181 microspheres formulated with or without sodium Expected Corresponding deposition in Percent DAS181 per stage size cut-off, respiratory Sodium Potassium Magnesium Calcium microns airways Sulfate Sulfate Sulfate Sulfate

Inhaler 19.86%. 28.58% 21.41% 16.71% Capsule 2.07% 2.30% 1.88% O.00% Throat + Cone >10 Oral cavity 11.67% 9.00% 12.91% 16.79% -1(S+ P) 8.6-10 Oral cavity 10.00% 3.43% 7.86% 14.87% –0(S+ P) 6.5-86 oropharynx 5.30% 3.08% 4.71% 7.779, 1(S+ P) 4.4-6.5 pharynx 6.97% 5.86% 6.58% 7.54% 2(S+ P) 3.3-44 trachea bronchi 7.55% 8.24% 6.90% 6.43% 3(S+P) 2.0-3.3 Secondary bronchi 19.57%. 20.21% 17.01% 12.65% US 2012/O 141590 A1 Jun. 7, 2012 51

TABLE 8-continued Aerodynamic Particle Size Distribution of DAS181 microspheres formulated with or without sodium Expected Corresponding deposition in Percent DAS181 per stage size cut-off, respiratory Sodium Potassium Magnesium Calcium microns airways Sulfate Sulfate Sulfate Sulfate 4(S+ P) 1.1-2.0 Terminal bronchi 12.39%. 14.00% 13.00% 10.39% 5(S+ P) O54-11 alveoli 2.80% 2.99% 4.31% 4.69% 6(S+ P) 0.25-0.54 alveoli 1.82% 2.31% 3.44% 2.16%

(0691. The dry powders also were incubated at +37° C. or Example 12 +53° C. for a duration as indicated in Table 9 and tested for Stability of DAS181 Microspheres sialidase activity using the 4-MU-NANA assay as described in Example 1 and incorporated by reference herein. The rela (0692. The stability of the DAS181 protein in the micro tive activity compared to non-lyophilized DAS181 micro spheres was assessed by measuring Sialidase activity over spheres stored at -80° C. is shown in Table 9. The results time using the 4-MU-NANA activity assay as described show that the stability of the microspheres prepared using the above in Example 1 and as incorporated by reference herein. The production of dry DAS181 microspheres was undertaken various metal Sulfates as counterions were comparable to that in a cocktail solution containing 10 mg/mL DAS181, 2 mM of sodium sulfate, with retention of almost all or all the Sodium sulfate, 5% V/v isopropanol. To some solutions, activity for over 2 months at 37°C. and retention of almost all 0.01% w/v sugar (sorbitol, mannitol, trehalose or sucrose) (sodium and potassium Sulfates) or over 85% (magnesium was added. The microspheres were formed by cooling the and zinc sulfates) of the activity for over 10 days at 53° C. solutions from +25°C. to -45°C. Upon freezing, the volatiles This experiment demonstrates that various non-sodium con (water and isopropanol) were removed by Sublimation, leav taining counterions can produce microspheres with desirable ing the dry powders containing microspheres. characteristics. (0693 A. Stability of DAS181 Microspheres without Sug aS TABLE 9 (0694. The DAS181 dry powder microspheres formulated Sialidase activity of DAS181 microsphere formulations: without sugars were stored at room temperature (25°C.) in a accelerated Stability studies. container next to Drierite desiccant (Hammond Drierite, Xenia, Ohio). The dry powder retained its original potency Percent Activity Remaining (as measured by sialidase activity using 4-MU-NANA Temperature according to Examplel and as incorporated by reference 37o C. 53o C. herein; results shown in Table 10) and aerodynamic particle Incubation Days size distribution (as measured by Andersen Cascade impac 42 Days 69 Days 11 Days 39 Days tion: Table 11) for at least 8 months. 2 mM Sodium Sulfate + 107.14%. 105.62%. 110.66%. 23.66% TABLE 10 0.268 mM CaCl, 2 mM Potassium Sulfate + 97.37%. 104.00% 101.54% 52.76% Specific activity of DAS181 dry powder. 0.268 mM CaCl, 2 mM Magnesium Sulfate + 123.81% 107.29% 85.93% 60.00% Test Time O 3 months 8 months 0.268 mM CaCl, 13.34 mM Calcium 116.67% 93.20% 87.12% 40.48% Sialidase Activity with 100% 102.0% 99.9% 2 mM Sulfate reference to time O

TABLE 11 Aerodynamic particle size distribution of DAS181 dry powder Expected Corresponding deposition in ACI size cut-off, respiratory Component microns airways Time O 3 Months 8 Months

Throat + >10 Oral cavity 1957 2.43 26.OOO.30 18.57 - 4.14 Cone Stage -1 8.6-10 Oral cavity 1787 0.51 12.87 - 1.56 15.13 - 241 Stage -0 6.5-86 oropharynx 10.27- 0.93 7.07 0.32 9.80 18O Stage 1 4.4-6.5 pharynx 8.57 0.49 8.8O. O.26 7.73 O.S7 US 2012/O 141590 A1 Jun. 7, 2012 52

TABLE 11-continued Aerodynamic particle size distribution of DAS181 dry powder Expected Corresponding deposition in ACI size cut-off, respiratory Component microns airways Time O 3 Months 8 Months Stage 2 3.3-44 trachea bronchi 10.67 + 0.23 10.70- 0.35 9.3O 0.82 Stage 3 2.0-3.3 Secondary 21.10 - 0.75 21.80 - 0.52 21.90 - 0.87 bronchi Stage 4 1.1-2.0 Terminal 10.10 - 0.75 10.63 - 0.80 14.50 - 3.22 bronchi Stage 5 O54-11 alveoli 147 O.23 1.73 - 0.06 2.37 (0.06 Stage 6 0.25-0.54 alveoli O.33 OO6 0.400.10 O.73 0.06

Table 11: Aerodynamic particle distribution was assessed by Andersen Cascade Impaction and expressed as % of totalDAS181 protein recovered, Capsules were filled with 10 mg of DAS181 dry powder and actuated using Cyclohaler dry powder inhaler as delivery device. Airflow rate was 60 Liters per minute. Assays were performed in triplicate, mean and standard deviation are shown,

(0695 B. Stability of DAS181 Microspheres Formulated the protective effect of the sugars, with Trehalose and Sucrose with Sugars providing the best protection. Results are expressed as % of (0696. The sialidase activity of DAS181 in the dry powder oligomers at 0 months, 1 month, 2 months or 3 months. microsphereformulations containing Sugars and in the unlyo philized microsphere formulations stored at -80° C., were TABLE 12A measured using the fluorescent substrate 4-MU-NANA as described in Example 1 and as incorporated by reference % of oligomers herein. The dry powder formulations containing no Sugar or O months 1 month 2 months 3 months various sugars as indicated below in Table 12 were stored at Mannitol O.O 6.O1 9.79 12.59 +42°C. for 4 weeks (forced degradation). The results are Trehalose O.O 3.75 S.82 6.79 shown in Table 12. Relative to unlyophilized formulations Sucrose O.O 3.92 5.67 8.16 stored at -80° C., the formulation containing no Sugar Sorbitol O.O 4.88 7.35 9.59 retained almost 80% of its activity. The addition of various sugars increase the stability so that about 88-98% of the 0699 The stabilizing effect of Sucrose and Trehalose on activity is retained, depending on the Sugar. DAS181 microsphereformulations was further confirmed by the following experiments. Formulations containing the Sug TABLE 12 ars and a Sugar-free control were produced and either left as Percent Sialidase Activity bulk powders (unencapsulated) or placed into clear size 3 Sugar Remaining after 4 weeks at 42°C. HPMC capsules. The samples were stored at 37° C. The percentage of high molecular weight DAS181 oligomers No Sugar 79.82 Sorbitol 91.23 (degradation products) was quantitated by size exclusion Mannitol 89.47 HPLC. The results in Table 12B (below) again demonstrate Trehalose 97.37 that Trehalose and Sucrose Sugars provided significant pro Sucrose 88.60 tection. Results are expressed as % of oligomers at 0 months, 1 month, 2 months or 8 months. 0697 The stabilizing effect of higher percentages of Sug ars on the DAS181 microspheres was investigated. The pres TABLE 12 B ence of glycine in the reaction cocktails served to prevent % of oligomers crystallization of the Sugars during the manufacture of the DAS181 microspheres. The study showed that up to 15% of HPMC Capsules Unencapsulated Trehalose, Sucrose, Sorbitol, or Mannitol, when combined with 5% glycine, can be incorporated into the DAS181 micro Sugar t = 0 t = 1 t = 2 t = 8 t = 1 t = 2 t = 8 sphere-forming reactions without forming crystals during Sucrose O.O 4.2 8.9 1S.O O.O 1.1 O.9 lyophilization. The microspheres were indistinguishable Trehalose O.O 4.8 8.6 14.3 O.O 1.O 1.O from the ones produced without Sugars, based on their None O.O S.O 9.4 18.6 O.9 2.4 3.5 appearance under light microscopy and scanning electron Formulations contained 15% wiw Sugar, 5% wiw Glycine, 2 mMAcetate pH 6.0, and 2 mM microscopy (SEM). The aerodynamic particle size distribu MgSO4 tion of the microspheres also remained unaffected. 0698 5 mg of the resulting DAS181 dry powder was Example 13 placed in clear size 3 HPMC capsules and stored at 37°C. The Preparation of Microspheres Using a Variety of percentage amount of high molecular weight DAS181 oligo Classes of Compounds mers (degradation products) was quantitatively analyzed by 0700. The methods provided herein can be used to prepare size exclusion HPLC. The results in Table 12A demonstrate microspheres using a variety of classes of compounds. In US 2012/O 141590 A1 Jun. 7, 2012

addition to proteins such as DAS181, BSA, trypsin, hemo globin, DNase I, lysozyme, ovalbumin, RNAse A, hexahisti TABLE 1.4 dine-tagged human proteinase inhibitor 8 (PI8, having the Combinations Used to Produce Microspheres of Different sequence of amino acids set forth in SEQ ID NO:15), red Compounds fluorescent protein (RFP) and green fluorescent protein A. Antibiotics (GFP), as described in the above Examples, this Example Microsphere demonstrates that the method can be used to prepare micro Counterion Antisolven pH Quality spheres of: Compound: Tobramycin 0701 A. The antibiotics Vancomycin, Tobramycin, Kanamycin and Ampicillin 2 mMNa-Citrate 59 S.O isopropano 0702 B. A nucleic acid siRNA 2 mMNa- 59 4.0 Glutamate isopropano 0703. C. A virus Tobacco Mosaic Virus 2 mM Arginine 59 7.0 0704. D. The peptides—leuprolide and somatostatin HCJNaOH isopropano 2 mM Itaconic Acid- 59 4.0 0705 The microspheres prepared from A-D above were Na isopropano compared to those of the protein, DAS181. 2 mMNa-Citrate 15% S.O isopropano 0706 Preparation of microspheres: for each of the com 2 mMNa- 15% 4.0 pounds listed in A-Dabove, and for DAS181, cocktail solu Glutamate isopropano tions containing 2 mg/ml of compound dissolved in an aque 2 mM Arginine 15% 7.0 HCJNaOH isopropano ous solvent and various counterions, antisolvents and pH, as 2 mM Arginine 15% 9.0 listed below, were prepared in a 96-well microtiter plate (0.1 HCJNaOH isopropano ml cocktail/well) at room temperature. Control Solutions con 2 mM Itaconic Acid- 15% 4.0 tained either solvent orantisolvent alone, with or without the Na isopropano 2 mM Itaconic Acid- 15% in 4.0 counterion. Cocktails were cooled by placing in a freezer. The Na propanol chilled plates were transferred onto pre-chilled (-45° C.) Compound: Kanamycin shelves of a Millrock Lab Series Lyophilizer, and the vacuum 2 mMNa-Citrate 59 S.O was applied. The frozen cocktail solutions were allowed to isopropano lyophilize for 16 hours. 2 mMNa- 59 4.0 Glutamate isopropano (0707. The lyophilized powders from the bottoms of the 2 mM Itaconic Acid- 59 4.0 wells were transferred onto glass slides and analyzed by light Na isopropano microscopy for appearance. The quality of the product micro 2 mM Itaconic Acid- 59 7.0 spheres was scored based on the uniformity of the micro Na isopropano 2 mMNa-Citrate 15% S.O spheres, the absence of undesirable non-microsphere par isopropano ticles (glass-like crystalline forms), and the absence of 2 mMNa- 15% 4.0 aggregates. Table 13 below provides an exemplary scoring Glutamate isopropano 2 mMNa- 15% 7.0 system, based on appearance. Glutamate isopropano 2 mM Arginine 15% 7.0 TABLE 13 HCJNaOH isopropano 2 mM Arginine 15% 9.0 Scoring System for Assessing Quality of Microspheres HCJNaOH isopropano 2 mM Itaconic Acid- 15% 4.0 Presence of Na isopropano Glass like 2 mM Itaconic Acid- 15% 7.0 Presence? Quality of crystals andfor Na isopropano Score Microspheres Aggregates 2 mMNa-Citrate 5% in S.O propanol O Absent Exclusive 2 mMNa- 5% in 4.0 1 Almost absent Almost exclusive Glutamate propanol 2 SC3C Highly dominant 2 mM Itaconic Acid- 5% in 7.0 3 observable Dominant Na propanol 4 Present in large quantities Present in smaller 2 mM Itaconic Acid- 15% in 4.0 relative to glass-like crystals or quantities than Na propanol aggregates the microspheres Compound: Ampicillin 5 Dominant Scarce 6 Almost uniform Observable, but 2 mMNa-Citrate 59 S.O minimal isopropanol 7 Uniform Observable, but 2 mMNa- 59 4.0 very minimal Glutamate isopropanol 8 Uniform Very few 9 Very uniform Almost absent 2 mM Itaconic Acid- 59 4.0 10 Perfect and uniform Absent Na isopropanol 2 mMNa-Citrate 15% S.O isopropanol 2 mMNa- 15% 4.0 Table 14 below shows the various combinations of com Glutamate isopropanol pound, solvent, antisolvent and counterion that were used to 2 mM Itaconic Acid- 15% 4.0 generate microspheres, and the aualitV of the resultina micro Na isopropanol spheres. US 2012/O 141590 A1 Jun. 7, 2012 54

Vancomycin in a 2 ml total Volume. All cocktail mixes tested TABLE 14-continued produced very high quality microspheres of Vancomycin as described below: Combinations Used to Produce Microspheres of Different Compounds A. Antibiotics Microsphere 20 mg Vancomycin Counterion Antisolvent pH Quality Microsphere 2 mMNa-Citrate 5% in- S.O 3f4 Counterion Antisolvent pH Quality propano 2 mMNa- 5% in- 4.0 3-5 5 mMNa-Citrate 15% in- S.O 9 Glutamate propanol propanol 2 mMNa- 15% in- 4.0 4 5 mM Na-glutamate 5% in- 7.0 9 Glutamate propanol propanol 2 mM Itaconic Acid- 15% in- 4.0 3 5 mMNa-Citrate 15% in- S.O 10 Na propanol 2 mM Itaconic Acid- 15% in- 7.0 7 propanol Na propanol 5 mM Na-glutamate 5% in- 7.0 9 Compound: Vancomycin propanol

2 mMNa-Citrate 59 S.O 4 isopropano 2 mMNa- 59 4.0 4 Glutamate isopropano 2 mMNa- 59 7.0 7 B. Nucleic Acid - siRNA Glutamate isopropano 2 mM Arginine 59 7.0 7 Microsphere HCJNaOH isopropano Counterion Antisolven pH Quality 2 mM Arginine 59 9.0 6 HCJNaOH isopropano Compound:siRNA 2 mM Itaconic Acid- 59 4.0 8 Na isopropano 2 mMNa-Citrate 59% 4.0 2 2 mM Itaconic Acid- 59 7.0 8 isopropano Na isopropano 2 mMNa-Citrate 59% S.O 2 2 mMNa-Citrate 15% S.O 4 isopropano isopropano 2 mMNa-Citrate 59% 7.0 2 2 mMNa- 15% 4.0 4 isopropano Glutamate isopropano 2 mMNa- 59% 4.0 3 2 mMNa- 15% 7.0 3 Glutamate isopropano Glutamate isopropano 2 mMNa- 59% 7.0 1 2 mM Arginine 15% 7.0 4 Glutamate isopropano HCJNaOH isopropano 2 mM Arginine 59% 7.0 1 2 mM Arginine 15% 9.0 4 HCJNaOH isopropano HCJNaOH isopropano 2 mM Arginine 59% 9.0 1 2 mM Itaconic Acid- 15% 4.0 7 HCJNaOH isopropano Na isopropano 2 mM Itaconic Acid- 59% 4.0 3 2 mM Itaconic Acid- 15% 7.0 3 Na isopropano Na isopropano 2 mM Itaconic Acid- 59% 7.0 O 2 mMNa-Citrate 5% in- S.O 9 Na isopropano O8O 2 mM Pivalic Acid 59% 4.0 1 2 mMNa- 5% in- 4.0 6 isopropano Glutamate O8O 2 mM Pivalic Acid 59% S.O 2 2 mMNa- 5% in- 7.0 8 isopropano Glutamate O8O 2 MPEI7SOOOO 59% 7 2 mM Arginine 5% in- 7.0 3 isopropano HCJNaOH O8O 2 MPEI 2SOOO 59% 7 2 mM Arginine 5% in- 9.0 4 isopropano HCJNaOH O8O 2 MPEI 2000 59% 3 2 mM Itaconic Acid- 5% in- 4.0 5 isopropano Na O8O 2 mMNa- 59% 4.0 1 2 mM Itaconic Acid- 5% in- 7.0 2 Sulfate, Na-Acetate isopropano Na O8O 2 mMNa- 59% 6.O 1 2 mMNa-Citrate 15% in- S.O 9 Sulfate, Na-Acetate isopropano O8O 2 mMNa-Citrate 15% 4.0 2 2 mMNa- 15% in- 4.0 7 isopropano Glutamate O8O 2 mMNa-Citrate 15% S.O 1 2 mMNa- 15% in- 7.0 4 isopropano Glutamate O8O 2 mMNa-Citrate 15% 7.0 O 2 mM Arginine 15% in- 9.0 4 isopropano HCJNaOH O8O 2 mMNa- 15% 7.0 1 2 mM Itaconic Acid- 5% in- 7.0 5 Glutamate isopropano Na O8O 2 mM Arginine 15% 7.0 4f S HCJNaOH isopropano 2 mM Arginine 15% 9.0 4 HCJNaOH isopropano 0708. The experiments with Vancomycin were also per- 2 mM Itaconic Acid- 15% 4.0 4 formed on a larger scale (20 mg Vancomycin), namely, at 2 Na isopropano mg/ml Vancomycin in a 5 ml total Volume; and at 10 mg/ml US 2012/O 141590 A1 Jun. 7, 2012 55

-continued

B. Nucleic Acid - siRNA C. Virus - Tobacco Mosaic Virus Compound: Tobacco Mosaic Virus Microsphere 0.5 mg/ml Counterion Antisolven pH Quality Microsphere 2 mM Pivalic Acid 59 4.0 3 Counterion Antisolven pH Quality isopropano N 59% 8 2 mM Pivalic Acid 59 S.O 3 Ole vo SOO8O spropano 2 mMNa-Citrate 59% 4.0 9 2 MPEI7SOOOO 59 6 isopropano SOO.80 2 mMNa-Citrate 59% S.O 4 2 MPEI 2SOOO 59 6 isopropano isopropano 2 mM Pivalic Acid- 59% S.O 6 2 mMNa- 59 4.0 1 Na isopropano Sulfate Na-Acetate isopropano 2 mMNa- 59% 7.0 7 2 mMNa- 59 6.O 3 Glutamate isopropano Sulfate Na-Acetate isopropano 2 mM Arginine 59% 7.0 8 Compound:siRNA (2 mg/ml) HCJNaOH SOO8O 2 mM Arginine 59% 9.0 8 None 59 10 HCJNaOH isopropano isopropano 2 mMNa- 59% 4.0 10 2 mM Arginine 59 7.0 7/8 sulfate Na-acetate isopropano HCJNaOH isopropano 2 mMNa 59%0. 6.O 7 2 mM Argini 59 9.0 4 sulfate Na-acetate SOO8O mM Arginine 1970 2 mMNa-Citrate None:* 4.0 5 HCUNaOH spropano 2 mMNa-Citrate None S.O 2 2 mM Itaconic Acid- 59 4.0 3 2 mM Pivalic Acid- None S.O 8 Na isopropano Na 2 MPEI 2SOOO 59 8 2 mMNa- None 7.0 3 isopropano Glutamate 1 mMPEI 2SOOO 59 7 2 mM Arginine None 7.0 6 isopropano HCJNaOH OS MPEI 2SOOO 59 7 2 mM Arginine None 9.0 2 isopropano HCJNaOH O.1 MPEI 2SOOO 59 7 2 mMNa- None 4.0 4 isopropano sulfate Na-acetate 2 mM Arginine 30% 7.0 5 2 mMNa- None 6.O 6 HCJNaOH isopropano sulfate Na-acetate 2 mM Arginine 30% 9.0 5.6 JNaOH As noted above, microspheres of good quality could beformed with tobacco mosaic virus, HCJNa SOO.80 even in the absence of antisolvent. Some crystallinity was observed, but depending on the 2 mM Itaconic Acid- 30% 4.0 8 choice of counterion (e.g., pivalic acid), uniform microspheres could be obtained without the Na isopropano addition of antisolvent. 2 MPEI 2SOOO 30% 7 isopropano 0709 D. Peptides Somatostatin and Leuprolide 1 mMPEI 2SOOO 30% 8 isopropano OS MPEI 2SOOO 30% 9 isopropano Microsphere O.1 MPEI 2SOOO 30% 6 Counterion Antisolven pH Quality isopropano Compound: siRNA (0.25 mg/ml) Compound: Somatostatin 2 mM Arginine 15% 7.0 O 2 mMNa-citrate 59 4.0 5 HCJNaOH isopropano SOO.80 2 mM Arginine 15% 9.0 O 2 mMNa-citrate 59 S.O 5 HCJNaOH isopropano is pro 8O 2 mM Arginine 30% 7.0 6 2 mMNa-citrate 5% 7.0 5 SOO.80 HCJNaOH SOO.80 2 mM Arginine 59 7.0 5 2 mM Arginine 30% 9.0 3 HCJNaOH isopropano HCJNaOH isopropano 2 mM Arginine 59 9.0 5 Compound:siRNA (5 mg/ml) HCJNaOH isopropano 2 mM Itaconic Acid- 59 4.0 5 2 mM Arginine 15% 7.0 3 Na isopropano HCJNaOH isopropano 2 mM Itaconic Acid- 59 7.0 3 Na isopropano 2 mM Itaconic acid- 15%0. 4.0 5 2 mM Pivalic Acid- 59 4.0 5 Na SOO.80 Na isopropano 2 mM Arginine 30% 7.0 4 2 mM Pivalic Acid- 59 7.0 6 HCJNaOH isopropano Na isopropano 2 mM Arginine 30% 4.0 4 2 mMNa- 59 4.0 6 HCJNaOH isopropano Glutamate isopropano 2 mMNa- 59 7.0 4 Glutamate isopropano US 2012/O 141590 A1 Jun. 7, 2012 56

-continued -continued Microsphere Microsphere Counterion Antisolvent Quality Counterion Antisolven pH Quality

2 MPEI7SOOOO 2 mM Arginine 59% 7.0 4 2 MPEI 2SOOO HCJNaOH isopropano 2 MPEI 2000 2 mM Arginine 59% 9.0 5 2 mMNa 59 4.0 HCJNaOH isopropano sulfate, Na-acetate isopropano 2 mM Itaconic Acid 59% 4.0 5 2 mMNa 59 6.O isopropano sulfate, Na-acetate isopropano Na 2 mMNa-citrate 59 4.0 2 mM Itaconic Acid 59% 7.0 5 isopropano Na isopropano 2 mMNa-citrate 59 2 mM Pivalic Acid 59% 4.0 7 isopropano Na isopropano 2 mMNa-citrate 59 7.0 2 mM Pivalic Acid 59% 7.0 7 isopropano Na isopropano 2 mM Arginine 59 7.0 2 mMNa 59% 4.0 7 HCJNaOH isopropano Glutamate isopropano 2 mM Arginine 59 9.0 2 mMNa 59% 7.0 8 HCJNaOH isopropano Glutamate isopropano 2 mM Itaconic Acid 59 4.0 2 MPEI7SOOOO 8 isopropano Na 2 MPEI 2SOOO 4 2 mM Itaconic Acid 59 7.0 2 MPEI 2000 4 Na isopropano 2 mMNa 15% 4.0 7 2 mM Pivalic Acid 59 4.0 Na isopropano sulfate Na-acetate isopropanol 2 mM Pivalic Acid 59 7.0 2 mMNa 15% 6.O 5 Na isopropano sulfate Na-acetate isopropanol 2 mMNa 59 4.0 Glutamate isopropano 2 mMNa 59 7.0 3 Glutamate isopropano 2 MPEI7SOOOO 2 MPEI 2SOOO E. DAS181 Protein 2 MPEI 2000 Compound: DAS181 2 mMNa 15% 4.0 : sulfate, Na-acetate isopropano Microsphere 2 mMNa 15% 6.O Counterion Antisolven pH Quality sulfate, Na-acetate SOO.80 Compound: Leuprolide 2 mMNa-citrate 59 4.0 7 isopropano 2 mMNa-citrate 59 4.0 2 mMNa-citrate 59 S.O 7 isopropano isopropano 2 mMNa-citrate 59 2 mMNa-citrate 59 7.0 5 isopropano isopropano 2 mMNa-citrate 59 7.0 2 mM Arginine 59 7.0 4 isopropano HCJNaOH isopropano 2 mM Arginine 59 7.0 2 mM Arginine 59 9.0 5 HCJNaOH isopropano HCJNaOH isopropano 2 mM Arginine 59 9.0 2 mM Itaconic Acid 59 4.0 5 HCJNaOH isopropano Na isopropano 2 mM Itaconic Acid 59 4.0 2 mM Itaconic Acid 59 7.0 5 Na isopropano Na isopropano 2 mM Itaconic Acid 59 7.0 2 mM Pivalic Acid 59 4.0 5 Na isopropano Na isopropano 2 mM Pivalic Acid 59 4.0 2 mM Pivalic Acid 59 7.0 7 Na isopropano Na isopropano 2 mM Pivalic Acid 59 7.0 2 mMNa 59 4.0 6 Na isopropano Glutamate isopropano 2 mMNa 59 4.0 2 mMNa 59 7.0 6 Glutamate isopropano Glutamate isopropano 2 mMNa 59 7.0 2 MPEI7SOOOO 8 Glutamate isopropano 2 MPEI 2SOOO 10 2 MPEI7SOOOO 2 MPEI 2000 7 2 MPEI 2SOOO 2 mMNa 59 4.0 8 2 MPEI 2000 sulfate Na-acetate isopropanol 2 mMNa 59 4.0 2 mMNa 59 6.O 7 sulfate, Na-acetate isopropano sulfate Na-acetate isopropanol 2 mMNa 59 6.O 2 mMNa-citrate 15% 4.0 5.6 sulfate, Na-acetate isopropano isopropanol 2 mMNa-citrate 15% 4.0 5.6 2 mMNa-citrate 15% S.O 7 isopropano isopropanol 2 mMNa-citrate 15% 2 mMNa-citrate 15% 7.0 6 isopropano isopropanol 2 mMNa-citrate 15% 7.0 2 mM Arginine 15% 7.0 4 isopropano HCJNaOH isopropanol US 2012/O 141590 A1 Jun. 7, 2012 57

theory, depending on the compound of interest, Such counte -continued rions potentially could act as “primers' or “carriers’ that help to nucleate and/or facilitate the formation of higher quality E. DAS181 Protein microspheres, relative to those obtained with “non-nucleat Compound: DAS181 ing counterions. For example, formation of a compound Microsphere (see, for example, in Table 14 above, microspheres formed Counterion Antisolven pH Quality from siRNA and DAS181, using PEI as counterion). 2 mM Arginine 59 9.0 7 0711. The results further demonstrate that under certain HCJNaOH isopropano conditions, microspheres can be formed in the absence of 2 mM Itaconic Acid- 59 4.0 7 counterion and/or antisolvent. For example, in the case of Na isopropano siRNA, very high quality (scale of 10) microspheres were 2 mM Itaconic Acid- 59 7.0 7 Na isopropano obtained when no counterion was added. Similarly, tobacco 2 mM Pivalic Acid- 59 4.0 6 mosaic virus formed microspheres in the absence of counte Na isopropano rion and, in Some instances, in the absence of antisolvent. 2 mM Pivalic Acid- 59 7.0 7 Na isopropano Example 14 2 mMNa- 59 4.0 6 Glutamate isopropano Size and Quality of Microspheres as a Function of 2 mMNa- 59 7.0 5 Glutamate isopropano Concentration of the Cocktail Components (Com 2 MPEI7SOOOO 8 pound, Counterion, Antisolvent) 2 MPEI 2SOOO 9 2 MPEI 2000 6 0712. This Example demonstrates that the size and quality 2 mMNa- 15% 4.0 7 of the microspheres of small molecule compounds, like those sulfate, Na-acetate isopropanol of macromolecules (see Examples 2-4), can readily be opti 2 mMNa- 15% 6.O 8 mized by varying parameters, such as the concentrations of sulfate, Na-acetate isopropanol compound, counterion, and/or antisolvent, in a variety of 2 mMNa-citrate 5% in- S.O 10 O8O permutations in high-throughput format. By conducting these 2 mM Arginine 5% in- 7.0 3 reactions in high-throughput format, conditions that are opti HCJNaOH O8O mal for microsphere formation of any compound can rapidly 2 mM Arginine 5% in- 9.0 7 be identified. HCJNaOH O8O 2 mM Itaconic Acid- 5% in- 4.0 5 0713 96-well plates containing cocktail solutions of Tet Na O8O racycline, Kanamycin or Ampicillin under various concen 2 mM Itaconic Acid- 5% in- 7.0 2 tration conditions were set up as described in Example 1. Na O8O Arginine was used as counterion and isopropanol was used as 2 mMNa- 5% in- 4.0 6 antisolvent. Concentrations of each of the cocktail compo Glutamate O8O nents—the compound, the counterion and the antisolvent— 2 mMNa- 5% in- 7.0 4 Glutamate O8O were varied as shown below in Table 15, and the effect on 2 mMNa-citrate 15% in- S.O 8.9 microsphere quality assessed. O8O 2 mM Arginine 15% in- 9.0 6 TABLE 1.5 HCJNaOH O8O 2 mM Itaconic Acid- 15% in- 4.0 4 Arginine Na O8O Concentration Isopropanol Microsphere 2 mM Itaconic Acid- 15% in- 7.0 2 (mg/ml) (%) Quality Na O8O Tetracycline 2 mMNa- 15% in- 4.0 6.7 Concentration Glutamate O8O (mg/ml) 2 mMNa- 15% in- 7.0 4 Glutamate O8O 25 60 O 1 25 60 10 3 25 60 2O 4 Results: These experiments demonstrate that by selecting the 25 60 30 7 25 60 40 5 appropriate combination of: (a) type and (b) concentration of 25 60 50 6 compound, counterion and antisolvent, microspheres of good 2O 48 60 6 quality (at least 6, as high as 10) can be obtained using a wide 15 36 70 1 25 60 50 5 variety of macromolecules and Small molecules. Depending 25 50 50 9 on the particular combination of compound, counterion and 25 40 50 9 25 30 50 9 antisolvent, the quality of the microspheres often was as good 25 2O 50 7 or better than the quality of microspheres obtained using the 25 10 50 7 sialidase fusion protein, DAS181, under comparable condi 25 5 50 7 25 O 50 9 tions. O 15 25 7 0710. In control cocktail reactions containing no com 5 15 25 9 10 15 25 8.9 pounds, it was noted that some counterions, such as polyeth 15 15 25 8.9 yleneimine (PEI) and Na-acetate/sulfate, could form micro 2O 15 25 7 spheres under certain conditions. Without being bound by any US 2012/O 141590 A1 Jun. 7, 2012 58

30 mg/ml arginine giving the Smallest size distribution as TABLE 15-continued visualized by light microscopy. It was interesting to note that in the absence of counterion, Small microspheres with very Arginine Concentration Isopropanol Microsphere little size variation were observed. Their overall quality was (mg/ml) (%) Quality high, although some amount of aggregation was present. 0716 Microsphere quality was also assessed by increas 25 15 25 8 30 15 25 9 ing tetracycline concentration at constant antisolvent and 31.25 15 25 7 arginine concentrations. In the absence of tetracycline, hygro Kanamycin scopic arginine microspheres were formed. As the tetracy Concentration cline concentration was increased, the microsphere size was (mg/ml) found to increase to a maximum at a concentration of 25-30 25 60 O O mg/ml tetracycline, then decreased again as the concentration 25 60 10 O of tetracycline was further increased. At 25-30 mg/ml tetra 25 60 2O O cycline, aggregation also was minimal; the aggregation 25 60 30 3 25 60 40 O increased as the tetracycline concentration was further 25 60 50 O increased. 2O 48 60 6 0717 Kanamycin: The formation of microspheres from 25 60 50 O kanamycin required at least 25-30% antisolvent (isopro 25 50 50 O 25 40 50 2 panol); below this concentration, hygroscopic crystals were 25 30 50 4 formed. At 60% isopropanol, better results were obtained, 25 2O 50 7 although there was precipitation prior to freezing, which 25 10 50 10 compromised microsphere quality. 25 5 50 7 25 O 50 8 0718 When the kanamycin microspheres were studied as O 15 25 O a function of varying arginine concentration, it was found that 5 15 25 4 decreasing the arginine concentration resulted in higher qual 10 15 25 8 15 15 25 7 ity, Smaller microspheres. At less than 10 mg/ml arginine, 2O 15 25 7 however, the microspheres became larger and tended to 25 15 25 8 agglomerate more. In the absence of arginine, high quality 30 15 25 7 microspheres were again obtained, although some aggrega 31.25 15 25 9 Ampicillin tion was present. Concentration 0719. When varying the concentration of kanamycin, it (mg/ml) was found that as the concentration of kanamycin was increased, the quality of the microspheres increased and the 25 60 O O 25 60 10 3 size decreased. 25 60 2O O 0720 Ampicillin: Although microspheres were obtained 25 60 30 O using amplicillin, their hygroscopic nature made it difficult to 25 60 40 O 25 60 50 6 unambiguously assign their quality. In general, several con 15 36 70 O ditions produced distinct microspheres, with the best quality 25 60 50 2 microspheres being observed at high (50%) antisolvent con 25 2O 50 O centration. 25 10 50 1 25 5 50 1 0721 This example demonstrates that a variety of small 25 O 50 2 molecule antibiotics can produce microspheres by the meth ods provided herein. The example also demonstrates that under certain conditions, the addition of a counterion may not be necessary for microparticle formation. This was observed Results: for all three antibiotics tested. Without being bound by theory, 0714 Tetracycline: With tetracycline, the absence of anti it is possible that the compounds themselves function as Solvent resulted in few, ifany, microspheres being formed. As counterions, or that the preparations used in the experiments the concentration of antisolvent was increased, the quality of contained excipients/impurities/bulking agents that served as the microspheres increased, reaching a maximum at about counterions. 30% isopropanol. Increasing the isopropanol concentration beyond 30% resulted in a decrease in overall microsphere Example 15 quality and the formation of larger, chunky agglomerations of microspheres and crystalline solids. At isopropanol concen Preparation of Microspheres from Water-Insoluble trations of 60% and 70%, the cocktail mixture was found to Molecules: Paclitaxel precipitate before freezing, resulting in large amounts of aggregates and crystals. A few microspheres were formed at 0722. The chemotherapeutic agent, Paclitaxel, has a log P the highest isopropanol concentrations, but they were not of value that is higher than 3 (log of the octanol/water partition uniform size. coefficient) (Bombuwala et al., Beilstein J. Org. Chem. 2006, 0715 Microsphere quality was then assessed as a function 2:13), which is a value that is representative of a large portion of counterion (arginine) concentration. It was found that at of the Small molecule drugs that are currently on the market. constant antisolvent and tertracycline concentration, the Hence, the identification of conditions for Paclitaxel micro microsphere size distribution decreases and its overall quality sphere formation should be applicable to a large number of increases as the concentration of arginine is decreased, with therapeutically relevant compounds. US 2012/O 141590 A1 Jun. 7, 2012 59

0723 Paclitaxel, being water-insoluble, was dissolved in microspheres were almost as high in quality as the best qual one of the following organic solvents: Isopropanol, t-Butyl ity microspheres observed at 90% t-butanol and 2 mM citrate, Alcohol or DMSO. A 20 mg/ml stock solution of paclitaxel in as discussed above. When the concentration of t-butanol was each of the organic solvents was used to generate cocktail lowered to 50% in the absence of citrate counterion, high solutions in 96-well plates, where the net concentration of quality microspheres were still present, although the aggre paclitaxel in each well (Le, reaction) was 2 mg/ml paclitaxel. gation increased. With ispropanol, a 20 mg/ml slurry was obtained and used as 0726. The results obtained with the organic solvent the stock solution, because the solubility of paclitaxel in DMSO showed higher amounts of crystallinity in general, isopropanol is lower than int-Butyl Alcohol and DMSO. 2 along with aggregated microspheres of lower quality, relative mMNa-citrate buffer, pH 5.0, was used as the antisolvent and to isopropanol and t-butanol. This could be due to the high counterion. The various experimental conditions are listed boiling point of DMSO, as the water in the solutions likely below in Table 16. The plates containing the cocktail solu evaporated/Sublimed first upon lyophilization, leaving nearly tions with varying concentrations of antisolvent/buffer rela pure DMSO solution from which the paclitaxel crystallized. tive to the concentration of organic solvent, were then placed Optical microscopy data did however reveal the presence of in a -80° C. freezer for lyophilization. microspheres, with the best microspheres being observed when 50% DMSO was used. TABLE 16 Example 16 Compound: Paclitaxel (2 mg/ml Citrate Buffer Effect of Drug, Antisolvent and Counterion Ratios (mM) Solvent (%) on the Quality of Microspheres 90% isopropanol 0727 Experiments were performed to evaluate the effect 75% isopropanol of antisolvent and counterion concentration variation on the 50% isopropanol 25% isopropanol formation of microspheres. The peptides leuprolide and 10% isopropanol Somatostatin, and the antibiotics Vancomycin and tobramy 100% isopropanol cin, were tested under a variety of conditions for forming 50% isopropanol 90% t-butanol microspheres. Table 17 describes the conditions under which 75% t-butanol the reactions were performed. Samples were analyzed in 50% t-butanol 96-well plates as described in the previous Examples. 25% t-butanol 10% t-butanol TABLE 17 100% t-butanol 50% t-butanol Compound: Leuprolide (2 mg/ml) 90% DMSO 75% DMSO Na-glutamate, pH Isopropanol Microsphere 50% DMSO 7.0 (mM) (%) Quality 25% DMSO 10% DMSO 2 50 9 100% DMSO 2 40 7 50% DMSO 2 30 8 2 2O 9 2 10 5 2 5 7 Results: 2 2.5 8 2 O 3 0724 With all three organic solvents, it was found that 17 5 5 paclitaxel precipitated if the solvent concentration was 25% 15 5 5 12.5 5 7 or less. Optical microscopy of the lyophilized samples 10 5 7 showed that with the organic solvent isopropanol, the micro 7.5 5 7 particles increased in quality as the concentration of isopro 5 5 6 panol was lowered from 90%, with the best microspheres 2.5 5 7 being formed in 50% isopropanol. When the concentration of O 5 6 isopropanol was lowered below 50%, crystals were observed, potentially due to precipitation of paclitaxel prior to freezing. Compound: Somatostatin (2 mg/ml) When 50% isopropanol was used with no citrate counterion, Na-Sulfate, Na the microspheres showed a higher tendency to aggregate than Acetate, pH 4.0 Isopropanol Microsphere in the presence of 2 mM citrate. With 100% isopropanol and (mM) (%) Quality no citrate counterion, the samples appeared to have high 2 50 9 crystallinity and were aggregated, although many Small 2 40 8 2 30 9 microparticles were also observed. 2 2O 7 0725. With t-butanol, the optimum solvent concentration 2 10 67 was higher than isopropanol, with best results being observed 2 5 6 2 2.5 34 at 90% t-butanol and aggregation and rod-like formations 2 O 3 increasing as the concentration of Solvent was decreased. At 17 5 6 25% and 10% t-butanol, significant crystallinity was 15 5 8 observed, likely due to precipitation of paclitaxel before 12.5 5 9 freezing. In 100% t-butanol with no citrate counterion, the US 2012/O 141590 A1 Jun. 7, 2012 60

was crystalline with only a few aggregated microspheres. TABLE 17-continued When the counterion concentration was varied at constant

10 5 7 antisolvent concentration (5%), at 17 mM Sulfate/Acetate 7.5 5 7 and 5% isopropanol, microspheres were present, but a high 5 5 6.7 degree of crystallinity also was observed. As the concentra 2.5 5 6 tion of counterion was decreased, the amount of crystals O 5 7 present decreased until well separated microspheres were Compound: Vancomycin (2 mg/ml) detected at 12.5 mM counterion concentration. As the sulfatef acetate concentration was further decreased, aggregation Na-citrate, pH 5.0 Isopropanol Microsphere increased again and microsphere size decreased. Somatosta (mM) (%) Quality tin was also found to form microspheres in the absence of 2 50 7 counterion, but they were aggregated and of varying (not 2 40 9 uniform) size. 2 30 8 0731. In the case of Vancomycin, with changing antisol 2 2O 10 2 10 7 vent concentration, small but well defined microspheres were 2 5 9 produced from 50% down to 2.5%. When the solvent concen 2 2.5 7 tration was further dropped down to 0% isopropanol, a high 2 O 7 degree of crystallinity was present, with a few aggregated 17 5 6 15 5 7 microspheres. When counterion concentration was varied, 12.5 5 7 the microspheres were found to be highly aggregated at 17 10 5 8 mM citrate, and the amount of aggregation decreased as the 7.5 5 9.8 counterion concentration was decreased. The best micro 5 5 9 spheres formed below 7.5 mM citrate, but as the counterion 2.5 5 9.8 concentration was further dropped down to Zero, the amount O 5 7 of aggregation again increased. Compound: Tobramycin (2 mg/ml) 0732. In the case oftobramycin, as the antisolvent concen Itaconic acid-Na, Isopropanol Microsphere tration was varied, with 50% isopropanol there was a signifi pH 4.0 (mM) (%) Quality cant amount of crystallinity and aggregation, although micro spheres were also detected. As the antisolvent concentration 2 50 7 was reduced from 40% to 10%, the microspheres formed 2 40 6.7 2 30 8.9 were found to be well-separated and of high quality. When the 2 2O 8 antisolvent concentration was further decreased from 5% to 2 10 9 0%, the amount of aggregation again increased and at 0% 2 5 7/8 there was a high degree of crystallinity along with significant 2 2.5 7/8 2 O 4f S numbers of aggregated microspheres. 17 5 Crystals 15 5 Crystals Example 17 12.5 5 Crystals 10 5 Crystals Aerodynamic Particle Size Distribution of Vancomy 7.5 5 Crystals cin Microspheres for Inhalation 5 5 Crystals 2.5 5 7 0733. As described herein, the methods provided herein O 5 8 can be used to produce microspheres in any desired size range, including a range of about 0.5 micron to about 6-8 0728 Results: microns for delivery via inhalation. 0729. In the leuprolide group, decreasing antisolvent con 0734 A. Preparation of Microspheres Vancomycin was centration reduced the aggregation of microspheres with an dissolved in aqueous buffer at a final concentration of 10 optimum at 10% isopropanol, and the aggregation increased mg/ml. The cocktail contained 5 mM sodium citrate pH 5.0 as again as the isopropanol concentration was further reduced counterion and 15% V/v n-propanol as anti-solvent. A 2 ml downto 0%. When the counterion concentration was varied at aliquot of cocktail was frozen in a 10-ml lyophilization vial constant antisolvent concentration (5%), 17 mM counterion placed in a -80° C. freezer for 1 hour. The frozen vial was showed a high degree of crystal formation. The crystal for transferred onto a -45° C. lyophilizer shelf and freeze dried mation decreased as the counterion (buffer) concentration for 36 hours. was decreased, until at 10 mM, the microspheres are evenly (0735. B. Aerodynamic Particle Size Distribution of sized and well separated. As the buffer concentration was Microspheres further decreased beyond 10 mmM, the aggregation began to 0736. The microspheres prepared as described in Example increase again, with a moderate degree of crystallinity being 5 were tested by Cascade Impaction using a New Generation observed at 0 mM glutamate. Impactor. The deposition of pharmaceuticals in the respira 0730. In the case of somatostatin, even-sized well-sepa tory tract can be predicted by the aerodynamic behavior of rated microspheres were observed at 50% isopropanol. The particles (microspheres) on the stages/collection plates of the level of aggregation increased as the concentration of antisol cascade impactor. vent was decreased, to an optimum of 10% isopropanol. (0737. The microspheres (10 mg) were loaded into HPMC Below 10% isopropanol, crystals began to appear and con (hydroxypropyl methylcellulose) capsule. The capsule was tinued to increase as the antisolvent concentration was placed into a CycloHaler (PharmaChemie) dry powder decreased to 0% isopropanol, where a majority of the sample inhaler and Subjected to cascade impaction. The collection US 2012/O 141590 A1 Jun. 7, 2012 plates of the impactor representing various areas/stages of nitrogen gas tank attached to the backfill system on the lyo deposition post-inhalation (trachea, primary and secondary philizer. Each of the reaction tubes was flushed with N. In bronchi, terminal bronchi, alveoli, etc.) were coated with addition, the backfill valve on the lyophilizer was left open to silicon spray to prevent bouncing of the microspheres. The continually flush a low humidity atmosphere over the microspheres from the stages and collection plates were samples. The microscope used to visualize the resulting recovered into a phosphate buffered saline containing 0.1% microspheres was contained within a plastic bag that was Tween, and the amount of deposited Vancomycin recovered continually purged with dry N. Sample tubes were placed from each stage and collection plate was quantified by mea under the bag to equilibrate for approximately 30 seconds, Suring absorbance at 280 nm. before opening and spreading onto the glass slide. 0738 Results: The geometric size of microspheres was 0742 Prostaglandin is unstable at pH values lower than 8: assessed by light microscopy and found to be in the range of therefore, the following basic buffers were used in this experi 1.0-3.0 microns. As shown in Table 18 below, the aerody ment: Polyethyleneimine (PEI), Triethylamine (TEA) and namic particle size was consistent with the observed geomet Arginine. Ciprostene is not highly soluble in aqueous solu ric size. The results demonstrate that methods provided tions, therefore n-propanol was added to the buffer, in herein can produce microspheres for delivery into deep lungs, amounts that rendered the compound soluble. The solvent/ and that the microspheres produced by methods provided antisolvent system for the prostaglandins was water/n-pro herein have good disagglomeration and flowability properties panol/t-butanol (water/aqueous buffer being more of the (provide a higher delivered dose). “antisolvent component for Ciprostene, which has poor solubility in water, and n-propanol/Tert-Butyl alcohol (t-bu TABLE 18 tanol, tBA) being more of the “antisolvent component for PGI2, which has higher solubility in water). The results are Results of Cascade Impaction Analyses of Vancomycin Microspheres summarized in Table 19 below: Component of Corresponding Expected Deposition Percent the Cascade Size Cut-Off in Respiratory Deposition of TABLE 19 Impactor (microns) Airways Vancomycin Buffer? Microsphere Capsule + NA NA 37.57 Counterion pH (% n-propanol) (% t-butanol) Quality device Throat >10 oral cavity 10.48 Compound: Prostaglandin I2 1 >8.06 Oral cavity pharynx 3.33 2 4.46-8.06 pharynx 7.71 2 mM Arginine 9 20 O 7 3 2.82-4.46 tracheabronchi 15.47 2 mM Arginine 9 30 O 5 4 1.66-2.82 secondary bronchi 16.69 2 mM Arginine 9 2O 5 7/8 5 0.94-1.66 terminal bronchi 6.38 2 mM Arginine 9 2O 30 5 alveoli 2 mM Arginine 9 2O 55 5.6 6 0.55-0.94 alveoli 1.59 2 mM Arginine 9 2O 70 4 7 0.34-0.55 alveoli O.S1 2 mM TEA 11 2O O O 8 <0.34 alveoli 0.27 2 mM TEA 11 30 O 2.3 2 mM TEA 11 2O 5 7/8 2 mM TEA 11 2O 30 8 2 mM TEA 11 2O 55 7 Example 18 2 mM TEA 11 2O 70 8 2 MPEI 10.75 2O O 7 Preparation of Microspheres Using Prostaglandin 2 MPEI 10.75 30 O 8 2 MPEI 10.75 2O 5 7 0739 Prostaglandins are a group of hormone-like com 2 MPEI 10.75 2O 30 5 pounds that are implicated in numerous physiological pro 2 MPEI 10.75 2O 55 7 cesses and, therefore, have clinical applications. One of the 2 MPEI 10.75 2O 70 6 prostaglandins, the prostacyclin PGI2 is a drug that is cur Compound: Ciprostene rently marketed for pulmonary hypertension. The API half 2 mM Arginine 9 2O O 7/8 life of this drug at physiological pH is on the order of minutes, 2 mM Arginine 9 30 O 6 2 mM Arginine 9 2O 5 8 requiring the drug to be administered through continuous 2 mM Arginine 9 2O 30 6.7 infusion in order to have an appreciable effect. Therefore, it is 2 mM Arginine 9 2O 55 8 desirable to create a PGI formulation that is inhalable and 2 mM Arginine 9 2O 70 7 works directly at the target site of action in the lungs, avoiding 2 mM TEA 11 2O O 4f S 2 mM TEA 11 30 O 3f4 the pharmacokinetic effects associated with clearance rates 2 mM TEA 11 2O 5 8.7 and stability in the bloodstream. This example demonstrates 2 mM TEA 11 2O 30 7 that the methods provided herein can be used to prepare high 2 mM TEA 11 2O 55 6.7 quality, inhalable microspheres of prostaglandins. 2 mM TEA 11 2O 70 5.6 2 MPEI 10.75 2O O 6 0740. The experiments were performed using PGI, and an 2 MPEI 10.75 30 O 6.7 analog of PGI, Ciprostene, at a concentration of 2 mg/ml. 2 MPEI 10.75 2O 5 9 Cocktail solutions were mixed at room temperature, then 2 MPEI 10.75 2O 30 8 cooled by placing in a freezer. The chilled plates were trans 2 MPEI 10.75 2O 55 7 ferred onto pre-chilled (-45° C.) shelves of a Millrock Lab 2 MPEI 10.75 2O 70 6 Series Lyophilizer, and the vacuum was applied. The frozen cocktail solutions were allowed to lyophilize for 16 hours. 0741. Because the resulting prostaglandin microspheres Results: are hygroscopic, upon microsphere initiation, the humidity 0743. With PGI, several conditions were identified for was maintained at low levels during the experiments, using a good quality microsphere formation, with several ratings US 2012/O 141590 A1 Jun. 7, 2012 62 above 6 and a maximum rating of 8. When the buffer/coun although there was evidence that microspheres were begin terion was Arginine, lower concentrations of n-propanol and ning to form. The DAS181 cocktail showed high quality t-butanol favored better microsphere formation alcohol con microspheres, with a rating of 9, when flash frozen in the centrations, crystal formation was observed. When the buffer/ lyophilization bottle. The quality of the DAS181 micro counterion was TEA, on the other hand, higher concentra spheres, however, was reduced to a rating of 5 in the faster tions of t-butanol favored higher quality microsphere freezing PCR tube experiment; a significant amount of rod like crystals were observed, although there were some formation. When the buffer? counterion was PEI, the best microparticles present. With Tobacco Mosaic Virus, high quality microspheres were obtained at a higher concentration quality microspheres, with a rating of 9, were formed in both of n-propanol (30%), and in the absence oft-butanol. flash freeze cases. It therefore appears that the formation of 0744 Ciprostene is a more stable analog of PGI, and it Tobacco Mosaic Virus microspheres, under the conditions also appeared to be less hygroscopic. With Arginine buffer, no tested, was not highly affected by the rate of freezing. particular concentration-dependent trend was observed, but 0749. With Vancomycin, on the other hand, the quality of several Solvent conditions produced high quality micro the microspheres decreased as the freezing rate was spheres with ratings of 8 (see, e.g. 20% n-Propanol/5% t-bu increased. While the Vancomycin cocktail produced a micro tanol and 20% n-Propanol/55% t-butanol). With TEA, the sphere rating of 9/10 under normal freezing conditions, as quality of microspheres obtained with n-propanol in the described in Example 13, the 200 ul flash freeze sample absence of t-butanol was low. The quality of microspheres provided lower quality microspheres with a rating of 7 and increased as t-butanol was added to the cocktail solution, with observed aggregation. The PCR tube flash freeze produced a maximum at about 5% t-butanol. As the concentration of ever lower quality microspheres, with a rating of 5, higher t-butanol was increased even further, increasing amounts of amounts of aggregation and a significant amount of rod-like aggregation was observed. PEI proved to be the best counte crystals. Thus, in the case of Vancomycin, faster freeze rates rion for ciprostene, with a maximum microsphere quality resulted in lower quality microspheres. Similarly, with Tetra rating of 9 at 20% n-propanoV5cYO t-butanol. As the con cycline, while microsphere ratings of 8/9 were obtained under centration of t-butanol was further increased, increasing normal freezing conditions (see Example 14), both flash amounts of aggregation were observed. freeze conditions produced lower quality microspheres of 0745. The results demonstrate that high quality micro rating 5/6, with significant aggregation. 0750. The results demonstrate that the freezing rate can spheres of prostaglandin can be formed under a variety of have an impact on the quality of microspheres generated conditions, which should facilitate a stable formulation for according to the methods provided herein. The impact, how pulmonary delivery. ever, is dependent on the compound forming the micro spheres. As shown in this example, for Some compounds, Example 19 such as Paclitaxel, DAS181, Vancomycin and Tetracycline, if Effect of Cooling Rate on the Quality of Micro the freezing rate is too rapid, the microspheres can get trapped spheres in crystalline phases or aggregate before having the opportu nity to grow to a reasonable size. 0746. This example demonstrates that a controlled cooling rate, during which the cocktail solutions from which the Example 20 microspheres are produced are maintained at specific tem Efficiency of Nucleic Acid Incorporation into Micro peratures for defined periods of time, as opposed to flash spheres freezing, produces higher quality microspheres with desired 0751. To assess the process yield for nucleic acid incorpo characteristics. Flash freeze experiments were conducted ration into microspheres, the following experiment was con with five different cocktails that previously produced excel ducted. One mg of yeast tRNA (Sigma, Type X-SA) in 0.5 ml lent microspheres under standard freezing conditions per Volume (2 mg/ml final concentration in the cocktail) was formed according to the methods provided herein. The com combined with isopropanol (IPA: 40% final concentration) pound/counterion/antisolvent conditions were as follows: and sodium citrate (100 mM final concentration) at pH 8.0. 1) Paclitaxel/citrate pH 5.0/90% t-butanol (see Example 15) Formation of microparticles from the resulting cocktail was 2) DAS181/citrate pH 5/5% n-propanol (see Example 13) induced by placing the cocktail on ice. The microspheres 3) Tobacco Mosaic Virus/Na sulfate-Na acetate pH 4/5% were fixed by the addition of 10 ml (20 volumes) of IPA, and isopropanol (see Example 13) pelleted by centrifugation at 5000 rpm for 3 min. The pellet 4) Vancomycin/citrate pH 5/5% n-propanol (see Example 13) was dried in a vacuum. Microscopic analysis confirmed the 5) Tetracycline/Arginine/25-30% isopropanol (see Example formation of high quality microspheres, 1-2 micron in size, 14) and the absence of aggregated material or crystals. 0747 Experiments were performed with 200ul of each of (0752. The amount of tRNA recovered in the pellet and the the above cocktail solutions in a 2 ml lyophilization bottle supernatant was quantitated by UV absorption at 260 nm. It (first flash freeze condition), and 25 ul of each of the above was found that 78% of the tRNA was packaged into the cocktail solutions in a PCR tube (second flash freeze condi microparticles and 22% tRNA remained in the supernatant. tion). The samples in the lyophilization bottles took approxi This result demonstrated that tRNA, and likely other nucleic mately 15 seconds to freeze. The samples in the PCR tubes acids such as DNA and siRNA, can be efficiently condensed took approximately 3 seconds or less to freeze. and packaged into a microsphere formulation. Example 21 Results: siRNA that is Incorporated into Microspheres 0748 Microscopic analysis of the samples showed that in Retains its Activity most cases, the freezing rate has a significant effect on the 0753 Experiments were performed to assess if the method formation of microspheres. The Paclitaxel samples were of producing microspheres as provided herein inhibits the mostly crystalline in both cases after the flash freeze, activity of the molecules incorporated in the microspheres. US 2012/O 141590 A1 Jun. 7, 2012

(0754) Preparation of siRNA Microspheres 0755. The exemplary molecule used in this experiment is TABLE 20-continued double stranded GAPDH siRNA (sense sequence 5'-UGGU siRNA Gene Silencing Activity when used alone or when incorporated UUACAUGUUCCAAUAUU-3'(SEQID. NO: 27); antisense into microspheres. sequence 5'-UAUUGGAACAUGUAAACCAUU-3' (SEQ IDNO: 28); with two “UU” overhangs at each3'-end). Micro Sample spheres containing GAPDH siRNA in various cocktail for No. Formulation Cocktail % Activity mulations, as described below, were produced: 6 10 mM (Lysine, Citric acid, 1:1 molar ratio), 15% 13020 (0756) 1: 2 mM Arginine, pH 7.0, 15% IPA, 2 mg/ml IPA, 1 mg/ml siRNA siRNA 7 10 mM Alanine, 15% IPA, 1 mg/ml siRNA 125 - 20 4 neg Same as 4, but no siRNA (negative control) 35 (0757 2: 2 mMPEI (25,000 molwt, branched, Sigma), pH 5 neg Same as 5, but no siRNA (negative control) 11 : 12 10, 15% IPA, 2 mg/ml siRNA 6 neg Same as 6, but no siRNA (negative control) 3O3S (0758 3:2 mM ltaconic Acid, pH 8.0, 15% IPA, 2 mg/ml #7 neg Same as 7, but no siRNA (negative control) 31 34 siRNA GAPDH siRNA containing microspheres, generated by the methods described herein, were reconstituted in water to 10 uM siRNA. The negative controls are composed of each (0759 4: 10 mM (Glutamic acid, Lysine, Alanine, 3:2:5 formulation without the siRNA. For the positive control, lyophilized GAPDH siRNA was reconstituted to 10 uMsiRNA. Each siRNA sample was transfected into Hep-2 cells using molar ratio), 5% IPA, 1 mg/ml. lipid-based siPORT TMNeoFXTM transfection reagent (Applied Biosystems#AM4510). At 48 hr post transfection, GAPDH enzyme activity was measured using the KDalert TM 0760 5: 10 mM (Lysine, Citric acid, 1:4 molar ratio), 15% GAPDHAssay Kit (Applied Biosystems #AM1639). Fluorescence readings in the negative IPA, 1 mg/ml siRNA, controls (no siRNA used in transfections) were used to set the baseline. The changes of fluorescentreading in the positive controls (siRNA not subjected to lyophilization) were set 0761. 6: 10 mM (Lysine, Citric acid, 1:1 molar ratio), 15% as 100% activity for siRNA. IPA, 1 mg/ml siRNA 0762 7: 10 mM Alanine, 15% IPA, 1 mg/ml siRNA Example 22 Control formulations contained all cocktail ingredients with the exception of siRNA. A lyophilized siRNA control con Microspheres Containing Nucleic Acids as Active tained no excipients and 15% IPA. Agents and Gelatin as a Carrier 0763 The resulting cocktails were chilled to form micro spheres and frozen in a single step by placing the vial onto the 0766. This Example demonstrates that the methods pro shelf of a -80° C. freezer. Lyophilization was performed vided herein can be used to prepare microspheres containing overnight at shelf temperature of +10°C. and a vacuum of 150 gelatin, and the gelatin can act as a carrier for other active mTorr. agents in the microspheres. The gelatin-containing micro 0764 Activity of siRNA in Microsphere Formulations spheres are stable, and they retain their stability when nucleic 0765. The siRNA microspheres isolated from the lyo acids are incorporated along with the gelatin. Microspheres philization were then reconstituted and transfected to Hep-2 were prepared containing gelatin from a variety of sources as cells. As a positive control, the same amount of GAPDH follows: siRNA in the original buffer was lyophilized, reconstituted, 0767 A. Gelatin from bovine skin, Type B (Sigma, and transfected, without formation of microspheres. At 48 hr G9382) post transfection, the level of GAPDH in the Hep-2 cells was 0768 B. Gelatin from porcine skin, Type A (Sigma, measured using a fluorescent enzymatic assay. The results G2500) (Table 20) demonstrated that siRNAs processed into micro 0769 C. Gelatin from coldwater fish skin (Sigma, G.7041) spheres had gene-silencing activity that was equivalent to or, 0770 Preparation of microspheres containing gelatin: for in Some instances, even greater than that of the corresponding each of the gelatin compounds listed in A-C above, cocktail positive control (i.e., 100% or more gene-silencing activity). Solutions containing from 2.5 mg/ml to 25 mg/ml of gelatin Microscopic analyses confirmed the formation of hiah Qual dissolved in aqueous solvent, counter ions at different pH, ity microspheres. and IPA as antisolvent at different concentrations, as listed below, were prepared in a 96-well microtiter plate (0.1 ml TABLE 20 cocktail/well) at room temperature. The cocktails in the siRNA Gene Silencing Activity when used alone or when incorporated 96-well plates were cooled by placing in a freezer. The chilled into microSpheres. plates were transferred onto pre-chilled (-45° C.) shelves of a Millrock Lab Series Lyophilizer, and a vacuum was applied. Sample The frozen cocktail solutions were allowed to lyophilize for No. Formulation Cocktail % Activity 16 hours. Lyophilized siRNA positive control (no 10O2O microsphere) (0771. The lyophilized powders from the bottoms of the 1 2 mM Arginine, pH 7.0, 15% IPA, 2 mg/ml siRNA 7S 25 wells were transferred onto glass slides and analyzed by light 2 mM PEI (25,000 mol wit, branched, Sigma), 11S 15 microscopy for appearance. The quality of the product micro pH 10, 15% IPA, 2 mg/ml siRNA spheres was scored based on the uniformity of the micro 3 2 mM Itaconic Acid, pH 8.0, 15% IPA, 1102 spheres, the absence of undesirable non-microsphere par 2 mg/ml siRNA 1 neg Same as 1, but no siRNA (negative control) O ticles (glass-like crystalline forms), and the absence of 2 neg Same as 2, but no siRNA (negative control) O aggregates. The scoring system as described in Table 13 was 3 neg Same as 3, but no siRNA (negative control) O used. 4 10 mM (Glutamic acid, Lysine, Alanine, 3:2:5 13S 25 molar ratio), 5% IPA, 1 mg/ml siRNA 0772 Table 21 below shows the various combinations of 5 10 mM (Lysine, Citric acid, 1:4 molar ratio), 15% 12S25 compound, solvent, antisolvent and counterion that were IPA, 1 mg/ml siRNA used to generate microspheres, and the quality of the resulting microspheres. US 2012/O 141590 A1 Jun. 7, 2012 64

TABLE 21 TABLE 21-continued Gelatin Microspheres Gelatin Microspheres Concentration of Microsphere Concentration of Microsphere Compound Counterion Antisolvent pH Quality Compound Counterion Antisolven pH Quality Compound: Gelatin from 2.5 mg/m 20 mM Tris 20% 8 8 bovine skin, Type B isopropano 2.5 mg/m 20 mM Tris 30% 8 7 2.5 mg/ml 20 mM Citric Acid 10% 3.5 isopropano isopropano 5 mg/m 20 mM Tris 10% 8 5 2.5 mg/ml 20 mM Citric Acid 20% 3.5 isopropano isopropano 5 mg/m 20 mM Tris 30% 8 5 2.5 mg/ml 20 mM Citric Acid 30% 3.5 isopropano isopropano 10 mg/m 20 mM Tris 10% 8 1 10 mg/ml 20 mM Citric Acid 59 3.5 isopropano isopropano 10 mg/m 20 mM Tris 20% 8 5 10 mg/ml 20 mM Citric Acid 10% 3.5 isopropano isopropano 10 mg/m 20 mM Tris 30% 8 1 10 mg/ml 20 mM Citric Acid 20% 3.5 isopropano isopropano 25 mg/m 20 mM Tris 20% 8 9 10 mg/ml 20 mM Citric Acid 30% 3.5 isopropano isopropano 25 mg/m 20 mM Tris 30% 8 9 25 mg/ml 20 mM Citric Acid 30% 3.5 isopropano isopropano Compound: Gelatin from porcine skin, Type A 0773 Preparation of microspheres containing gelatin and 2.5 mg/m 20 mM Citric Aci 59 3.5 nucleic acids: for each of the three gelatin compounds listed isopropano in A-C above, cocktail solutions containing 15 mg/ml of 2.5 mg/m 20 mM Citric Aci 10% 3.5 isopropano gelatin and various concentrations of tRNA dissolved in 2.5 mg/m 20 mM Citric Aci 20% 3.5 aqueous solvent, with counterions at different pH, and IPA as isopropano antisolvent at different concentrations, as listed below, were 5 mg/m 20 mM Citric Aci 10% 3.5 isopropano prepared in a 96-well microtiterplate (0.1 ml cocktail?well) at 5 mg/m 20 mM Citric Aci 20% 3.5 room temperature. tRNA used in this experiment was type isopropano X-SA, from Bakers Yeast (Sigma, R8759). The cocktail solu 5 mg/m 20 mM Citric Aci 30% 3.5 tions were cooled by placing in a freezer. The chilled plates isopropano 10 mg/m 20 mM Citric Aci 20% 3.5 were transferred onto pre-chilled (-45° C.) shelves of a Mill isopropano rock Lab Series Lyophilizer, and a vacuum was applied. The 10 mg/m 20 mM Citric Aci 30% 3.5 frozen cocktail solutions were allowed to lyophilize for 16 isopropano hours. Compound: Gelatin from cold water fish skin (0774. The lyophilized powders from the bottoms of the wells were transferred onto glass slides and analyzed by light 2.5 mg/m 20 mM Citric Aci 59 3.5 isopropano microscopy for appearance. The quality of the product micro 2.5 mg/m 20 mM Citric Aci 10% 3.5 spheres was scored based on the uniformity of the micro isopropano spheres, the absence of undesirable non-microsphere par 2.5 mg/m 20 mM Citric Aci 20% 3.5 isopropano ticles (glass-like crystalline forms), and the absence of 2.5 mg/m 20 mM Citric Aci 30% 3.5 aggregates. isopropano 5 mg/m 20 mM Citric Aci 59 3.5 isopropano 5 mg/m 20 mM Citric Aci 10% 3.5 Concentration of Microsphere isopropano tRNA Counterion Antisolvent pH Quality 5 mg/m 20 mM Citric Aci 20% 3.5 isopropano Compound: Gelatin from bovine 5 mg/m 20 mM Citric Aci 30% in 3.5 skin, Type B with tRNA propanol 10 mg/m 20 mM Citric Aci 59 3.5 2 mg/ml 10 mM Citric Acid 10% 3.5 7 isopropano isopropanol 10 mg/m 20 mM Citric Aci 20% 3.5 2 mg/ml 10 mM Citric Acid 20% 3.5 5 isopropano isopropanol 10 mg/m 20 mM Citric Aci 30% 3.5 2 mg/ml 10 mM Citric Acid 30% 3.5 4 isopropano isopropanol 25 mg/m 20 mM Citric Aci 20% 3.5 2 mg/ml 10 mM Citric Acid 40% 3.5 5 isopropano isopropanol 25 mg/m 20 mM Citric Aci 30% 3.5 1 mg/ml 10 mM Citric Acid 10% 3.5 1 isopropano isopropanol 2.5 mg/m 20 mM Tris 59 1 mg/ml 10 mM Citric Acid 20% 3.5 3 isopropano isopropanol 2.5 mg/m 20 mM Tris 10% 1 mg/ml 10 mM Citric Acid 30% 3.5 5 isopropano isopropanol US 2012/O 141590 A1 Jun. 7, 2012 65

-continued -continued

Concentration of Microsphere Concentration of Microsphere tRNA Counterion Antisolven pH Quality tRNA Counterion Antisolven pH Quality 1 mg/m 0 mM Citric Acid 40% 3.5 5 2 mg/m OmM Citric Aci 40% 3.5 5 isopropano isopropano 0.5 mg/m 0 mM Citric Acid 10% 3.5 3 1 mg/m OmM Citric Aci 10% 3.5 4 isopropano isopropano 0.5 mg/m 0 mM Citric Acid 20% 3.5 1 mg/m OmM Citric Aci 20% 3.5 4 isopropano isopropano 0.5 mg/m 0 mM Citric Acid 30% 3.5 1 mg/m OmM Citric Aci 30% 3.5 6 isopropano isopropano 0.5 mg/m 0 mM Citric Acid 40% 3.5 1 mg/m OmM Citric Aci 40% 3.5 4 isopropano isopropano 0.1 mg/m 0 mM Citric Acid 30% 3.5 0.5 mg/m OmM Citric Aci 10% 3.5 10 isopropano isopropano 0.1 mg/m 0 mM Citric Acid 40% 3.5 0.5 mg/m OmM Citric Aci 20% 3.5 8 isopropano isopropano 2 mg/m 0 mM Tris 30% 0.5 mg/m OmM Citric Aci 30% 3.5 8 isopropano isopropano 2 mg/m 0 mM Tris 40% 0.5 mg/m OmM Citric Aci 40% 3.5 7 isopropano isopropano 1 mg/m 0 mM Tris 40% 0.1 mg/m OmM Citric Aci 20% 3.5 7 isopropano isopropano 0.5 mg/m 0 mM Tris 40% 0.1 mg/m OmM Citric Aci 30% 3.5 7 isopropano isopropano Compound: Gelatin from porcine 0.1 mg/m OmM Citric Aci 40% 3.5 7 skin, Type A with tRNA isopropano 2 mg/m 0 mM Tris 20% 8 6 2 mg/m OmM Citric Aci 10% 3.5 isopropano isopropano 2 mg/m 0 mM Tris 30% 8 5 2 mg/m OmM Citric Aci 20% 3.5 isopropano isopropano 2 mg/m 0 mM Tris 40% 8 6 2 mg/m OmM Citric Aci 30% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 10% 8 4 2 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 20% 8 5 1 mg/m OmM Citric Aci 10% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 30% 8 7 1 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 40% 8 4 0.5 mg/m OmM Citric Aci 10% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 10% 8 2 0.5 mg/m OmM Citric Aci 20% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 20% 8 8 0.5 mg/m OmM Citric Aci 30% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 30% 8 8 0.5 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 40% 8 4 0.1 mg/m OmM Citric Aci 30% 3.5 isopropano isopropano 0.1 mg/m 0 mM Tris 10% 8 2 0.1 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 0.1 mg/m 0 mM Tris 20% 8 4 2 mg/m 0 mM Tris 30% isopropano isopropano 0.1 mg/m 0 mM Tris 30% 8 3 2 mg/m 0 mM Tris 40% isopropano isopropano 0.1 mg/m 0 mM Tris 40% 8 5 1 mg/m 0 mM Tris 30% isopropano isopropano 1 mg/m 0 mM Tris 40% isopropano 0775. Results: These experiments demonstrate that by 0.5 mg/m 0 mM Tris 30% selecting the appropriate parameters, stable gelatin micro isopropano spheres can be obtained. Further, active agents such as nucleic 0.5 mg/m 0 mM Tris 40% isopropano acids can be incorporated into the gelatin matrix to produce a 0.1 mg/m 0 mM Tris 40% drug product with defined potency. isopropano Compound: Gelatin from cold Example 23 water fish skin with tRNA Preparation of Microspheres Using a Polysaccharide 2 mg/ml 10 mM Citric Acid 10% 3.5 as a Carrier isopropanol 2 mg/ml 10 mM Citric Acid 20% 3.5 0776. This Example demonstrates that the methods pro isopropanol vided herein can be used to prepare microspheres containing 2 mg/ml 10 mM Citric Acid 30% 3.5 polysaccharides. The polysaccharides in turn can be carriers isopropanol for therapeutic agents or active agents incorporated into the microspheres. The following compounds were tested: US 2012/O 141590 A1 Jun. 7, 2012 66

(0777 A) Dextran Sulfate Sodium Salt (Sigma, D 6924) 0778 B) Hydroxypropyl-B-cyclodextrin (Tokyo Chemi -continued cal Industry Co., Ltd, HO979) Concentration Microsphere 0779 Preparation of microspheres: for the compounds A) of Compound Counterion Antisolven pH Quality and B) above, cocktail solutions containing from 0.5 mg/ml to 10 mg/m 10 mM Citric Aci 30% 5.2 1 10 mg/ml of compound, with counterions at different pH, and isopropano IPA as antisolvent at different concentrations, as listed below, 5 mg/m 10 mM Citric Aci 59 5.2 2 isopropano were prepared in a 96-well microtiter plate (0.1 ml cocktail/ 5 mg/m 10 mM Citric Aci 10% 5.2 3 well) at room temperature. Cocktails were cooled by placing isopropano in a freezer. The chilled plates were transferred onto pre 5 mg/m 10 mM Citric Aci 20% 5.2 1 chilled (-45° C.) shelves of a Millrock Lab Series Lyo isopropano 10 mg/m 30 mM Citric Aci 20% 5.2 1 philizer, and a vacuum was applied. The frozen cocktail solu isopropano tions were allowed to lyophilize for 16 hours. 10 mg/m 30 mM Citric Aci 30% 5.2 1 0780. The lyophilized powders from the bottoms of the isopropano 5 mg/m 30 mM Citric Aci 59 5.2 3 wells were transferred onto glass slides and analyzed by light isopropano microscopy for appearance. The quality of the product micro 5 mg/m 30 mM Citric Aci 10% 5.2 1 spheres was scored based on the uniformity of the micro isopropano spheres, the absence of undesirable non-microsphere par 5 mg/m 30 mM Citric Aci 20% 8 1 ticles (glass-like crystalline forms), and the absence of isopropano 5 mg/m 30 mM Citric Aci 30% 5.2 3 aggregates. isopropano 10 mg/m 30 mM Tris 59 8 3 isopropano 10 mg/m 30 mM Tris 10% 8 1 Concentration Microsphere isopropano of Compound Counterion Antisolvent pH Quality 10 mg/m 30 mM Tris 20% 8 3 isopropano Compound: Dextran 10 mg/m 30 mM Tris 30% 8 4 Sulfate isopropano 5 mg/m 30 mM Tris 59 8 3 5 mg/m OmM Citric Acid 59 3.5 4 isopropano isopropano 5 mg/m 30 mM Tris 20% 8 4 5 mg/m 0 mM Citric Acid 10% 3.5 5 isopropano isopropano 5 mg/m 30 mM Tris 30% 8 2 5 mg/m 0 mM Citric Acid 20% 3.5 2 isopropano isopropano 10 mg/m 0 mM Tris 59 2 5 mg/m 0 mM Citric Acid 30% 3.5 6 isopropano isopropano 10 mg/m 0 mM Tris 30% 1 1 mg/m OmM Citric Acid 59 3.5 2 isopropano isopropano 5 mg/m 0 mM Tris 59 2 1 mg/m 0 mM Citric Acid 10% 3.5 4 isopropano isopropano 5 mg/m 0 mM Tris 10% 5 1 mg/m 0 mM Citric Acid 20% 3.5 3 isopropano isopropano 5 mg/m 0 mM Tris 20% 5 5 mg/m 0 mM Tris 59 8 2 isopropano isopropano 5 mg/m 0 mM Tris 30% 2 5 mg/m 0 mM Tris 10% 8 2 isopropano isopropano 1 mg/m 0 mM Tris 59 5 5 mg/m 0 mM Tris 20% 8 2 isopropano isopropano 1 mg/m 0 mM Tris 10% 4 10 mg/m 30 mM Citric Aci 59 3.5 2 isopropano isopropano 1 mg/m 0 mM Tris 20% 4 10 mg/m 30 mM Citric Aci 10% 3.5 3 isopropano isopropano 1 mg/m 0 mM Tris 30% 3 10 mg/m 30 mM Citric Aci 20% 3.5 2 isopropano isopropano 10 mg/m 30 mM Tris 10% 1 5 mg/m 30 mM Citric Aci 59 3.5 2 isopropano isopropano 10 mg/m 30 mM Tris 30% 5 5 mg/m 30 mM Citric Aci 10% 3.5 3 isopropano isopropano 5 mg/m 30 mM Tris 59 1 5 mg/m 30 mM Citric Aci 20% 3.5 2 isopropano isopropano 5 mg/m 30 mM Tris 30% 2 5 mg/m 30 mM Citric Aci 30% 3.5 2 isopropano isopropano Compound: 1 mg/m 30 mM Citric Aci 59 3.5 2 isopropano Hydroxypropyl-B- 1 mg/m 30 mM Citric Aci 20% 3.5 1 cyclodextrin isopropano 10 mg/m 10 mM Citric Aci 59 5.2 1 5 mg/ml 10 mM Citric Acid 59 3.5 1 isopropano isopropanol 10 mg/m 10 mM Citric Aci 10% 5.2 4 5 mg/ml 10 mM Citric Acid 20% 3.5 2 isopropano isopropanol 10 mg/m 10 mM Citric Aci 20% 5.2 2 5 mg/ml 10 mM Citric Acid 30% 3.5 1 isopropano isopropanol US 2012/O 141590 A1 Jun. 7, 2012 67

-continued TABLE 24 Concentration Microsphere Amino Acid Microspheres of Compound Counterion Antisolvent pH Quality Amino Acids: 1 mg/ml 10 mM Citric Acid 10% 3.5 1 isopropanol Antisolvent pH Microsphere Quality 1 mg/ml 10 mM Citric Acid 20% 3.5 4 isopropanol Compound: Alanine 1 mg/ml 10 mM Citric Acid 30% 3.5 5 isopropanol 5% isopropano 7 9 0.5 mg/ml 10 mM Citric Acid 59 3.5 2 10% isopropano 7 2 isopropanol 20% isopropano 7 8 5 mg/ml 10 mM Tris 20% 8 1 30% isopropano 7 5 isopropanol 5% isopropano 6 7 Compound: Glutamic Acid

5% isopropano 3.2 6 Results: These experiments demonstrate that by selecting the 10% isopropano 3.2 3 appropriate combination of: (a) type and (b) concentration of 20% isopropano 3.2 6 compound, counterion and antisolvent, polysaccharide Compound: Tryptophan microspheres can be obtained. 5% isopropano 7 5 10% isopropano 7 4 Example 24 20% isopropano 7 7 30% isopropano 7 4 20% isopropano 6 2 Amino Acid Microspheres 30% isopropano 6 7 Compound: Methionine 0781. This Example demonstrates that the methods pro 5% isopropano 7 3 vided herein can be used to prepare microspheres containing 10% isopropano 7 4 various amino acids, which could be active agents or thera 20% isopropano 7 1 peutic agents themselves, or serve as carriers for other active 30% isopropano 7 2 agents and therapeutic agents. Microspheres of the following 5% isopropano 5.7 2 10% isopropano 5.7 7 amino acids were prepared: 30% isopropano 5.7 1 0782. A. Alanine Compound: Phenylalanine

0783 B. Glutamic Acid 10% isopropano 7 6 0784 C. Tryptophan 20% isopropano 7 8 0785. D. Methionine 30% isopropano 7 5 5% isopropano 5.5 6 0786 E. Phenylalanine 10% isopropano 5.5 5 0787 F. Glycine 20% isopropano 5.5 3 30% isopropano 5.5 7 0788 G. Lycine Compound: Glycine 0789 Preparation of amino acid microspheres: for each of 5% isopropano 7 7 the compounds listed in A-G above, cocktail solutions con 10% isopropano 7 6 taining 20 mM amino acid dissolved in aqueous solvent, at 20% isopropano 7 4 different pH, and ispropanol (IPA) as antisolvent at different 30% isopropano 7 4 concentrations, as listed below, were prepared in a 96-well 5% isopropano 6 5 microtiter plate (0.1 ml cocktail/well) at room temperature. 10% isopropano 6 5 20% isopropano 6 3 Cocktails were cooled by placing in a freezer. The chilled 30% isopropano 6 6 plates were transferred onto pre-chilled (-45° C.) shelves of Compound: Lysine a Millrock Lab Series Lyophilizer, and the vacuum was applied. The frozen cocktail solutions were allowed to lyo 5% isopropano 7 3 philize for 16 hours. 20% isopropano 7 7 30% isopropano 7 3 0790. The lyophilized powders from the bottoms of the 5% isopropano 5.5 4 wells were transferred onto glass slides and analyzed by light 10% isopropano 5.5 5 microscopy for appearance. The quality of the product micro 30% isopropano 5.5 7 spheres was scored based on the uniformity of the micro spheres, the absence of undesirable non-microsphere par ticles (glass-like crystalline forms), and the absence of Results: These experiments demonstrate that by selecting the aggregates. The scoring system as described in Table 13 was appropriate combination of: (a) type and (b) concentration of used. amino acid, counterion and antisolvent, microspheres made 0791 Table 24 below shows the various combinations of of amino acids can be obtained. compound, Solvent, and antisolvent that were used to gener 0792. Since modifications will be apparent to those of skill ate microspheres, and the quality of the resulting micro in this art, it is intended that this invention be limited only by spheres. the scope of the appended claims. US 2012/O 141590 A1 Jun. 7, 2012 68

SEQUENCE LISTING

< 16 Os NUMBER OF SEO ID NOS: 28

SEQ ID NO 1 LENGTH: 901 TYPE : PRT ORGANISM: Actinomyces viscosus

<4 OOs, SEQUENCE: 1.

Met Thir Ser His Ser Pro Phe Ser Arg Arg Arg Lell Pro Ala Luell Luell 1. 5 15

Gly Ser Luell Pro Lell Ala Ala Thir Gly Luell Ile Ala Ala Ala Pro Pro 25 3O

Ala His Ala Wall Pro Thir Ser Asp Gly Luell Ala Asp Wall Thir Ile Thir 35 4 O 45

Glin Wall Asn Ala Pro Ala Asp Gly Luell Tyr Ser Wall Gly Asp Wall Met SO 55 6 O

Thir Phe Asn Ile Thir Lell Thir Asn Thir Ser Gly Glu Ala His Ser Tyr 65 70

Ala Pro Ala Ser Thir Asn Lell Ser Gly Asn Wall Ser Arg Trp 85 90 95

Arg Asn Wall Pro Ala Gly Thir Thir Lys Thir Asp Thir Gly Luell Ala 105 11 O

Thir His Thir Wall Thir Ala Glu Asp Luell Ala Gly Gly Phe Thir Pro 115 12 O 125

Glin Ile Ala Glu Wall Lys Ala Wall Glu Tyr Ala Gly Ala Luell 13 O 135 14 O

Ser Thir Pro Glu Thir Ile Gly Ala Thir Ser Pro Wall Ala Asn 145 150 155 160

Ser Luell Arg Wall Glu Ser Ile Thir Pro Ser Ser Ser Glin Glu Asn Tyr 1.65 17O 17s

Luell Gly Asp Thir Wall Ser Thir Wall Arg Wall Arg Ser Wall Ser 18O 185 19 O

Asp Thir Ile Asn Wall Ala Ala Thir Glu Ser Ser Phe Asp Asp Luell 195

Gly Arg Glin His Trp Gly Gly Luell Pro Gly Lys Gly Ala Wall 21 O 215 22O

Tyr Asn Pro Lell Thir His Thir Ile Thir Glin Ala Asp Wall Asp 225 23 O 235 24 O

Ala Gly Arg Trp Thir Pro Ser Ile Thir Luell Thir Ala Thir Gly Thir Asp 245 250 255

Gly Ala Thir Luell Glin Thir Lell Thir Ala Thir Gly Asn Pro Ile Asn Wall 26 O 265 27 O

Wall Gly Asp His Pro Glin Ala Thir Pro Ala Pro Ala Pro Asp Ala Ser 27s 285

Thir Glu Luell Pro Ala Ser Met Ser Glin Ala Glin His Lell Ala Ala Asn 29 O 295 3 OO

Thir Ala Thir Asp Asn Tyr Arg Ile Pro Ala Ile Thir Thir Ala Pro Asn 3. OS 310 315

Gly Asp Luell Luell Ile Ser Asp Glu Arg Pro Asp Asn Gly Asn 3.25 330 335

Gly Gly Ser Asp Ala Pro Asn Pro Asn His Ile Wall Glin Arg Arg Ser US 2012/O 141590 A1 Jun. 7, 2012 69

- Continued

34 O 345 35. O Thr Asp Gly Gly Lys Thir Trp Ser Ala Pro Thr Tyr Ile His Glin Gly 355 360 365 Thr Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val 37 O 375 38O Asp His Glin Thr Gly Thr Ile Phe Asin Phe His Val Lys Ser Tyr Asp 385 390 395 4 OO Glin Gly Trp Gly Gly Ser Arg Gly Gly. Thir Asp Pro Glu Asn Arg Gly 4 OS 41O 415 Ile Ile Glin Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thir Trp 42O 425 43 O Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Llys Pro Trp Thr 435 44 O 445 Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin Ile Gln His Gly Pro 450 45.5 460 His Ala Gly Arg Lieu Val Glin Glin Tyr Thir Ile Arg Thr Ala Gly Gly 465 470 47s 48O Ala Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Lys Thir Trip 485 490 495 Glin Ala Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Llys Val Val SOO 505 51O Glu Lieu. Ser Asp Gly Ser Lieu Met Lieu. ASn Ser Arg Ala Ser Asp Gly 515 52O 525 Ser Gly Phe Arg Llys Val Ala His Ser Thr Asp Gly Gly Glin Thir Trp 53 O 535 54 O Ser Glu Pro Val Ser Asp Lys Asn Lieu Pro Asp Ser Val Asp Asn Ala 5.45 550 555 560 Glin Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala 565 st O sts Llys Val Lieu. Lieu Lleu Ser His Ser Pro Asn Pro Arg Pro Trp Ser Arg 58O 585 59 O Asp Arg Gly. Thir Ile Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thr 595 6OO 605 Thir Ser Llys Val Phe His Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala 610 615 62O Val Glin Ser Asp Gly Ser Ile Gly Lieu. Lieu. Ser Glu Asp Ala His Asn 625 630 635 64 O Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn Trp 645 650 655 Lieu. Gly Glu Gln Cys Gly Glin Llys Pro Ala Glu Pro Ser Pro Ala Pro 660 665 67 O Ser Pro Thr Ala Ala Pro Ser Ala Ala Pro Thr Glu Lys Pro Ala Pro 675 68O 685

Ser Ala Ala Pro Ser Ala Glu Pro Thr Glin Ala Pro Ala Pro Ser Ser 69 O. 695 7 OO

Ala Pro Glu Pro Ser Ala Ala Pro Glu Pro Ser Ser Ala Pro Ala Pro 7 Os 71O 71s 72O

Glu Pro Thir Thir Ala Pro Ser Thr Glu Pro Thr Pro Ala Pro Ala Pro 72 73 O 73 Ser Ser Ala Pro Glu Gln Thr Asp Gly Pro Thr Ala Ala Pro Ala Pro 740 74. 7 O US 2012/O 141590 A1 Jun. 7, 2012 70

- Continued

Glu Thir Ser Ser Ala Pro Ala Ala Glu Pro Thr Glin Ala Pro Thir Wall 7ss 760 765 Ala Pro Ser Val Glu Pro Thr Glin Ala Pro Gly Ala Gln Pro Ser Ser 770 775 78O Ala Pro Llys Pro Gly Ala Thr Gly Arg Ala Pro Ser Val Val Asn Pro 78s 79 O 79. 8OO Lys Ala Thr Gly Ala Ala Thr Glu Pro Gly Thr Pro Ser Ser Ser Ala 805 810 815 Ser Pro Ala Pro Ser Arg Asn Ala Ala Pro Thr Pro Llys Pro Gly Met 82O 825 83 O Glu Pro Asp Glu Ile Asp Arg Pro Ser Asp Gly Thr Met Ala Glin Pro 835 84 O 845 Thr Gly Gly Ala Ser Ala Pro Ser Ala Ala Pro Thr Glin Ala Ala Lys 850 855 860 Ala Gly Ser Arg Lieu. Ser Arg Thr Gly Thr Asn Ala Lieu. Lieu. Ile Lieu 865 87O 87s 88O Gly Lieu Ala Gly Val Ala Val Val Gly Gly Tyr Lieu. Lieu. Lieu. Arg Ala 885 890 895 Arg Arg Ser Lys Asn 9 OO

<210 SEQ ID NO 2 &211s LENGTH: 394 212. TYPE: PRT <213> ORGANISM: Actinomyces viscosus <4 OOs, SEQUENCE: 2 Gly Asp His Pro Glin Ala Thr Pro Ala Pro Ala Pro Asp Ala Ser Thr 1. 5 1O 15

Glu Lieu Pro Ala Ser Met Ser Glin Ala Gln His Lieu Ala Ala Asn. Thr 2O 25 3O Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile Thir Thr Ala Pro Asn Gly 35 4 O 45 Asp Lieu. Lieu. Ile Ser Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly SO 55 6 O Gly Ser Asp Ala Pro Asn Pro Asn His Ile Val Glin Arg Arg Ser Thr 65 70 7s 8O Asp Gly Gly Lys Thr Trp Ser Ala Pro Thr Tyr Ile His Glin Gly Thr 85 90 95 Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp 1OO 105 11 O His Glin Thr Gly Thr Ile Phe Asin Phe His Val Lys Ser Tyr Asp Glin 115 12 O 125 Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg Gly Ile 13 O 135 14 O Ile Glin Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thir Trp Thr 145 150 155 160 His Arg Thir Ile Thr Ala Asp Ile Thir Lys Asp Llys Pro Trp Thir Ala 1.65 17O 17s Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin Ile Gln His Gly Pro His 18O 185 19 O Ala Gly Arg Lieu Val Glin Glin Tyr Thir Ile Arg Thr Ala Gly Gly Ala US 2012/O 141590 A1 Jun. 7, 2012 71

- Continued

195 2OO 2O5 Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Llys Thir Trp Glin 21 O 215 22O Ala Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Llys Val Val Glu 225 23 O 235 24 O Lieu. Ser Asp Gly Ser Lieu Met Lieu. Asn. Ser Arg Ala Ser Asp Gly Ser 245 250 255 Gly Phe Arg Llys Val Ala His Ser Thr Asp Gly Gly Glin Thr Trp Ser 26 O 265 27 O Glu Pro Val Ser Asp Lys Asn Lieu Pro Asp Ser Val Asp Asn Ala Glin 27s 28O 285 Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala Lys 29 O 295 3 OO Val Lieu. Lieu. Lieu. Ser His Ser Pro Asn Pro Arg Pro Trp Ser Arg Asp 3. OS 310 315 32O Arg Gly. Thir Ile Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thir Thr 3.25 330 335 Ser Llys Val Phe His Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala Val 34 O 345 35. O Glin Ser Asp Gly Ser Ile Gly Lieu. Lieu. Ser Glu Asp Ala His Asn Gly 355 360 365 Ala Asp Tyr Gly Gly Ile Trp Tyr Arg ASn Phe Thr Met ASn Trp Lieu. 37 O 375 38O Gly Glu Gln Cys Gly Glin Llys Pro Ala Glu 385 390

<210s, SEQ ID NO 3 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 Asn Gly Arg Arg Ile Cys Lieu. Asp Lieu. Glin Ala Pro Lieu. Tyr Lys Llys 1. 5 1O 15 Ile Ile Llys Llys Lieu. Lieu. Glu Ser 2O

<210s, SEQ ID NO 4 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 4 Gly Arg Glu Lieu. Cys Lieu. Asp Pro Lys Glu Asn Trp Val Glin Arg Val 1. 5 1O 15 Val Glu Lys Phe Lieu Lys Arg Ala Glu Asn. Ser 2O 25

<210s, SEQ ID NO 5 &211s LENGTH: 34 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 5 Glin Ile His Phe Phe Phe Ala Lys Lieu. Asn. Cys Arg Lieu. Tyr Arg Llys 1. 5 1O 15 US 2012/O 141590 A1 Jun. 7, 2012 72

- Continued

Ala Asn Llys Ser Ser Llys Lieu Val Ser Ala Asn Arg Lieu. Phe Gly Asp 2O 25 3O

Llys Ser

<210s, SEQ ID NO 6 &211s LENGTH: 34 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6 Glu Lieu. Arg Val Arg Lieu Ala Ser His Lieu. Arg Llys Lieu. Arg Lys Arg 1. 5 1O 15 Lieu. Lieu. Arg Asp Ala Asp Asp Lieu. Glin Lys Arg Lieu Ala Val Tyr Glin 2O 25 3O Ala Gly

<210s, SEQ ID NO 7 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 Arg Arg Lieu. Arg Arg Met Glu Ser Glu Ser Glu Ser 1. 5 1O

<210s, SEQ ID NO 8 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 8 Lys Arg Llys Llys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg 1. 5 1O 15 Llys Llys Lys Asn. Pro 2O

<210s, SEQ ID NO 9 &211s LENGTH: 4 OO 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 9 Met Gly Glu Lieu Pro Ala Ser Met Ser Glin Ala Glin His Lieu Ala Ala 1. 5 1O 15 Asn Thr Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile Thr Thr Ala Pro 2O 25 3O Asn Gly Asp Lieu. Lieu. Ile Ser Tyr Asp Glu Arg Pro Lys Asp Asn Gly 35 4 O 45 Asn Gly Gly Ser Asp Ala Pro Asn Pro Asn His Ile Val Glin Arg Arg SO 55 6 O Ser Thr Asp Gly Gly Lys Thr Trp Ser Ala Pro Thr Tyr Ile His Glin 65 70 7s 8O Gly Thr Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val 85 90 95 Val Asp His Glin Thr Gly. Thir Ile Phe Asin Phe His Val Lys Ser Tyr US 2012/O 141590 A1 Jun. 7, 2012 73

- Continued

1OO 105 11 O Asp Glin Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg 115 12 O 125 Gly Ile Ile Glin Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thr 13 O 135 14 O Trp. Thir His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Llys Pro Trp 145 150 155 160 Thir Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin Ile Gln His Gly 1.65 17O 17s Pro His Ala Gly Arg Lieu Val Glin Glin Tyr Thr Ile Arg Thr Ala Gly 18O 185 19 O Gly Ala Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Llys Thr 195 2OO 2O5 Trp Glin Ala Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Llys Val 21 O 215 22O Val Glu Lieu. Ser Asp Gly Ser Lieu Met Lieu. Asn. Ser Arg Ala Ser Asp 225 23 O 235 24 O Gly Ser Gly Phe Arg Llys Val Ala His Ser Thr Asp Gly Gly Glin Thr 245 250 255 Trp Ser Glu Pro Val Ser Asp Lys Asn Lieu Pro Asp Ser Val Asp Asn 26 O 265 27 O Ala Glin Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg 27s 28O 285 Ala Lys Val Lieu Lleu Lleu Ser His Ser Pro Asn Pro Arg Pro Trp Ser 29 O 295 3 OO Arg Asp Arg Gly. Thir Ile Ser Met Ser Cys Asp Asp Gly Ala Ser Trip 3. OS 310 315 32O Thir Thr Ser Llys Val Phe His Glu Pro Phe Val Gly Tyr Thr Thir Ile 3.25 330 335 Ala Val Glin Ser Asp Gly Ser Ile Gly Lieu. Lieu. Ser Glu Asp Ala His 34 O 345 35. O Asn Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn 355 360 365 Trp Lieu. Gly Glu Glin Cys Gly Glin Llys Pro Ala Lys Arg Llys Llys Llys 37 O 375 38O Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg Llys Llys Lys Asn Pro 385 390 395 4 OO

<210s, SEQ ID NO 10 &211s LENGTH: 422 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 10 Met Val Lys Arg Llys Llys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg 1. 5 1O 15 Asn Arg Llys Llys Lys Asn Pro Gly Gly Gly Gly Ser Gly Asp His Pro 2O 25 3O Glin Ala Thr Pro Ala Pro Ala Pro Asp Ala Ser Thr Glu Lieu Pro Ala 35 4 O 45 Ser Met Ser Glin Ala Glin His Lieu Ala Ala Asn. Thir Ala Thir Asp Asn US 2012/O 141590 A1 Jun. 7, 2012 74

- Continued

SO 55 6 O Tyr Arg Ile Pro Ala Ile Thir Thr Ala Pro Asn Gly Asp Lieu. Lieu. Ile 65 70 7s 8O Ser Tyr Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly Gly Ser Asp Ala 85 90 95 Pro Asn Pro Asn His Ile Val Glin Arg Arg Ser Thr Asp Gly Gly Lys 1OO 105 11 O Thir Trp Ser Ala Pro Thr Tyr Ile His Glin Gly Thr Glu Thr Gly Lys 115 12 O 125 Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp His Glin Thr Gly 13 O 135 14 O Thir Ile Phe Asin Phe His Val Lys Ser Tyr Asp Gln Gly Trp Gly Gly 145 150 155 160 Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg Gly Ile Ile Glin Ala Glu 1.65 17O 17s Val Ser Thr Ser Thr Asp Asn Gly Trp Thir Trp Thr His Arg Thr Ile 18O 185 19 O Thir Ala Asp Ile Thr Lys Asp Llys Pro Trp Thr Ala Arg Phe Ala Ala 195 2OO 2O5 Ser Gly Glin Gly Ile Glin Ile Glin His Gly Pro His Ala Gly Arg Lieu. 21 O 215 22O Val Glin Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala Val Glin Ala Val 225 23 O 235 24 O Ser Val Tyr Ser Asp Asp His Gly Llys Thr Trp Glin Ala Gly Thr Pro 245 250 255 Ile Gly Thr Gly Met Asp Glu Asn Llys Val Val Glu Lieu. Ser Asp Gly 26 O 265 27 O Ser Lieu Met Lieu. Asn. Ser Arg Ala Ser Asp Gly Ser Gly Phe Arg Llys 27s 28O 285 Val Ala His Ser Thr Asp Gly Gly Glin Thr Trp Ser Glu Pro Val Ser 29 O 295 3 OO Asp Lys Asn Lieu Pro Asp Ser Val Asp Asn Ala Glin Ile Ile Arg Ala 3. OS 310 315 32O Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala Lys Val Lieu. Lieu. Lieu. 3.25 330 335 Ser His Ser Pro Asn Pro Arg Pro Trp Ser Arg Asp Arg Gly. Thir Ile 34 O 345 35. O Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thr Thr Ser Llys Val Phe 355 360 365 His Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala Val Glin Ser Asp Gly 37 O 375 38O Ser Ile Gly Lieu Lleu Ser Glu Asp Ala His Asn Gly Ala Asp Tyr Gly 385 390 395 4 OO Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn Trp Lieu. Gly Glu Glin Cys 4 OS 41O 415 Gly Glin Llys Pro Ala Glu 42O

<210s, SEQ ID NO 11 &211s LENGTH: 415 212. TYPE: PRT <213> ORGANISM: Artificial Sequence US 2012/O 141590 A1 Jun. 7, 2012 75

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 11 Met Gly Glu Lieu Pro Ala Ser Met Ser Glin Ala Glin His Lieu Ala Ala 1. 5 1O 15 Asn Thr Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile Thr Thr Ala Pro 2O 25 3O Asn Gly Asp Lieu. Lieu. Ile Ser Tyr Asp Glu Arg Pro Lys Asp Asn Gly 35 4 O 45 Asn Gly Gly Ser Asp Ala Pro Asn Pro Asn His Ile Val Glin Arg Arg SO 55 6 O Ser Thr Asp Gly Gly Lys Thr Trp Ser Ala Pro Thr Tyr Ile His Glin 65 70 7s 8O Gly Thr Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val 85 90 95 Val Asp His Glin Thr Gly. Thir Ile Phe Asin Phe His Val Lys Ser Tyr 1OO 105 11 O Asp Glin Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg 115 12 O 125 Gly Ile Ile Glin Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thr 13 O 135 14 O Trp Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Llys Pro Trp 145 150 155 160 Thir Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin Ile Gln His Gly 1.65 17O 17s Pro His Ala Gly Arg Lieu Val Glin Glin Tyr Thr Ile Arg Thr Ala Gly 18O 185 19 O Gly Ala Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Llys Thr 195 2OO 2O5 Trp Glin Ala Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Llys Val 21 O 215 22O Val Glu Lieu. Ser Asp Gly Ser Lieu Met Lieu. Asn. Ser Arg Ala Ser Asp 225 23 O 235 24 O Gly Ser Gly Phe Arg Llys Val Ala His Ser Thr Asp Gly Gly Glin Thr 245 250 255 Trp Ser Glu Pro Val Ser Asp Lys Asn Lieu Pro Asp Ser Val Asp Asn 26 O 265 27 O Ala Glin Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg 27s 28O 285 Ala Lys Val Lieu Lleu Lleu Ser His Ser Pro Asn Pro Arg Pro Trp Ser 29 O 295 3 OO Arg Asp Arg Gly. Thir Ile Ser Met Ser Cys Asp Asp Gly Ala Ser Trip 3. OS 310 315 32O Thir Thr Ser Llys Val Phe His Glu Pro Phe Val Gly Tyr Thr Thir Ile 3.25 330 335 Ala Val Glin Ser Asp Gly Ser Ile Gly Lieu. Lieu. Ser Glu Asp Ala His 34 O 345 35. O Asn Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn 355 360 365 Trp Lieu. Gly Glu Glin Cys Gly Glin Llys Pro Ala Glu Pro Ser Pro Ala 37 O 375 38O US 2012/O 141590 A1 Jun. 7, 2012 76

- Continued

Pro Ser Pro Thir Ala Ala Pro Ser Ala Ala Lys Arg Llys Llys Lys Gly 385 390 395 4 OO Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg Llys Llys Lys Asn Pro 4 OS 41O 415

<210s, SEQ ID NO 12 &211s LENGTH: 404 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 12 Met Gly His His His His His His Leu Glu Gly Asp His Pro Glin Ala 1. 5 1O 15 Thr Pro Ala Pro Ala Pro Asp Ala Ser Thr Glu Lieu Pro Ala Ser Met 2O 25 3O Ser Glin Ala Gln His Lieu Ala Ala Asn. Thir Ala Thr Asp Asn Tyr Arg 35 4 O 45 Ile Pro Ala Ile Thr Thr Ala Pro Asn Gly Asp Leu Lieu. Ile Ser Tyr SO 55 6 O Asp Glu Arg Pro Lys Asp Asn Gly Asn Gly Gly Ser Asp Ala Pro Asn 65 70 7s 8O Pro Asn His Ile Val Glin Arg Arg Ser Thr Asp Gly Gly Lys Thir Trp 85 90 95 Ser Ala Pro Thr Tyr Ile His Glin Gly Thr Glu Thr Gly Lys Llys Val 1OO 105 11 O Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp His Glin Thr Gly Thr Ile 115 12 O 125 Phe Asin Phe His Val Lys Ser Tyr Asp Glin Gly Trp Gly Gly Ser Arg 13 O 135 14 O Gly Gly. Thir Asp Pro Glu Asn Arg Gly Ile Ile Glin Ala Glu Val Ser 145 150 155 160 Thir Ser Thr Asp Asn Gly Trp Thir Trp Thr His Arg Thr Ile Thr Ala 1.65 17O 17s Asp Ile Thir Lys Asp Llys Pro Trp Thir Ala Arg Phe Ala Ala Ser Gly 18O 185 19 O Glin Gly Ile Glin Ile Gln His Gly Pro His Ala Gly Arg Lieu Val Glin 195 2OO 2O5 Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala Val Glin Ala Val Ser Val 21 O 215 22O Tyr Ser Asp Asp His Gly Lys Thr Trp Glin Ala Gly Thr Pro Ile Gly 225 23 O 235 24 O Thr Gly Met Asp Glu Asn Llys Val Val Glu Lieu. Ser Asp Gly Ser Lieu. 245 250 255 Met Lieu. Asn. Ser Arg Ala Ser Asp Gly Ser Gly Phe Arg Llys Val Ala 26 O 265 27 O His Ser Thr Asp Gly Gly Glin Thr Trp Ser Glu Pro Val Ser Asp Llys 27s 28O 285 Asn Lieu Pro Asp Ser Val Asp Asn Ala Glin Ile Ile Arg Ala Phe Pro 29 O 295 3 OO Asn Ala Ala Pro Asp Asp Pro Arg Ala Lys Val Lieu Lleu Lieu. Ser His 3. OS 310 315 32O US 2012/O 141590 A1 Jun. 7, 2012 77

- Continued

Ser Pro Asn Pro Arg Pro Trp Ser Arg Asp Arg Gly Thr Ile Ser Met 3.25 330 335 Ser Cys Asp Asp Gly Ala Ser Trp Thir Thr Ser Lys Val Phe His Glu 34 O 345 35. O Pro Phe Val Gly Tyr Thr Thr Ile Ala Val Glin Ser Asp Gly Ser Ile 355 360 365 Gly Lieu. Lieu. Ser Glu Asp Ala His Asn Gly Ala Asp Tyr Gly Gly Ile 37 O 375 38O Trp Tyr Arg Asn Phe Thr Met Asn Trp Leu Gly Glu Gln Cys Gly Glin 385 390 395 4 OO Llys Pro Ala Glu

<210s, SEQ ID NO 13 &211s LENGTH: 416 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 13 Met Lys Arg Llys Llys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn 1. 5 1O 15 Arg Llys Llys Lys Asn Pro Gly Asp His Pro Glin Ala Thr Pro Ala Pro 2O 25 3O Ala Pro Asp Ala Ser Thr Glu Lieu Pro Ala Ser Met Ser Glin Ala Glin 35 4 O 45 His Lieu Ala Ala Asn. Thir Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile SO 55 6 O Thir Thr Ala Pro Asn Gly Asp Lieu. Lieu. Ile Ser Tyr Asp Glu Arg Pro 65 70 7s 8O Lys Asp Asn Gly Asn Gly Gly Ser Asp Ala Pro Asn Pro Asn His Ile 85 90 95 Val Glin Arg Arg Ser Thr Asp Gly Gly Llys Thir Trp Ser Ala Pro Thr 1OO 105 11 O Tyr Ile His Glin Gly Thr Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp 115 12 O 125 Pro Ser Tyr Val Val Asp His Glin Thr Gly Thr Ile Phe Asin Phe His 13 O 135 14 O Val Lys Ser Tyr Asp Glin Gly Trp Gly Gly Ser Arg Gly Gly. Thir Asp 145 150 155 160 Pro Glu Asn Arg Gly Ile Ile Glin Ala Glu Val Ser Thr Ser Thr Asp 1.65 17O 17s Asn Gly Trp Thir Trp Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys 18O 185 19 O Asp Llys Pro Trp Thr Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin 195 2OO 2O5 Ile Gln His Gly Pro His Ala Gly Arg Lieu Val Glin Glin Tyr Thr Ile 21 O 215 22O Arg Thr Ala Gly Gly Ala Val Glin Ala Val Ser Val Tyr Ser Asp Asp 225 23 O 235 24 O His Gly Lys Thr Trp Glin Ala Gly Thr Pro Ile Gly Thr Gly Met Asp 245 250 255 US 2012/O 141590 A1 Jun. 7, 2012 78

- Continued Glu Asn Llys Val Val Glu Lieu. Ser Asp Gly Ser Lieu Met Lieu. Asn. Ser 26 O 265 27 O Arg Ala Ser Asp Gly Ser Gly Phe Arg Llys Val Ala His Ser Thr Asp 27s 28O 285 Gly Gly Glin Thir Trp Ser Glu Pro Val Ser Asp Lys Asn Lieu Pro Asp 29 O 295 3 OO Ser Val Asp Asn Ala Glin Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro 3. OS 310 315 32O Asp Asp Pro Arg Ala Lys Val Lieu. Lieu. Lieu. Ser His Ser Pro Asn Pro 3.25 330 335 Arg Pro Trp Ser Arg Asp Arg Gly. Thir Ile Ser Met Ser Cys Asp Asp 34 O 345 35. O Gly Ala Ser Trp Thr Thr Ser Llys Val Phe His Glu Pro Phe Val Gly 355 360 365 Tyr Thir Thr Ile Ala Val Glin Ser Asp Gly Ser Ile Gly Lieu. Leu Ser 37 O 375 38O Glu Asp Ala His Asn Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn 385 390 395 4 OO Phe Thr Met Asn Trp Lieu. Gly Glu Gln Cys Gly Glin Llys Pro Ala Glu 4 OS 41O 415

<210s, SEQ ID NO 14 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Random primer sequence for N-terminal optimization (aa) 22 Os. FEATURE: <221s NAME/KEY: VARIANT <222s. LOCATION: 2, 3 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OOs, SEQUENCE: 14 Met Xaa Xala Lys Arg Llys Llys Lys Gly Gly 1. 5 1O

<210s, SEQ ID NO 15 &211s LENGTH: 374 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: protease inhibitor 8 (PI8) (Serpin B8) <4 OOs, SEQUENCE: 15 Met Asp Asp Lieu. Cys Glu Ala Asn Gly. Thir Phe Ala Ile Ser Lieu. Phe 1. 5 1O 15 Lys Ile Lieu. Gly Glu Glu Asp Asn. Ser Arg Asn Val Phe Phe Ser Pro 2O 25 3O Met Ser Ile Ser Ser Ala Leu Ala Met Val Phe Met Gly Ala Lys Gly 35 4 O 45 Ser Thr Ala Ala Glin Met Ser Glin Ala Lieu. Cys Lieu. Tyr Lys Asp Gly SO 55 6 O Asp Ile His Arg Gly Phe Glin Ser Lieu. Lieu. Ser Glu Val Asn Arg Thr 65 70 7s 8O Gly Thr Glin Tyr Lieu. Lieu. Arg Thr Ala Asn Arg Lieu. Phe Gly Glu Lys 85 90 95 US 2012/O 141590 A1 Jun. 7, 2012 79

- Continued Thr Cys Asp Phe Lieu Pro Asp Phe Lys Glu Tyr Cys Gln Llys Phe Tyr 1OO 105 11 O Glin Ala Glu Lieu. Glu Glu Lieu. Ser Phe Ala Glu Asp Thr Glu Glu. Cys 115 12 O 125 Arg Llys His Ile Asn Asp Trp Val Ala Glu Lys Thr Glu Gly Lys Ile 13 O 135 14 O Ser Glu Val Lieu. Asp Ala Gly Thr Val Asp Pro Lieu. Thir Lys Lieu Val 145 150 155 160 Lieu Val Asn Ala Ile Tyr Phe Lys Gly Lys Trp Asn. Glu Glin Phe Asp 1.65 17O 17s Arg Llys Tyr Thr Arg Gly Met Lieu. Phe Llys Thr Asn. Glu Glu Lys Llys 18O 185 19 O Thr Val Glin Met Met Phe Lys Glu Ala Lys Phe Lys Met Gly Tyr Ala 195 2OO 2O5 Asp Glu Val His Thr Glin Val Lieu. Glu Lieu Pro Tyr Val Glu Glu Glu 21 O 215 22O Lieu. Ser Met Val Ile Lieu. Lieu Pro Asp Asp Asn. Thir Asp Lieu Ala Val 225 23 O 235 24 O Val Glu Lys Ala Lieu. Thir Tyr Glu Lys Phe Lys Ala Trp Thr Asn. Ser 245 250 255 Glu Lys Lieu. Thir Lys Ser Llys Val Glin Val Phe Lieu Pro Arg Lieu Lys 26 O 265 27 O Lieu. Glu Glu Ser Tyr Asp Lieu. Glu Pro Phe Lieu. Arg Arg Lieu. Gly Met 27s 28O 285 Ile Asp Ala Phe Asp Glu Ala Lys Ala Asp Phe Ser Gly Met Ser Thr 29 O 295 3 OO Glu Lys Asn Val Pro Lieu. Ser Llys Val Ala His Lys Cys Phe Val Glu 3. OS 310 315 32O Val Asn. Glu Glu Gly. Thr Glu Ala Ala Ala Ala Thr Ala Val Val Arg 3.25 330 335 Asn Ser Arg Cys Ser Arg Met Glu Pro Arg Phe Cys Ala Asp His Pro 34 O 345 35. O Phe Leu Phe Phe Ile Arg Arg His Llys Thr Asn Cys Ile Leu Phe Cys 355 360 365 Gly Arg Phe Ser Ser Pro 37 O

<210s, SEQ ID NO 16 &211s LENGTH: 4176 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Plasmic vector PTrce 9A

<4 OOs, SEQUENCE: 16 gtttgacagc titat catcga Ctgcacggtg Caccalatgct tctggcgt.ca ggcagc.cat C 6 O ggaagctgtg gtatggctgt gcaggtogta aat cactgca taatt.cgtgt cqct Caaggc 12 O gcactic cc.gt totggataat gtttitttgcg ccgacat cat aacggttctg gcaaatatt c 18O tgaaatgagc tigttgacaat taatcatc.cg gct cqtataa ttgttggaat ttgagcgga 24 O taacaatttic acacaggaaa Cagac catgg aatticgagct cq9tacccgg ggat.cct ct a 3OO gagt cacct gcaggcatgc aagcttggct gttittggcgg atgagagaag attitt cago C 360

US 2012/O 141590 A1 Jun. 7, 2012 82

- Continued

Thr Asp Gly Gly Lys Thir Trp Ser Ala Pro Thr Tyr Ile His Glin Gly 85 90 95 Thr Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val 1OO 105 11 O Asp His Glin Thr Gly Thr Ile Phe Asin Phe His Val Lys Ser Tyr Asp 115 12 O 125 Glin Gly Trp Gly Gly Ser Arg Gly Gly. Thir Asp Pro Glu Asn Arg Gly 13 O 135 14 O Ile Ile Glin Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thir Trp 145 150 155 160 Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Llys Pro Trp Thr 1.65 17O 17s Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin Ile Gln His Gly Pro 18O 185 19 O His Ala Gly Arg Lieu Val Glin Glin Tyr Thir Ile Arg Thr Ala Gly Gly 195 2OO 2O5 Ala Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Lys Thir Trip 21 O 215 22O Glin Ala Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Llys Val Val 225 23 O 235 24 O Glu Lieu. Ser Asp Gly Ser Lieu Met Lieu. Asn. Ser Arg Ala Ser Asp Gly 245 250 255 Ser Gly Phe Arg Llys Val Ala His Ser Thr Asp Gly Gly Glin Thir Trp 26 O 265 27 O Ser Glu Pro Val Ser Asp Lys Asn Lieu Pro Asp Ser Val Asp Asn Ala 27s 28O 285 Glin Ile Ile Arg Ala Phe Pro Asn Ala Ala Pro Asp Asp Pro Arg Ala 29 O 295 3 OO Llys Val Lieu. Lieu Lleu Ser His Ser Pro Asn Pro Arg Pro Trp Ser Arg 3. OS 310 315 32O Asp Arg Gly. Thir Ile Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thr 3.25 330 335 Thir Ser Llys Val Phe His Glu Pro Phe Val Gly Tyr Thr Thr Ile Ala 34 O 345 35. O Val Glin Ser Asp Gly Ser Ile Gly Lieu. Lieu. Ser Glu Asp Ala His Asn 355 360 365 Gly Ala Asp Tyr Gly Gly Ile Trp Tyr Arg Asn Phe Thr Met Asn Trp 37 O 375 38O Lieu. Gly Glu Gln Cys Gly Glin Llys Pro Ala Lys Arg Llys Llys Lys Gly 385 390 395 4 OO Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg Llys Llys Lys Asn Pro 4 OS 41O 415

<210s, SEQ ID NO 18 &211s LENGTH: 116 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: Preprosomatostatin

<4 OOs, SEQUENCE: 18 Met Lieu. Ser Cys Arg Lieu. Glin Cys Ala Lieu Ala Ala Lieu. Ser Ile Val 1. 5 1O 15 US 2012/O 141590 A1 Jun. 7, 2012 83

- Continued

Lieu Ala Lieu. Gly Cys Val Thr Gly Ala Pro Ser Asp Pro Arg Lieu. Arg 2O 25 3O Glin Phe Lieu. Glin Llys Ser Lieu Ala Ala Ala Ala Gly Lys Glin Glu Lieu. 35 4 O 45 Ala Lys Tyr Phe Lieu Ala Glu Lieu. Lieu. Ser Glu Pro Asn. Glin Thr Glu SO 55 6 O Asn Asp Ala Lieu. Glu Pro Glu Asp Lieu. Ser Glin Ala Ala Glu Glin Asp 65 70 7s 8O Glu Met Arg Lieu. Glu Lieu. Glin Arg Ser Ala Asn. Ser Asn Pro Ala Met 85 90 95 Ala Pro Arg Glu Arg Lys Ala Gly Cys Lys Asn. Phe Phe Trp Llys Thr 1OO 105 11 O Phe Thir Ser Cys 115

<210s, SEQ ID NO 19 &211s LENGTH: 92 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: Prosomatostatin

<4 OOs, SEQUENCE: 19 Ala Pro Ser Asp Pro Arg Lieu. Arg Glin Phe Lieu Gln Lys Ser Lieu. Ala 1. 5 1O 15 Ala Ala Ala Gly Lys Glin Glu Lieu Ala Lys Tyr Phe Lieu Ala Glu Lieu 2O 25 3O Lieu. Ser Glu Pro Asn Glin Thr Glu Asn Asp Ala Lieu. Glu Pro Glu Asp 35 4 O 45 Lieu. Ser Glin Ala Ala Glu Glin Asp Glu Met Arg Lieu. Glu Lieu. Glin Arg SO 55 6 O Ser Ala Asn. Ser Asn Pro Ala Met Ala Pro Arg Glu Arg Lys Ala Gly 65 70 7s 8O Cys Lys Asn Phe Phe Trp Llys Thr Phe Thr Ser Cys 85 90

<210s, SEQ ID NO 2 O &211s LENGTH: 28 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: Somatostatin 28 (SS-28)

<4 OOs, SEQUENCE: 2O Ser Ala Asn. Ser Asn Pro Ala Met Ala Pro Arg Glu Arg Lys Ala Gly 1. 5 1O 15 Cys Lys Asn Phe Phe Trp Llys Thr Phe Thr Ser Cys 2O 25

<210s, SEQ ID NO 21 &211s LENGTH: 14 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <223> OTHER INFORMATION: Somatostatin 14 (SS-14)

<4 OOs, SEQUENCE: 21 US 2012/O 141590 A1 Jun. 7, 2012 84

- Continued Ala Gly Cys Lys Asn Phe Phe Trp Llys Thr Phe Thr Ser Cys 1. 5 1O

<210s, SEQ ID NO 22 &211s LENGTH: 9 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Leuprolide 22 Os. FEATURE: <221s NAME/KEY: MOD RES &222s. LOCATION: 1 <223> OTHER INFORMATION: pyroglutamic acid 22 Os. FEATURE: <221s NAME/KEY: MOD RES &222s. LOCATION: 6 <223> OTHER INFORMATION: d-leucine 22 Os. FEATURE: <221s NAME/KEY: MOD RES &222s. LOCATION: 10 <223> OTHER INFORMATION: Proline residue bound to ethylamide (NHEt) <4 OOs, SEQUENCE: 22 Glu. His Trp Ser Tyr Lieu Lleu. Arg Pro 1. 5

<210s, SEQ ID NO 23 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic siRNA NP1496 sense strand 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: 2O, 21 223 OTHER INFORMATION: n = cT

<4 OOs, SEQUENCE: 23 ggaucullalulu ulculu.cggagn in 21

<210s, SEQ ID NO 24 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic siRNA NP-1496 antisense strand 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: 2O, 21 223 OTHER INFORMATION: n = ct

<4 OOs, SEQUENCE: 24

Cuccgaagaa aluaagaluccin in 21

<210s, SEQ ID NO 25 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic siRNA PA-2O87 sense strand 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: 2O, 21 223 OTHER INFORMATION: n = cT

<4 OOs, SEQUENCE: 25 gcaauugagg agugcclugan in 21 US 2012/O 141590 A1 Jun. 7, 2012 85

- Continued

SEQ ID NO 26 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic siRNA PA-2087 antisense strand FEATURE: NAMEAKEY: misc feature LOCATION: 2O, 21 OTHER INFORMATION: n = cT

<4 OOs, SEQUENCE: 26 lucaggcaciuc Cucaaulugcn in 21

SEO ID NO 27 LENGTH: 21 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic siRNA. GAPDH sense strand

< 4 OOs SEQUENCE: 27 luggluluulacau guluccalaulau u. 21

SEQ ID NO 28 LENGTH: 21 TYPE : RNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic siRNA. GAPDH antisense strand <4 OOs, SEQUENCE: 28 ulauluggalaca lugulaalaccalu u. 21

What is claimed is: 9. The method of claim 1, wherein steps a) and b) are 1. A method of making microparticles of a compound, performed simultaneously, sequentially, intermittently, or in comprising: any order, followed by step c). a) adding a counterion to a solution containing the com 10. The method of claim 1, wherein steps b) and c) are pound in a solvent; performed simultaneously, sequentially, intermittently, or in b) adding an antisolvent to the solution; and c) gradually cooling the solution to a temperature below any order, preceded by step a). about 25°C., whereby a composition containing micro 11. The method of claim 1, wherein steps a) and c) are particles comprising the compound is formed, wherein performed simultaneously. steps a), b) and c) are performed simultaneously, 12. The method of claim 1, wherein the counterion and the sequentially, intermittently, or in any order. compound are identical to one another. 2. The method of claim 1, wherein the counterion is not a 13. The method of claim 1, wherein the compound and the polymer. counterion are different from one another. 3. The method of claim 1, wherein the antisolvent is not a 14. The method of claim 1, wherein the counterion and the polymer. antisolvent are identical to one another. 4. The method of claim 1, wherein prior to step a), the compound is dissolved in the solvent at a temperature of 15. The method of claim 1, wherein the compound is a about or at 30° C. or below. macromolecule with a molecular weight of about or at 1000 5. The method of claim 4, wherein prior to step a), the or 1000 to about or at five billion or five billion Daltons. compound is dissolved in the solvent at a temperature of 16. The method of claim 15, wherein the compound is a about or at 25° C. or below. macromolecule with a molecular weight of about or at 1000 6. The method of claim 1, wherein steps a) and b) are or 1000 to about or at one billion or one billion Daltons. performed at ambient temperature. 17. The method of claim 16, wherein the compound is a 7. The method of claim 1, wherein none of the solutions of macromolecule with a molecular weight of about or at 1000 steps a)-c) are heated and/or maintained at a temperature or 1000 to about or at 50 million or 50 million Daltons. above about or at 30° C. 18. The method of claim 17, wherein the compound is a 8. The method of claim 1, wherein the compound is not a macromolecule with a molecular weight of about or at 1000 protein or a polypeptide. or 1000 to about or at 10 million or 10 million Daltons. US 2012/O 141590 A1 Jun. 7, 2012

19. The method of claim 18, wherein the compound is a agents, antiviral agents, antitumor agents, antidepressants, macromolecule with a molecular weight of about or at 1000 psychotropic agents, cardiotonics, diuretics, antiarrythmic or 1000 to about or at one million or one million Daltons. agents, vasodilators, antihypertensive agents, antidiabetic 20. The method of claim 19, wherein the compound is a agents, anticoagulants, and cholesterol lowering agents. macromolecule with a molecular weight of about or at 1000 34. The method of claim 33, wherein the small molecule is or 1000 to about or at 500,000 or 500,000 Daltons. an antibiotic. 21. The method of claim 20, wherein the compound is a 35. The method of claim 34, wherein the antibiotic is macromolecule with a molecular weight of about or at 1000 selected from among aminoglycosides, ansamycins, carba or 1000 to about or at 100,000 or 100,000 Daltons. cephem, carbapenems, cephalosporins, macrollides, penicil 22. The method of claim 21, wherein the compound is a lins, quinolones, Sulfonamides and tetracyclines. macromolecule with a molecular weight of about or at 1000 36. The method of claim 35, wherein the antibiotic is a or 1000 to about or at 50,000 or 50,000 Daltons. penicillin or a tetracycline. 23. The method of claim 22, wherein the compound is a 37. The method of claim 35, wherein the antibiotic is an macromolecule with a molecular weight of about or at 1000 aminoglycoside. or 1000 to about or at 10,000 or 10,000 Daltons. 38. The method of claim37, wherein the aminoglycoside is 24. The method of claim 23, wherein the compound is a kanamycin or tobramycin. macromolecule with a molecular weight of about or at 1000 39. The method of claim 33, wherein the small molecule is or 1000 to about or at 5,000 or 5,000 Daltons. an antiviral agent. 25. The method of claim 24, wherein the compound is a 40. The method of claim 39, wherein the antiviral agent is macromolecule with a molecular weight of about or at 1000 for treatment of influenza, parainfluenza or respiratory Syn or 1000 to about or at 2,000 or 2000 Daltons. cytial virus-mediated infections. 26. The method of claim 1, wherein the compound is a 41. The method of claim 40, wherein the antiviral agent is Small molecule. Zanamivir or oseltamivir phosphate. 27. The method of claim 15, wherein the macromolecule is 42. The method of claim 33, wherein the small molecule is selected from among a polynucleotide, a nucleic acid, a a chemotherapeutic agent. polypeptide, a glycopeptide, a protein, a carbohydrate, a lipid, 43. The method of claim 42, wherein the chemotherapeutic a fatty acid, a polysaccharide, carbohydrate- or polysaccha agent is selected from among alkylating agents, anthracy ride-protein conjugates, virus, virus particles, Viroids, prions clines, cytoskeletal disruptors, epothilones, inhibitors of and mixtures thereof. topoisomerase II, nucleotide analogs, platinum-based agents, 28. The method of claim 2, wherein the compound is a retinoids and Vinca alkaloids. macromolecule, and the macromolecule is selected from 44. The method of claim 43, wherein the chemotherapeutic among a polynucleotide, a nucleic acid, a polypeptide, a agent is a cytoskeletal disruptor. glycopeptide, a protein, a carbohydrate, a lipid, a fatty acid, a 45. The method of claim 44, wherein the cytoskeletal dis polysaccharide, carbohydrate- or polysaccharide-protein ruptor is paclitaxel. conjugates, virus, virus particles, viroids, prions and mixtures 46. The method of claim 33, wherein the small molecule is thereof. a prostaglandin. 29. The method of claim 15, wherein the macromolecule is 47. The method of claim 27, wherein the macromolecule is selected from among hormones, prostaglandins, antibiotics, a nucleic acid. chemotherapeutic agents, hematopoietics, anti-infective 48. The method of claim 47, wherein the nucleic acid is agents, antiulcer agents, antiallergic agents, antipyretics, selected from among DNA, RNA and PNA. analgesics, anti-inflammatory agents, antidementia agents, 49. The method of claim 48, wherein the nucleic acid is antiviral agents, antitumor agents, antidepressants, psycho RNA. tropic agents, cardiotonics, diuretics, antiarrhythmic agents, 50. The method of claim 49, wherein the RNA is selected vasodilators, antihypertensive agents, antidiabetic agents, from among siRNA, tRNA, snRNA and ribozymes. anticoagulants, and cholesterol lowering agents. 51. The method of claim 50, wherein the RNA is siRNA. 30. The method of claim 27, wherein the macromolecule is 52. The method of claim 27, wherein the macromolecule is conjugated to a small molecule. a virus. 31. The method of claim 30, wherein the small molecule is selected from among haptens, hormones, prostaglandins, 53. The method of claim 52, wherein the virus is tobacco antibiotics, chemotherapeutic agents, hematopoietics, anti mosaic virus. infective agents, antiulcer agents, antiallergic agents, anti 54. The method of claim 27, wherein the macromolecule is pyretics, analgesics, anti-inflammatory agents, antidementia a glycopeptide. agents, antiviral agents, antitumor agents, antidepressants, 55. The method of claim 54, wherein the glycopeptide is psychotropic agents, cardiotonics, diuretics, antiarrhythmic Vancomycin. agents, vasodilators, antihypertensive agents, antidiabetic 56. The method of claim 27, wherein the macromolecule is agents, anticoagulants, and cholesterol lowering agents. a peptide. 32. The method of claim 26, wherein the small molecule 57. The method of claim 56, wherein the peptide is leupro has a molecular weight of about or at 50 to about or at 1000 lide. Daltons. 58. The method of claim 56, wherein the peptide is soma 33. The method of claim 26, wherein the small molecule is to statin selected from among haptens, hormones, prostaglandins, 59. The method of claim 1, wherein the compound is water antibiotics, chemotherapeutic agents, hematopoietics, anti insoluble. infective agents, antiulcer agents, antiallergic agents, anti 60. The method of claim 1, wherein steps a), b) and c) are pyretics, analgesics, anti-inflammatory agents, antidementia performed sequentially in the order: a), then b), then c). US 2012/O 141590 A1 Jun. 7, 2012

61. The method of claim 1, wherein the solvent is miscible 84. The method of claim 1, wherein the pH of the solution or partially miscible with the antisolvent. is from about 4.0 or 4.0 to about 9.0 or 9.0. 62. The method of claim 1, further comprising, after step 85. The method of claim 1, wherein the resulting micro c), separating the microparticles from the Solution to remove particle composition further comprises micro-carriers, acid components other than the microparticles. resistant coating agents, protease-resistant coating agents, 63. The method of claim 62, wherein the composition enteric coating agents, bulking agents, excipients, inactive consists essentially of the microparticles comprising the com ingredients, stability enhancers, taste and/or odor modifiers pound. or masking agents, Vitamins, Sugars, therapeutic agents, anti 64. The method of claim 62, wherein the separation is oxidants, immuno-modulators, trans-membrane transport effected by sedimentation or by filtration. modifiers, anti-caking agents, chitosans or flowability 65. The method of claim 62, wherein the separation is enhancers. effected by freeze-drying. 86. The method of claim 85, wherein the resulting micro 66. The method of claim 1, wherein the antisolvent is particle further comprises a micro-carrier, and the micro selected from among water, buffered solutions, aliphatic carrier is selected from among amino acids, carboxylic acids, alcohols, aromatic alcohols, chloroform, polyhydric Sugar proteins, nucleic acids, polysaccharides, and materials that alcohols, aromatic hydrocarbons, aldehydes, ketones, esters, are capable of forming hydrogels. ethers, dioxanes, alkanes, alkenes, conjugated dienes, dichlo 87. The method of claim 86, wherein the micro-carrier is a romethane, carbon tetrachloride, dimethylformamide material that is capable of forming a hydrogel. (DMF), dimethyl sulfoxide (DMSO), acetonitrile, ethyl 88. The method of claim 87, wherein the micro-carrier is acetate, polyols, polyimides, polyimines, polyesters, polyal gelatin or dextran. dehydes and mixtures thereof. 89. The method of claim 88, wherein the compound is a 67. The method of claim 66, wherein the antisolvent is an protein. aliphatic alcohol or an aromatic alcohol. 90. The method of claim 88, wherein the compound is a 68. The method of claim 67, wherein the antisolvent is an nucleic acid. aliphatic alcohol. 91. The method of claim 90, wherein the nucleic acid is 69. The method of claim 68, wherein the aliphatic alcohol siRNA and the micro-carrier is gelatin. is isopropanol. 92. The method of claim 1, wherein the compound is a 70. The method of claim 1, wherein the counterion is micro-carrier. selected from among an anionic compound, a cationic com 93. The method of claim 92, wherein the micro-carrier is pound and a Zwitterionic compound. selected from among amino acids, carboxylic acids, proteins, 71. The method of claim 70, wherein the counterion is an nucleic acids, polysaccharides, and materials that are capable anionic compound. of forming hydrogels. 72. The method of claim 71, wherein the anionic com 94. The method of claim 93, wherein the micro-carrier is a pound is selected from among Sodium citrate, Sodium Sulfate, material that is capable of forming a hydrogel. Zinc sulfate, magnesium sulfate, potassium Sulfate and cal 95. The method of claim 94, wherein the micro-carrier is cium sulfate. gelatin. 73. The method of claim 72, wherein the anionic com 96. The method of claim 95, wherein the micro-carrier is pound is sodium Sulfate. dextran. 74. The method of claim 70, wherein the counterion is 97. The method of claim 1, wherein the amount of com selected from among citric acid, itaconic acid and pivalic pound in the microparticles relative to the total amount of acid. compound in the solution of step a) is about 5% or 5% to 75. The method of claim 70, wherein the counterion is an greater than about 99% or 99%, w/w. amino acid. 98. The method of claim 97, wherein the amount of com 76. The method of claim 75, wherein the compound is pound in the microparticles relative to the total amount of glycine or arginine. compound in the solution of step a) is about 10% or 10% to 77. The method of claim 15, wherein the counterion is a about 85% or 85%, w/w. polymer, and the macromolecule is selected from among a 99. The method of claim 98, wherein the amount of com polynucleotide, a nucleic acid, a carbohydrate, a lipid, a fatty pound in the microparticles relative to the total amount of acid, a polysaccharide, carbohydrate- or polysaccharide-pro compound in the solution of step a) is about 25% or 25% to tein conjugates, a virus, virus particles, Viroids, prions and about 55% or 55%, w/w. mixtures thereof. 100. The method of claim 97, wherein the amount of com 78. The method of claim 77, wherein the polymer is the pound in the microparticles relative to the total amount of counterion and the antisolvent. compound in the solution of step a) is about 80% or 80% to 79. The method of claim 78, wherein the polymer is poly greater than about 99% or 99%, w/w. ethylene glycol (PEG) or polyethyleneimine (PEI). 101. The method of claim 1, wherein the temperature is 80. The method of claim 1, wherein the counterion is a between about or at 4°C. to about or at -200° C. polymer. 102. The method of claim 101, wherein the temperature is 81. The method of claim 80, wherein the polymer is the between about or at 2°C. to about or at -170° C. counterion and the antisolvent. 103. The method of claim 102, wherein the temperature is 82. The method of claim 81, wherein the polymer is poly between about 0°C. or 0°C. to about -2°C. or -2°C. to from ethylene glycol (PEG) or polyethyleneimine (PEI). about -150° C. or -150° C. to about -165° C. or -165° C. 83. The method of claim 1, wherein the microparticles are 104. The method of claim 1, wherein the resulting compo obtained by precipitation, by phase separation or by colloid sition has a shelflife of from about or at one week to about or formation. at 1 month, from about or at 1 month to about or at six months, US 2012/O 141590 A1 Jun. 7, 2012

from about or at six months to about oratone year, from about 121. The method of claim 120, wherein the size of the or at 1 year to about or at 2 years, or from about or at 2 years microparticles is from about or at 1.0 um to about or at 2.0, to about or at 5 years at a temperature of about or at 55°C., 50° 3.0, 4.0 or 5.0 um. C., 45° C., 44° C., 42°C., 40° C., 39° C., 38°C., 37° C. or 122. A composition, comprising microparticles of a com below. pound and a counterion, wherein the compound and the coun 105. The method of claim 1, wherein the solution and/or terion are different from one another. the resulting composition further comprises an active agent. 123. The composition of claim 122, wherein the compound 106. The method of claim 105, wherein the active agent is is a macromolecule with a molecular weight of about or at selected from among antibiotics, chemotherapeutic agents, 1000 or 1000 to about or at five billion or five billion Daltons. antidiabetics, anticonvulsants, analgesics, antiparkinsons, 124. The composition of claim 123, wherein the compound anti-inflammatories, calcium antagonists, anesthetics, anti is a macromolecule with a molecular weight of about or at microbials, antimalarials, antiparasitics, antihypertensives, 1000 or 1000 to about or at one billion or one billion Daltons. antihistamines, antipyretics, alpha-adrenergic agonists, 125. The composition of claim 124, wherein the compound alpha-blockers, biocides, bactericides, bronchial dilators, beta-adrenergic blocking drugs, contraceptives, cardiovascu is a macromolecule with a molecular weight of about or at lar drugs, calcium channel inhibitors, depressants, diagnos 1000 or 1000 to about or at 50 million or 50 million Daltons. tics, diuretics, electrolytes, enzymes, hypnotics, hormones, 126. The composition of claim 125, wherein the compound hypoglycemics, hyperglycemics, muscle contractants, is a macromolecule with a molecular weight of about or at muscle relaxants, neoplastics, glycoproteins, nucleoproteins, 1000 or 1000 to about or at 5 million or 5 million Daltons. lipoproteins, ophthalmics, psychic energizers, sedatives, Ste 127. The composition of claim 126, wherein the compound roids, sympathomimetics, parasympathomimetics, tranquil is a macromolecule with a molecular weight of about or at izers, urinary tract drugs, vaccines, vaginal drugs, nonsteroi 1000 or 1000 to about or at one million or one million Dal dal anti-inflammatory drugs, angiotensin converting tOnS. enzymes, polynucleotides, polypeptides, polysaccharides, 128. The composition of claim 127, wherein the compound enzymes, hormones, vitamins, minerals, and nutritional is a macromolecule with a molecular weight of about or at Supplements. 1000 or 1000 to about or at 200,000 or 200,000 Daltons. 107. The method of claim 1, wherein the moisture content 129. The composition of claim 128, wherein the compound of the microparticles is adjusted whereby at least about 90% is a macromolecule with a molecular weight of about or at or 90% of the activity of the compound is retained after 1000 or 1000 to about or at 50,000 or 50,000 Daltons. storage for about six months to about 1 year at a temperature 130. The composition of claim 129, wherein the compound of about 25°C. is a macromolecule with a molecular weight of about or at 108. The method of claim 1, wherein the moisture content 1000 or 1000 to about or at 10,000 or 10,000 Daltons. of the microparticles is from about or at 0.01% to about or at 131. The composition of claim 130, wherein the compound 20%. is a macromolecule with a molecular weight of about or at 109. The method of claim 1, wherein the concentration of 1000 or 1000 to about or at 3,000 or 3000 Daltons. counterion added to the solution is from about or at 0 mM or 132. The composition of claim 122, wherein the compound 0 mM to about or at 100 mM or 100 mM. is a small molecule. 110. The method of claim 109, wherein the concentration 133. The composition of claim 123, wherein the macro of counterion added to the solution is from about or at 0 mM molecule is selected from among a polynucleotide, a nucleic or 0 mM to about or at 50 nnM or 50 mM. acid, a polypeptide, a glycopeptide, a protein, a carbohydrate, 111. The method of claim 110, wherein the concentration a lipid, a fatty acid, a polysaccharide, carbohydrate- or of counterion added to the solution is from about or at 1 mM polysaccharide-protein conjugates, virus, virus particles, or 1 mM to about or at 5 mM or 5 mM. Viroids, prions and mixtures thereof. 112. The method of claim 111, wherein the concentration 134. The composition of claim 123, wherein the macro of counterion added to the solution is about or at 2 mM. molecule is selected from among hormones, prostaglandins, 113. The method of claim 1, wherein the gradual cooling is antibiotics, chemotherapeutic agents, hematopoietics, anti by chilling. infective agents, antiulcer agents, antiallergic agents, anti 114. The method of claim 1, wherein the gradual cooling is pyretics, analgesics, anti-inflammatory agents, antidementia by an endothermic reaction. agents, antiviral agents, antitumor agents, antidepressants, 115. The method of claim 1, wherein the gradual cooling is psychotropic agents, cardiotonics, diuretics, antiarrhythmic at a rate of from about or at 0.01° C./min or 0.01° C./min to agents, vasodilators, antihypertensive agents, antidiabetic about or at 20° C./min or 20° C./min. agents, anticoagulants, and cholesterol lowering agents. 116. The method of claim 115, wherein the gradual cooling 135. The composition of claim 123, wherein the macro is at a rate of about or at 0.2°C./minto about or at 5°C/min. molecule is conjugated to a small molecule. 117. The method of claim 116, wherein the gradual cooling 136. The composition of claim 135, wherein the small is at a rate of about or at 0.5°C/minto about or at 2°C./min molecule is selected from among haptens, hormones, pros 118. The method of claim 117, wherein the gradual cooling taglandins, antibiotics, chemotherapeutic agents, hematopoi is at a rate of about or at 1° C./min. etics, anti-infective agents, antiulcer agents, antiallergic 119. The method of claim 1, wherein the size of the micro agents, antipyretics, analgesics, anti-inflammatory agents, particles is from about or at 0.001 um or 0.001 um to about or antidementia agents, antiviral agents, antitumor agents, anti at 50 um or 50 Lum. depressants, psychotropic agents, cardiotonics, diuretics, 120. The method of claim 119, wherein the size of the antiarrhythmic agents, vasodilators, antihypertensive agents, microparticles is from about or at 0.5um or 0.5um to about or antidiabetic agents, anticoagulants, and cholesterol lowering at 10 um or 10 Lum. agents. US 2012/O 141590 A1 Jun. 7, 2012

137. The composition of claim 132, wherein the small 161. The composition of claim 160, wherein the counterion molecule has a molecular weight of about or at 50 to about or is glycine or arginine. at 1000 Daltons. 162. The composition of claim 122, wherein the counterion 138. The composition of claim 137, wherein the small is polyethylene glycol (PEG) or polyethyleneimine (PEI). molecule is selected from among haptens, hormones, pros 163. The composition of claim 122, wherein the resulting taglandins, antibiotics, chemotherapeutic agents, hematopoi microparticle composition further comprises micro-carriers, etics, anti-infective agents, antiulcer agents, antiallergic acid-resistant coating agents, protease-resistant coating agents, antipyretics, analgesics, anti-inflammatory agents, agents, enteric coating agents, bulking agents, excipients, antidementia agents, antiviral agents, antitumor agents, anti inactive ingredients, stability enhancers, taste and/or odor depressants, psychotropic agents, cardiotonics, diuretics, modifiers or masking agents, vitamins, Sugars, therapeutic antiarrhythmic agents, vasodilators, antihypertensive agents, agents, anti-oxidants, immuno-modulators, trans-membrane antidiabetic agents, anticoagulants, and cholesterol lowering transport modifiers, anti-caking agents, chitosans or flowabil agents. ity enhancers. 139. The composition of claim 138, wherein the small 164. The composition of claim 163, wherein the resulting molecule is an antibiotic. microparticle composition further comprises a micro-carrier. 140. The composition of claim 139, wherein the antibiotic 165. The composition of claim 164, wherein the micro is selected from among aminoglycosides, ansamycins, carba carrier is selected from among amino acids, carboxylic acids, cephem, carbapenems, cephalosporins, macrollides, penicil proteins, nucleic acids, polysaccharides, and materials that lins, quinolones, Sulfonamides and tetracyclines. are capable of forming hydrogels. 141. The composition of claim 138, wherein the compound 166. The composition of claim 165, wherein the micro is an antiviral agent. carrier is a material that is capable of forming a hydrogel. 142. The composition of claim 141, wherein the antiviral 167. The composition of claim 166, wherein the micro agent is for treatment of influenza, parainfluenza, or respira carrier is gelatin or dextran. tory syncytial virus-mediated infections. 168. The composition of claim 167, wherein the compound 143. The composition of claim 142, wherein the antiviral is a protein. agent is Zanamivir or oseltamivir phosphate. 169. The composition of claim 167, wherein the compound 144. The composition of claim 138, wherein the compound is a nucleic acid. is a chemotherapeutic agent. 170. The composition of claim 169, wherein the nucleic 145. The composition of claim 144, wherein the chemo acid is siRNA and the micro-carrier is gelatin. therapeutic agent is paclitaxel. 171. The composition of claim 122, wherein the compound 146. The composition of claim 138, wherein the compound is a micro-carrier. is a prostaglandin. 172. The composition of claim 171, wherein the micro 147. The composition of claim 133, wherein the macro carrier is selected from among amino acids, carboxylic acids, molecule is a nucleic acid. proteins, nucleic acids, polysaccharides, and materials that 148. The composition of claim 147, wherein the nucleic are capable of forming hydrogels. acid is selected from among DNA, RNA and PNA. 173. The composition of claim 172, wherein the micro 149. The composition of claim 148, wherein the nucleic carrier is a material that is capable of forming a hydrogel. acid is RNA. 174. The composition of claim 173, wherein the micro 150. The composition of claim 149, wherein the RNA is carrier is gelatin or dextran. selected from among siRNA, tRNA, snRNA and ribozymes. 175. The composition of claim 122 that has a shelf life of 151. The composition of claim 150, wherein the RNA is from about or at one week to about or at 1 month, from about siRNA. or at 1 month to about or at six months, from about or at six 152. The composition of claim 133, wherein the macro months to about oratone year, from about or at 1 year to about molecule is a virus. or at 2 years, or from about or at 2 years to about or at 5 years 153. The composition of claim 133, wherein the macro at a temperature of about or at 55° C., 50° C., 45° C., 44°C., molecule is a peptide. 42°C., 40° C., 39° C., 38°C., 37° C. or below. 154. The composition of claim 153, wherein the peptide is 176. The composition of claim 122 that further comprises leuprolide or Somatostatin. an active agent. 155. The composition of claim 122, wherein the compound 177. The composition of claim 176, wherein the active is water-insoluble. agent is selected from among antibiotics, chemotherapeutic 156. The composition of claim 122, wherein the counterion agents, antidiabetics, anticonvulsants, analgesics, antiparkin is selected from among an anionic compound, a cationic Sons, anti-inflammatories, calcium antagonists, anesthetics, compound and a Zwitterionic compound. antimicrobials, antimalarials, antiparasitics, antihyperten 157. The composition of claim 156, wherein the counterion sives, antihistamines, antipyretics, alpha-adrenergicagonists, is an anionic compound. alpha-blockers, biocides, bactericides, bronchial dilators, 158. The composition of claim 157, wherein the anionic beta-adrenergic blocking drugs, contraceptives, cardiovascu compound is selected from among sodium citrate, sodium lar drugs, calcium channel inhibitors, depressants, diagnos Sulfate, Zinc sulfate, magnesium Sulfate, potassium sulfate tics, diuretics, electrolytes, enzymes, hypnotics, hormones, and calcium sulfate. hypoglycemics, hyperglycemics, muscle contractants, 159. The composition of claim 122, wherein the counterion muscle relaxants, neoplastics, glycoproteins, nucleoproteins, is selected from among citric acid, itaconic acid and pivalic lipoproteins, ophthalmics, psychic energizers, sedatives, Ste acid. roids, sympathomimetics, parasympathomimetics, tranquil 160. The composition of claim 122, wherein the counterion izers, urinary tract drugs, vaccines, vaginal drugs, nonsteroi is an amino acid. dal anti-inflammatory drugs, angiotensin converting US 2012/O 141590 A1 Jun. 7, 2012 90 enzymes, polynucleotides, polypeptides, polysaccharides, 195. The method of claim 194, wherein the administering enzymes, hormones, vitamins, minerals, and nutritional is performed orally, intravenously, intranasally, parenterally, Supplements. Subcutaneously, transdermally, topically, intraarticularly, 178. The composition of claim 122, wherein the amount of intramuscularly or by inhalation. compound in the microparticles is from about or at 0.1% to 196. A method of making microparticles of siRNA, com about or at 99% or greater, ww. prising: (a) adding an antisolvent to a solution of siRNA in an 179. The composition of claim 178, wherein the amount of aqueous solvent; and compound in the microparticles is from about 90% to about (b) gradually cooling the solution to a temperature below 99% W/w. about 25°C., whereby a composition containing micro 180. The composition of claim 122, wherein the moisture particles comprising siRNA is formed, wherein steps (a) content of the microparticles is adjusted whereby at least and (b) are performed simultaneously, sequentially, about 90% or 90% of the activity of the compound is retained intermittently, or in any order. after storage for about or at six months to about or at 1 year at 197. The method of claim 196, further comprising: a temperature of about 25°C. (c) adding a counterion, wherein steps (a), (b) and (c) are 181. The composition of claim 122, wherein the amount of performed simultaneously, sequentially, intermittently, counterion in the microparticles is from about 0.01% or or in any order. 0.01% to about 60% or 60% w/w. 198. The method of claim 196, wherein the antisolvent is 182. The composition of claim 181, wherein the amount of isopropanol. counterion in the microparticles is from about 0.1% or 0.1% 199. The method of claim196, wherein the solventis water. to about 10% or 10% w/w. 200. A composition, comprising microparticles of siRNA. 183. The composition of claim 182, wherein the amount of 201. The composition of claim 200, further comprising a counterion in the microparticles is from about 0.2% or 0.2% counterion. to about 5% or 5% w/w. 202. A method of making microparticles of a virus, com 184. The composition of claim 183, wherein the amount of prising: counterion in the microparticles is from about 6% or 6% to (a) adding an antisolvent to a solution of virus in an aque about 12% or 12%. ous solvent; and 185. The composition of claim 122 that is for oral admin (b) gradually cooling the solution to a temperature below istration or ingestion. about 25°C., whereby a composition containing micro 186. The composition of claim 122 that is for intravenous, particles comprising a virus is formed, wherein steps (a) intranasal, parenteral, pulmonary, Subcutaneous, ophthalmic and (b) are performed simultaneously, sequentially, or intramuscular administration. intermittently, or in any order. 203. The method of claim 202, further comprising: 187. The composition of claim 122 that is for inhalation. (c) adding a counterion, wherein steps (a), (b) and (c) are 188. The composition of claim 122, wherein the size of the performed simultaneously, sequentially, intermittently, microparticles is from about 0.001 um or 0.001 um to about or in any order. 10 turn or 10 um. 204. The method of claim 202, wherein the antisolvent is 189. An article of manufacture, comprising the composi isopropanol. tion of claim 122, a packaging material for the composition 205. A method of making microparticles of a virus, com and a label that indicates that the composition is for a thera prising: peutic, nutraceutical or cosmetic indication. (a) adding a counterion to a solution of virus in an aqueous 190. The article of claim 189, wherein the composition is Solvent; and for a therapeutic indication. (b) gradually cooling the solution to a temperature below 191. The article of claim 190, wherein the therapeutic about 25°C., whereby a composition containing micro indication is influenza, parainfluenza or respiratory disorders. particles comprising a virus is formed, wherein steps (a) 192. The article of claim 191, further comprising an inhaler and (b) are performed simultaneously, sequentially, for pulmonary administration of the composition. intermittently, or in any order. 193. A method of preventing or treating an infectious dis 206. The method of claim 205, wherein the solventis water. ease, comprising administering a therapeutically effective 207. A composition, comprising microparticles of a virus. amount of the composition of claim 122 to a Subject. 208. The composition of claim 207, further comprising a 194. The method of claim 193, wherein the infectious counterion. disease is selected from among influenza, parainfluenza, res piratory syncytial virus.