(12) Patent Application Publication (10) Pub. No.: US 2009/0098207 A1 Malakhov Et Al

US 20090098207A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0098207 A1 Malakhov et al. (43) Pub. Date: Apr. 16, 2009

(54) TECHNOLOGY FOR THE PREPARATION OF Related U.S. Application Data MCROPARTICLES (60) Provisional application No. 60/961,872, filed on Jul. 24, 2007. (75) Inventors: Michael Malakhov, San Diego, CA s (US); Fang Fang, San Diego, CA Publication Classification (US) (51) Int. Cl. A6IR 9/14 (2006.01) Correspondence Address: A6IR 8/02 (2006.01) FISH & RICHARDSON, PC (52) U.S. Cl...... 424/489 P.O. BOX 1022 MINNEAPOLIS, MN 55440-1022 (US) (57) ABSTRACT Microspheres are produced by contacting a solution of a (73) Assignee: NexBio, Inc., San Diego, CA (US) macromolecule or Small molecule in a solvent with an anti Solvent and a counterion, and chilling the solution. The (21) Appl. No.: 12/179,520 microspheres are useful for preparing pharmaceuticals, nutraceuticals, cosmetic products and the like of defined (22) Filed: Jul. 24, 2008 dimensions. US 2009/00982O7 A1 Apr. 16, 2009

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 004WO1/6504PC, 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 2009/00982O7 A1 Apr. 16, 2009

include any and all permutations and combinations of the or at 1000 or 1000 to about or at 200,000 or 200,000 Daltons; ingredients, steps and/or parameters described below. about or at 1000 or 1000 to about or at 100,000 or 100,000 0010. The methods provided herein can include the steps Daltons; about or at 1000 or 1000 to about or at 50,000 or of: 50,000 Daltons; about or at 1000 or 1000 to about or at 25,000 0.011 a) adding a counterion to a solution containing the or 25,000 Daltons; about or at 1000 or 1000 to about or at compound in a solvent; 15,000 or 15,000 Daltons; about or at 1000 or 1000 to about 0012 b) adding an antisolvent to the solution; and or at 10,000 or 10,000 Daltons; about or at 1000 or 1000 to 0013 c) gradually cooling the solution to a temperature about or at 5,000 or 5,000 Daltons; about or at 1000 or 1000 below about 25°C., to about or at 3,000 or 3000 Daltons; or about or at 1000 or whereby a composition containing microparticles of the com 1000 to about or at 2,000 or 2000 Daltons. pound is formed. In the method, steps a), b) and c) can be performed simultaneously, sequentially, intermittently, or in 0018. The macromolecule can be a polynucleotide, a any order. nucleic acid, a polypeptide, a glycopeptide, a protein, a car 0014. In some examples, the counterion is not a polymer. bohydrate, a lipid, a fatty acid, a polysaccharide, carbohy In further examples, the antisolvent is not a polymer. The drate- or polysaccharide-protein conjugate, virus, virus par temperature at which the steps are performed also can be ticle, viroid, prion or mixture thereof. In other examples, the altered. In some embodiments, the compound is dissolved in macromolecule is a hormone, prostaglandin, antibiotic, che the solvent at a temperature of about or at 30° C. or below motherapeutic agent, hematopoietic, anti-infective agent, prior to step a). In other embodiments, the compound is antiulcer agent, antiallergic agent, antipyretic, analgesic, dissolved in the solvent at a temperature of about or at 25°C. anti-inflammatory agent, antidementia agent, antiviral agent, or below. In one aspect, none of the solutions of steps a)-c) are antitumor agent, antidepressant, psychotropic agents, cardio heated and/or maintained at a temperature above about or at tonics, diuretic, antiarrhythmic agent, vasodilator, antihyper 30°C. In some examples, the compound in these methods is tensive agent, antidiabetic agent, anticoagulant, or choles not a protein or polypeptide. terol lowering agent. 0015. In some embodiments, the compounds can be 0019. In one embodiment, the macromolecule is conju heated to temperatures of above ambient temperature to dis gated to a small molecule. In some embodiments, the Small Solve the compound in the solvent/antisolvent system, then molecule has a molecular weight of about or at 50 to about or cooled to a temperature at which microspheres are formed. at 1000 Daltons. The small molecule can be selected from For example, for some macromolecules and small molecules, among haptens, hormones, prostaglandins, antibiotics, che the compound can be heated in solution to about or at 35°C., motherapeutic agents, hematopoietics, anti-infective agents, 37° C., 40° C. 450 C., 50° C., 60° C., 650 C., 70° C., 75° C., antiulcer agents, antiallergic agents, antipyretics, analgesics, 80° C., 85°C., 90° C.,950 C., 100° C. 1250 C., 150° C. 1750 anti-inflammatory agents, antidementia agents, antiviral C., 200° C. or greater, then cooled to a temperature of, for agents, antitumor agents, antidepressants, psychotropic example, about or at 190° C., 170° C., 150° C., 125° C., 100° agents, cardiotonics, diuretics, antiarrhythmic agents, vasodi C., 80° C., 75° C., 60° C., 50° C., 40° C., 30° C., 20° C. 150 lators, antihypertensive agents, antidiabetic agents, antico C. or lower, at which the microspheres are formed. agulants, and cholesterol lowering agents. For example, the 0016. The order in which the steps are performed can be Small molecule can be an antibiotic, and can be selected from varied. For example, steps a) and b) can be performed simul among aminoglycosides, ansamycins, carbacephem, carbap taneously, sequentially, intermittently, or in any order, fol enems, cephalosporins, macrollides, penicillins, quinolones, lowed by step c). In other examples, steps b) and c) are Sulfonamides and tetracyclines. In instances where the anti performed simultaneously, sequentially, intermittently, or in biotic is an aminoglycoside, the aminoglycoside can be kana any order, preceded by step a). In a further example, steps a) mycin or tobramycin. In instances where the Small molecule and c) are performed simultaneously. In other embodiments, is an antiviral agent, the antiviral agent can be for treatment of steps a), b) and c) are performed sequentially in the order: a). influenza, parainfluenza or respiratory syncytial virus-medi thenb), then c). In some embodiments, the counterion and the ated infections. In some examples, the antiviral agent is Zan compound are identical to one another. In other embodi amivir or oseltamivir phosphate. In embodiments where the ments, the compound and the counterion are different from Small molecule is a chemotherapeutic agent, the chemothera one another. In other examples, the counterion and the anti peutic agent can be selected from among alkylating agents, Solvent are identical to one another. anthracyclines, cytoskeletal disruptors, epothilones, inhibi 0017. The compound can be a small molecule or a mac tors of topoisomerase II, nucleotide analogs, platinum-based romolecule. In instances where the compound is a macromol agents, retinoids and Vinca alkaloids. In some examples, the ecule, the macromolecule can have a molecular weight of chemotherapeutic agent is a cytoskeletal disruptor, and the about or at 1000 or 1000 to about or at five billion or five cytoskeletal disruptor is paclitaxel. I other examples, the billion Daltons; about or at 1000 or 1000 to about or at one Small molecule is a prostaglandin. billion or one billion Daltons; about or at 1000 or 1000 to 0020. In some embodiments, the macromolecule in the about or at 50 million or 50 million Daltons; about or at 1000 methods presented herein is a nucleic acid. The nucleic acid or 1000 to about or at 20 million or 20 million Daltons; about can be selected from among DNA, RNA and PNA. In or at 1000 or 1000 to about or at 15 million or 15 million instances where the nucleic acid is RNA, the RNA can be Daltons; about or at 1000 or 1000 to about or at 10 million or siRNA, snRNA, tRNA or a ribozyme. In some examples, the 10 million Daltons; about or at 1000 or 1000 to about or at 5 macromolecule is a virus, and the virus is tobacco mosaic million or 5 million Daltons; about or at 1000 or 1000 to about virus. In other embodiments, the macromolecule is a glyco or at one million or one million Daltons; about or at 1000 or peptides, and the glycopeptide is Vancomycin. In further 1000 to about or at 500,000 or 500,000 Daltons; about or at embodiments, the macromolecule is a peptide. For example, 1000 or 1000 to about or at 300,000 or 300,000 Daltons; about the peptide can be leuprolide or somatostatin. US 2009/00982O7 A1 Apr. 16, 2009

0021. The solvent used in the methods provided hereincan or 85%, w/w; about 20% or 20% to about 60% or 60%, w/w: be miscible or partially miscible with the antisolvent. The about 25% or 25% to about 55% or 55%, w/w; about 30% or 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 compound is 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 US 2009/00982O7 A1 Apr. 16, 2009

at 0.01° C./min or 0.01° C./min to about or at 20° C./min or agents, antiallergic agents, antipyretics, analgesics, anti-in 20°C/min: from about or at 0.05°C/min or about or at 0.1° flammatory agents, antidementia agents, antiviral agents, 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, US 2009/00982O7 A1 Apr. 16, 2009

taste and/or odor modifiers or masking agents, vitamins, Sug um to about 5.0 um or 5.0 um; from about 1.0 um or 1.0 um to ars, therapeutic agents, anti-oxidants, immuno-modulators, about 5.0 um or 5.0 um; or from about 1.0 um to about 2.0, trans-membrane transport modifiers, anti-caking agents, chi 3.0, 4.0 or 5.0 um. tosans or flowability enhancers. In some aspects, the compo 0046. Also provided herein are articles of manufacture sition has a shelflife of from about or at one week to about or containing the composition provided herein, a packaging at 1 month, from about or at 1 month to about or at six months, material for the composition and a label that indicates that the from about or at six months to about oratone year, from about composition is for a therapeutic, nutraceutical or cosmetic or at 1 year to about or at 2 years, or from about or at 2 years indication. In some examples, the composition used in the to about or at 5 years at a temperature of about or at 55°C., 50° article is for a therapeutic indication, Such as, for example, C., 45° C., 44° C., 42°C., 40° C., 39° C., 38°C., 37° C. or cancer, influenza, parainfluenza or respiratory disorders. The below. article of can further contain an inhaler for pulmonary admin 0041. The compositions provided herein also can contain istration of the composition. In some embodiments, the an active agent. The active agent can be selected from among inhaler is a dry powder inhaler, a metered dose inhaler or an antibiotics, chemotherapeutic agents, antidiabetics, anticon electrostatic delivery device. Vulsants, analgesics, antiparkinsons, anti-inflammatories, 0047 Provided herein are methods of preventing or treat calcium antagonists, anesthetics, antimicrobials, antimalari ing an infectious disease, by administering a therapeutically als, antiparasitics, antihypertensives, antihistamines, anti effective amount of the composition provided herein to a pyretics, alpha-adrenergicagonists, alpha-blockers, biocides, Subject. In some aspects, the infectious disease is selected bactericides, bronchial dilators, beta-adrenergic blocking from among arboviral infections, botulism, brucellosis, can drugs, contraceptives, cardiovascular drugs, calcium channel didiasis, campylobacteriosis, chickenpox, chlamydia, chol inhibitors, depressants, diagnostics, diuretics, electrolytes, era, coronovirus infections, staphylococcus infections, cox enzymes, hypnotics, hormones, hypoglycemics, hyperglyce sackie virus infections, Creutzfeldt-Jakob disease, mics, muscle contractants, muscle relaxants, neoplastics, gly cryptosporidiosis, cyclospora infection, cytomegalovirus coproteins, nucleoproteins, lipoproteins, ophthalmics, psy infections, Epstein-Barr virus infection, dengue fever, diph chic energizers, sedatives, Steroids, sympathomimetics, theria, ear infections, encephalitis, influenza virus infections, parasympathomimetics, tranquilizers, urinary tract drugs, parainfluenza virus infections giardiasis, gonorrhea, Haemo vaccines, vaginal drugs, nonsteroidal anti-inflammatory philus influenzae infections, hantavirus infections, viral hepa drugs, angiotensin converting enzymes, polynucleotides, titis, herpes simplex virus infections, HIV/AIDS, helico polypeptides, polysaccharides, enzymes, hormones, vita 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 2009/00982O7 A1 Apr. 16, 2009

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 0066. 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: 0067. 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., whereby a composition containing a naturally occurring or chemically synthesized organic or microparticles of a virus is formed, where steps (a) and (b) are inorganic molecule that is greater than or equal to about a performed simultaneously, sequentially, intermittently, or in 1000 Daltons to about or greater than 1, 2, 3, 5, 7, 10 or more any order. trillion Daltons. A "macromolecule' as used herein includes 0062. 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 0063 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 0064. 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. 0065. The term “molecule' is used interchangeably herein 0068. 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 2009/00982O7 A1 Apr. 16, 2009

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 0071 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, 0069. 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 0072 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 0073. 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 0070. 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 (0074 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 2009/00982O7 A1 Apr. 16, 2009

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%, 007.9 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 0080. 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 0075. The term “consists essentially of or “consisting or electric force. essentially of as used herein refers to an entity from which I0081. 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 0076. 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 0082. 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 0083. 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 I0084 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 0077. 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 I0085. 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 0078. 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 2009/00982O7 A1 Apr. 16, 2009 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 I0086. 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 0087. 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 0092. 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)-propionamido hexanoate, 3-(2-py microparticles, also are considered “fluid as defined herein. ridyldithio)-propionylhydrazide, Ellman's reagent, dichloro 0093. 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, 0088. 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 0094. 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 0089. The term “catalytic domain” of a protein as used particles provided herein. herein refers to a protein or polypeptide in which the only 0.095 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 0096. 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 0097. 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. 0090. As used herein, the term “nucleic acid refers to 0098. 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 (0099. 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 0100. The term 'agglomerates’ refers to the association of uridine. one or more particles, such as microspheres, loosely held 0091. 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 2009/00982O7 A1 Apr. 16, 2009 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 0101 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, 0106. 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 0102 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 0107 As used herein, “hydrophobic” refers to a substance 50%, 60%, 70%, 80%, 85%, 90% 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. (0103) As used herein, the terms “activity” or “function” 0108. 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 0104. As used herein, “functional activity” also is inter of water or aqueous solution, such as a buffer. changeable with “activity.” “biological activity” or “func 0109 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 0110. 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 0105. 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 2009/00982O7 A1 Apr. 16, 2009

0111. 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 0.115. 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 0116. 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 0117. 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, 0112 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- 0113. 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 0118. 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. 0114. 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 0119. 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 nitrites, phenols and thiols. active agents specifically mentioned herein, including, but 0.120. 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 2009/00982O7 A1 Apr. 16, 2009

or 50%, at least about 60% or 60%, at least about 70% or 70%, 0.126 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 0121. 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 I0127. 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 I0128. 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 0122. 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 I0129. The rate of gradual cooling is empirically deter vent are immiscible also can be used. mined based on the type of macromolecule, solvents, coun 0123. As used herein, the term “p' 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 0124. 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 0.130 Microparticles of desired size also can be formed, polyethyleneimine (PEI). for example, by rapidly chilling the cocktail (e.g. using a heat 0.125. 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 I0131 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 2009/00982O7 A1 Apr. 16, 2009

C., -150° C., -160° C., -165° C., -170° C., -175° C., -180° 0.137 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 0132) 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 0133. 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 0.138. 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 0134. 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 0.139. 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 0135. 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 0140. 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 0.136 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 0141 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 2009/00982O7 A1 Apr. 16, 2009

novirus infections, staphylococcus infections, coxsackie 0147 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 0.148. 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 0149. 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 0142. 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 0143. 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 0150. 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 0144. 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 0145 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 0151. In one embodiment, the microspheres formed by tion of microspheres; and contacting the compound with a counterion and antisolvent 0146 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., 0152. 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 2009/00982O7 A1 Apr. 16, 2009 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 0159. The formation and characteristics of the micro ing microspheres. spheres produced by the methods provided herein can empiri 0153. 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 0154 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 (0160 Molecules terion, a Substantial number of the charged groups, in some 0.161 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 0155 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 0156. 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 0162. 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 0157. 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 0163 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. 0164 Alkyl refers to straight or branched chain substi 0158. 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 2009/00982O7 A1 Apr. 16, 2009

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 0.165 “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, Co or more unsaturated bonds, and those that are Substituted. alkyl, C-haloalkyl, C-hydroxy-alkyl, C-aminoalkyl, Examples of cycloalkyls include, but are not limited to, cyclo Co-alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, propane, cyclobutane, cyclopentane, cyclopentene, cyclo Sulfamoyl, or trifluoromethyl. Examples of heteroaryl groups pentadiene, cyclohexane, cyclohexene, 1.3-cyclohexadiene, include, but are not limited to, unsubstituted and mono- or 1,4-cyclohexadiene, cycloheptane and cycloheptene. di-substituted derivatives of furan, benzofuran, thiophene, 0166 “Heterocyclic' compounds, which are rings where benzothiophene, pyrrole, pyridine, indole, oxazole, benzox at least one atom forming the ring is a carbonatom and at least azole, isoxazole, benzisoxazole, thiazole, benzothiazole, one atom forming the ring is a heteroatom. isothiazole, imidazole, benzimidazole, pyrazole, indazole, 0167 "Bicyclic ring, which refers to two rings that are tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, fused. Bicyclic rings include, for example, decaline, pental purine and pyrazine, furazan, 1.2.3-oxadiazole, 1,2,3-thiadia ene, naphthalene, aZulene, heptalene, isobenzofuran, Zole, 1,2,4-thiadiazole, triazole, benzotriazole, pteridine, chromene, indolizine, isoindole, indole, purine, indoline, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinno indene, quinolizine, isoquinoline, quinoline, phthalazine, line, phthalazine, quinazoline and quinoxaline. Substituents naphthyrididine, quinoxaline, cinnoline, pteridine, isochro can be, for example, halo, hydroxy, cyano, O—C-alkyl, man, chroman and various hydrogenated derivatives thereof. Co-alkyl, hydroxy-C-alkyl and amino C-alkyl. Bicyclic rings can be optionally Substituted. Each ring is 0171 “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 0168 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, 0172 “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, 0173 “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. 0.174 The Substituent groups on organic compounds can 0169 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” 0170 “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 2009/00982O7 A1 Apr. 16, 2009

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 0175 Macromolecules and Small Molecules prostaglandins. 0176 The compounds used to form microparticles accord 0.178 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 0179 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 0177. 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 0180 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 0181 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 2009/00982O7 A1 Apr. 16, 2009

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 0.190 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; Aminopropylon, Aminopyrine, Ammonium Salicylate, Anti pyrine, Antipyrine Salicylate, Antrafenine, ApaZone, Aspirin, 0182 f-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 0183 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, 0184 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 0185 Alcohol deterrents such as Calcium Cyanamide Cit ine, Simetride, Sodium Salicylate, Sulfamipyrine, Suprofen, rated, Disulfuram, Nadide and Nitrefazole; Talniflumate, Tenoxicam, Terofenamate, Tetradrine, Tinori 0186 Aldose reductase inhibitors such as Epalrestat, dine, Tolfenamic Acid, Tolpronine, Tramadol, Viminol, Xen Ponalrestat, Sorbinil and Tolrestat; bucin and Zomepirac: 0187 Anabolics such as Androisoxazole, Androstenediol, 0191 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 0188 Analgesics (dental) such as Chlorobutanol, Clove 17B-Cypionate, Testosterone Enanthate, Testosterone Nico and Eugenol; tinate, Testosterone Pheynylacetate, Testosterone Propionate 0189 Analgesics (narcotic) such as Alfentanil, Allylpro and Tiomesterone; dine, Alphaprodine, Anileridine, Benzylmorphine, Bezitra 0.192 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 2009/00982O7 A1 Apr. 16, 2009

Myrtecaine, Naepaine, Octacaine, Orthocaine, Oxethazaine, 0202 Antiandrogens such as Bifluranol, Cyoctol, Cypro Parethoxycaine, Phenacaine Hydrochloride, Phencyclidine, terone, Delmadinone Acetate, Flutimide, Nilutamide and Phenol, Piperocaine, Piridocaine, Polidocanol, Pramoxine, Oxendolone; Prilocaine, Procaine, Propanidid, Propanocaine, Propara 0203 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, 0193 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 0204 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 0194 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, 0.195 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 0205 Antiarteriosclerotics such as Pyridinol Carbamate; Zium Iodide, Tetrachloroethylene, Tetramisole, thiabenda 0206 Antiarthritic/Antirheumatics such as Allocupreide Zole, Thymol, Thymyl N-lsoamylcarbamate, Triclofenol pip Sodium, Auranofin, Aurothioglucose, Aurothioglycanide, erazine and Urea Stibamine; Azathioprine, Calcium 3-Aurothio-2-propanol-1-sulfonate, 0196. Anthelmintics (Onchocerca) such as Ivermectin and Celecoxib, Chloroquine, Clobuzarit, Cuproxoline, Diacerein, Suramin Sodium; Glucosamine, Gold Sodium Thiomalate, Gold Sodium Thio 0.197 Anthelmintics (Schistosoma) such as Amoscanate, Amphotalide, Antimony Potassium Tartrate, Antimony sulfate, Hydroxychloroquine, KebuZone, Lobenzarit, Melit Sodium Gluconate, Antimony Sodium Tartrate, Antimony tin, Methotrexate, Myoral and Penicillamine: Sodium Thioglycollate, Antimony Thioglycollamide, Becan 0207 Antibacterial (antibiotic) drugs including: Ami thone, Hycanthone, Lucanthone Hydrochloride, Niridazole, noglycosides such as Amikacin, Apramycin, Arbekacin, Oxamniquine, Praziquantel, Stibocaptate, Stibophen and Bambermycins, Butirosin, Dibekacin, Dihdrostreptomycin, Fortimicin(s), Gentamicin, Ispamicin, Kanamycin, Microno Urea Stibamine; micin, Neomycin, Neomycin Undecylenate, Netilmicin, 0198 Anthelmintic (Trematodes) such as Anthiolimine Paromomycin, Ribostamycin, Sisomicin, Spectinomycin, and Tetrachloroethylene; Streptomycin, Streptonicozid, Vancomycin (also considered 0199 Antiacne drugs such as Adapelene, Algestone a glycopeptide) and Tobramycin; Acetophenide, AZelaic Acid, Benzoyl Peroxide, Cyoctol, Cyproterone, Motretinide, Resorcinol, Retinoic Acid, Tetro 0208 Amphenicols such as AZidamfenicol, Chloram quinone and Tretinonine; phenicol, Chloramphenicol Palmitate, Chloramphenicol 0200 Antiallergics such as Amlexanox, Astemizole, Pantothenate, Florfenicol and Thiamphenicol; AZelastine, Cromolyn, Fenpiprane. Histamine, Ibudilast, 0209 Ansamycins such as Rifamide, Rifampin, Rifamy Nedocromil, Oxatomide, Pentigetide, Poison Ivy Extract, cin and Rifaximin: Poison Oak Extract, Poison Sumac Extract, Repirinast, Tra 0210 B-Lactams, including: Carbapenems such as Imi nilast, TraxanoX and Urushiol: penem; 0201 Antiamebics such as Arsthinol, Bialamicol, Carbar 0211 Cephalosporins such as Cefactor, Cefadroxil, Cefa sone, Cephaeline, Chlorbetamide, Chloroquine, Chlorphe mandole, Cefatrizine, Cefazedone, Cefazolin, Cefixime, Cef noxamide, Chlortetracycline, Dehydroemetime, Dibro menoxime, 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, Cephalosporin, Zole, Sulfarside, Teclozan, Tetracycline. Thiocarbamizine, Cephalothin, Cephapirin Sodium, Cephradine and Piveefal Thiocarbarsone and Tinidazole; eX1n, US 2009/00982O7 A1 Apr. 16, 2009 20

0212 Cephamycins such as Cefbuperazone, Cefnmetazole, sulfathiazole, Sulfabenzamide, Sulfacetamide, Sulfachlorpy Cefninox, Cefetan and Cefoxitin; ridazine, Sulfachrysoidine, Sulfacytine, Sulfadiazine, 0213 Monobactams such as Aztreonam, Carumonam and Sulfadicramide, Sulfadimethoxine, Sulfadoxine, Sulfaethi Tigemonam, dole, Sulfaguanidine, Sulfaguanol, Sulfalene, Sulfaloxic 0214 Oxacephems such as Flomoxef and Moxolactam; Acid, Sulfamerazine, Sulfameter, Sulfamethazine, Sulfame 0215 Penicillins such as Amidinocillin, Amdinocillin thizole, Sulfamethomidine, Sulfamethoxazole, Sul Pivoxil, Amoxicillin, Ampicillin, Apalcillin, Aspoxicillin, famethoxypyridazine, Sulfametrole, Sulfamidochrysoidine, AZidocillan, AZiocillan, 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, Meziocillin, Nafcillin Sodium, Oxacil Sulfasymazine, Sulfathiazole, Sulfathiourea, Sulfatolamide, lin, Penamecillin, Penethamate Hydriodide, Penicillin G Sulfisomidine and Sulfisoxazole; Benethamine, Penicillin G BenZathine, Penicillin G Benzhy 0225. 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, V Hydrabamine, Penimepicycline, Phenethicillin Potassium, N'digalactoside, Sulfoxone Sodium and Thiazolsulfone; and Piperacillin, Pivapicillin, Propicillin, Quinacillin, Sulbenicil 0226 others such as Clofoctol, Hexedine, Methenamine, lin, Talampicillin, Temocillin and Ticarcillin; Methenamine Anhydromethylene-citrate, Methenamine 0216 Lincosamides Such as Clindamycin and Lincomy Hippurate, Methenamine Mandelate, Methenamine Sul cin; fosalicylate, Nitroxoline and Xibornol: 0217 Macrollides such as Azithroimycin, Carbomycin, 0227 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 0218 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, 0219 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, 0220 other antibiotics such as Cycloserine, Mupirocin Mecloxamine, Mepenzolate Bromide, Metcaraphen, Meth and Tuberin; antheline Bromide, Methixene, Methscopolamine Bromide, 0221) 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 0222 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 0223 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: 0224 Sulfonamides such as Acetyl Sulfamethoxypyra 0228 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 2009/00982O7 A1 Apr. 16, 2009

Lomactil, Magnesium Bromide, Magnesium Sulfate, Mephe 0242 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, 0243 Antifungal drugs (synthetic), including: Ally Sodium Bromide, Sodium Valproate, Solanum, Strontium lamines such as Naftifine and Terbinafine; Bromide, Suclofenide, Sulthiame, Tetrantoin, Tiagabine, Tri 0244 Imidazoles such as Bifonazole, Butoconazole, methadione, Valproic Acid, Valpromide, Vigabatrin and Chlordantoin, Chlormidazole, Cloconazole, Clotrimazole, Zonisamide: Econazole, Enilconazole, Fenticonazole, Isoconazole, Keto 0229 Antidepressants, including: Bicyclics such as conazole, Miconazole, Omoconazole, Oxiconazole, Nitrate, Binedaline, CaroXaZone, Citalopram, Dimethazan, Indal Sulconazole and Tioconazole; pine, Fencamine, Fluvoxamine Maleate, Indeloxazine 0245 Triazoles such as Fluconazole, Itraconazole and Hydrochcloride, Nefopam, Nomifensine, Oxitriptan, Terconazole; and Oxypertine, Paroxetine, Sertraline. Thiazesim, Trazodone, 0246 others such as Acrisorcin, Amorolfine, Biphe Venlafaxine and Zometapine; namine, Bromosalicylchloranilide, Buclosamide, Calcium 0230 Hydrazides/Hydrazines such as Benmoxine, Ipro Propionate, Chlophenesin, Ciclopirox, Cloxyquin, Coparaf clozide, Iproniazid, Isocarboxazid, Nialamide, Octamoxin finate, Diamthazole, Dihydrochloride. Exalamide, Flucy and Pheneizine; tosine, Halethazole, Hexetidine, Loflucarban, Nifuratel, 0231. Pyrrolidones such as Cotinine, Rolicyprine and Potassium Iodide, Propionic Acid, Pyrithione, Salicylanilide, Rolipram; Sodium Propionate, Sulbentine, Tenonitrozole, Tolciclate, 0232 Tetracyclics such as Maprotiline, Metralindole, Tolindate, Tolnaftate, Tricetin, Ujothion, Undecylenic Acid Mianserin and Oxaprotiline: and Zinc Propionate; 0233 Tricyclics such as Adinazolam, Amitriptyline, Ami 0247 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, 0248 Antigonadotropins such as Danazol, Gestrinone and Propizepine, Protriptyline, Quinupramine, Tianeptine and Paroxypropione; Trimipramine; and 0249 Antigout drugs such as Allopurinol, Carprofen, 0234 others such as Adrafinil, Benactyzine, Bupropion, Colchicine, Probenecid and Sulfinpyrazone: Butacetin, Deanol, Deanol Aceglumate, Deanol Acetamido 0250 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 0251 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, 0235 Antidiabetics, including: Biguanides such as Orphenadrine, Phenyltoloxamine, Piprinhydrinate and Seta Buformin, Metformin and Phenformin; S1ne, 0236 Hormones Such as Glucagon, Insulin, Insulin Injec 0252 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 0237 Sulfonylurea derivatives such as Acetohexamide, rilene, Phenbenzamine, Pyrilamine, Talastine, Thenyidi 1-Butyl-3-metanily lurea, Carbutamide, Chlorpropamide, amine, Thonzylamine Hydrochloride, Tripelennamine and Glibornuride, Gliclazide, Glipizide, Gliquidone, Glisoxepid, Zolamine; Glyburide, Glybuthiazol(e), Glybuzole, Glyhexamide, Gly 0253 piperazines such as Cetirizine, Chlorcyclizine, Cin midine, Glypinamide, Phenbutamide, Tolazamide, Tolbuta narizine, Clocinizine and Hydroxy Zine; mide and Tolcyclamide; and 0254 Tricyclics, including: Phenothiazines such as 0238 others such as Acarbose, Calcium Mesoxalate and Ahistan, Etymemazine, Fenethazine, N-Hydroxyethyl Miglitol; promethazine Chloride, Isopromethazine, Mequitazine, 0239 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 0255 others such as AZatadine, Clobenzepam, Cyprohep eramide, Mebiquine, Trillium and Uzarin; tadine, Deptropine, Isothipendyl, Loratadine and Pro 0240 Antidiuretics such as Desmopressin, Felypressin, thipendyl; and Lypressin, Ornipressin, Oxycinchophen, Pituitary—Poste 0256 other antihistamines such as Antazoline, Astemi rior, Terlipressin and Vasopressin; Zole, AZelastine, Cetoxime, Clemizole, Clobenztropine, 0241 Antiestrogens such as Delmadinone Acetate, Diphenazoline, Diphenhydramine, Fluticasone Propionate, Ethamoxytriphetol, Tamoxifen and Toremifene: Mebhydroline, Phenindamine, Terfenadine and Tritoqualine: US 2009/00982O7 A1 Apr. 16, 2009 22

0257 Antihyperlipoproteinemics, including: Aryloxyal 0272 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, 0273 Sulfonamide derivatives such as Ambuside, Clopa Etonfibrate, Fenofibrate, Gemfibrozil, Nicofibrate, Pirifi mide, Furosemide, Indapamide, QuinethaZone, Tripamide brate, Ronifibrate, Simfibrate and Theofibrate; and Xipamide; and 0258 Bile acid sequesterants such as Cholestyramine 0274 others such as Ajmaline, Y-Aminobutyric Acid, Bufeniode, Candesartan, Chlorthalidone, Cicletaine, Ciclosi Resin, Colestipol and Polidexide: domine, Cryptenamine Tannates, Eprosartan, Fenoldopam, 0259 HMG CoA reductase inhibitors such as Fluvastatin, Floseduinan, Indoramin, Irbesartan, Ketanserin, Losartan, Lovastatin, Pravastatin Sodium and Simvastatin: Metbutamate, Mecamylamine, Methyldopa, Methyl 4-Py 0260 Nicotinic acid derivatives Aluminum Nicotinate, ridyl Ketone Thiosemicarbarzone, Metolazone, Minoxidil, Acipimox, Niceritrol, Nicoclonate, Nicomol and Oxiniacic Muzolimine, Pargyline, Pempidine, Pinacidil, Piperoxan, Acid; Primaperone, Protoveratrines, Raubasine, Rescimetol, Ril 0261) Thyroid hormones and analogs such as Etiroxate, menidene, Saralasin, Sodium Nitroprusside, Ticrynafen, Tri Thyropropic Acid and Thyroxine; and others such as Acifran, methaphan Camsylate, Tyrosinase, Urapidil and Valsartan; Azacosterol, Benfluorex, B-Benzalbutyramide, Carnitine, 0275 Antihyperthyroids such as 2-Amino-4-methylthiaz Chondroitin Sulfate, Clomestone, Detaxtran, Dextran Sulfate ole, 2-Aminothiazole, Carbimazole, 3,5-Dibromo-L-ty Sodium, 5,8,11,14, 17-Eicosapentaenoic Acid, Eritadenine, rosine, 3,5-Diiodotyrosine, Hinderin, Iodine, lothiouracil, Furazbol, Meglutol, Melinamide, Mytatrienediol, Ornithine, Methimazole, Methylthiouracil, Propylthiouracil, Sodium Perchlorate. Thibenzazoline. Thiobarbital and 2-Thiouracil; Y-Oryzanol, Pantethine, Penataerythritol Tetraacetate, C.-Phe 0276 Antihypotensive drugs such as Amezinium Methyl nylbutyramide, Pirozadil, Probucol, C-Sitosterol, Sultosilic Sulfate, Angiotensin Amide, Dimetofrine, Dopamine, Etife Acid, piperazine Salt, Tiadenol, Triparanol and Xenbucin; lmin, Etilefrin, Gepefrine, Metaraminol, Midodrine, Norepi 0262 Antihypertensive drugs, including: Arylethanola nephrine, Pholedrinead and Synephrine; mine derivatives such as Amosulalol, Bufuralol, Dilevalol, 0277 Antihypothyroid drugs such as Levothyroxine Labetalol, Pronethalol, Sotalol and Sulfinalol; Sodium, Liothyronine, Thyroid, Thyroidin, Thyroxine, 0263 Aryloxypropanolamine derivatives such as Acebu Tiratricol and TSH: tolol, Alprenolol, Arotinolol, Atenolol, Betaxolol, Bevan 0278 Anti-inflammatory (non-steroidal) drugs, includ tolol, Bisoprolol, Bopindolol, Bunitrolol, Bupranolol. Buto ing: Aminoarylcarboxylic acid derivatives such as Enfenamic filolol, Carazolol, Cartezolol, Carvedilol, Celiprolol, Acid, Etofenamate, Flufenamic Acid, Isonixin, Meclofe Cetamolol, Epanolol, Indenolol, Mepindolol, Metipranolol, namic Acid, Mefanamic Acid, Niflumic Acid, Talnifiumate, Metoprolol, Moprolol, Nadolol, Nipradilol, Oxprenolol, Pen Terofenamate and Tolfenamic Acid; butolol, Pindolol, Propranolol, Talinolol, Tetraolol, Timolol 0279 Arylacetic acid derivatives such as Acemetacin, and Toliprolol; Alclofenac, Amfenac, Bufexamac, Cinmetacin, Clopirac, 0264 Benzothiadiazine derivatives such as Althiazide, Diclofenac Sodium, Etodolac, Felbinac, Fenclofenac, Fen Bendroflumethiazide, Benzthiazide, Benzylhydrochlorothi clorac, Fenclozic Acid, Fentiazac, Glucametacin, Ibufenac, azide, Buthiazide, Chlorothiazide, Chlorthalidone, Cyclo Indomethacin, IsofeZolac, ISOXepac, Lonazolac, Metiazinic penthiazide, Cyclothiazide, Diazoxide, Epithiazide, Ethiaz Acid, Oxametacine, Proglumetacin, Sulindac, Tiaramide, ide, Fenguizone, Hydrochlorothiazide, Hydroflumethiazide, Tolimetin and Zomepirac: Methyclothiazide, Meticrane, Metolazone, Paraflutizide, 0280 Arylbutyric acid derivatives such as Bumadizon, Polythiazide, Tetrachlormethiazide and Trichlormethiazide; Butibufen, Fenbufen and Xenbucin; 0265 N-Carboxyalkyl (peptide/lactam) derivatives such 0281 Arylcarboxylic acids such as Clidanac, Ketorolac as Alacepril, Captopril, CilaZapril, Delapril, Enalapril, Enala and Tinoridine; prilat, Fosinopril, Lisinopril, Moveltipril, Perindopril, 0282 Arylpropionic acid derivatives such as Alminopro Quinapril and Ramipril; fen, Benoxaprofen, Bucloxic Acid, Carprofen, Fenoprofen, 0266 Dihydropyridine derivatives such as Amlodipine, Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam, Felodipine, Isradipine, Nicardipine, Nifedipine, Nilvadipine, Indoprofen, Ketoprofen, Loxoprofen, Miroprofen, Nisoldipine and Nitrendipirne; Naproxen, Oxaprozin, Piketoprofen, Pirprofen, Pranoprofen, Protizinic Acid, Suprofen and Tiaprofenic Acid; 0267 Guanidine derivatives such as Bethanidine, 0283 Pyrazoles such as Difenamizole and Epirizole; Debrisoquin, Guanabenz, Guanacline, Guanadrel, GuanaZo 0284. Pyrazolones such as Apazone, BenZpiperylon, dine, Guanethidine, Guanfacine, Guanochlor, Guanoxabenz Feprazone, Mofebutazone, Morazone, Oxyphenbutazone, and Guanoxan; Phenylbutazone, Pipebuzone, Propyphenazone, Ramifena 0268 Hydrazines and phthalazines such as Budralazine, Zone, SuxibuZone and Thiazolinobutazone; Cadralazine, Dihydralazine, Endralazine, Hydracarbazine, 0285 Salicylic acid derivatives such as Acetaminosalol, Hydralazine, Pheniprazine, Pildralazine and Todralazine; Aspirin, Benorylate, Bromosaligenin, Calcium Acetylsalicy 0269. Imidazole derivatives such as Clonidine, Lofexi late. Diflunisal, Etersalate, Fendosal, Gentisic Acid, Glycol dine, Phentolamine, Phentolamine Mesylate, Tiamenidine Salicylate, Imidazole Salicylate, Lysine Acetylsalicylate, and Tolonidine; Mesalamine, Morpholine Salicylate, 1-Naphthyl Salicylate, 0270 Quaternary ammonium compounds Azamethonium Olsalazine, Parsalmide, PhenylAcetylsalicylate, Phenyl Sali Bromide, Chlorisondamine Chloride, Hexamethonium, Pen cylate, Salacetamide, Salicylamine O-Acetic Acid, Salicyl tacynium Bis(methyl sulfate), Pentamethonium Bromide, sulfuric Acid, Salsalate and Sulfasalazine; Pentolinium Tartate, Phenactopinium Chloride and Trime 0286. Thiazinecarboxamides such as Droxicam, Isoxi thidiunum Methosulfate; cam, Piroxicam and Tenoxicam, and 0271 Quinazoline derivatives such as Alfuzosin, 0287 others such as e-Acetamidocaproic Acid, S-Adeno BunaZosin, Doxazosin, Prasosin, Terazosin and TrimaZosin; Sylmethionine, 3-Amino-4-hydroxybutyric Acid, Amix US 2009/00982O7 A1 Apr. 16, 2009 etrine, Bendazac, BenZydamine, Bucolome. Difempiramide, 0299 Pyrimidine analogs such as Ancitabine, AZacitidine, Ditazol, EmorfaZone, GuaiaZulene, Nabumetone, Nime 6-AZauridine, Carmofur, Cytarabine, Doxifluridine, Enocit sulide, Orgotein, Oxaceprol, Paranyline, Perisoxal, Pifoxime, abine, Floxuridine Fluoroouracil and Tegafur; ProquaZone, Proxazole and Tenidap: 0300 Enzymes such as L-Asparaginase; and 0288 Antimalarial drugs such as Acedapsone, Amodi 0301 others such as Aceglatone, Amsacrine, Bestrabucil, aquin, Arteether, Artemether, Artemisinin, Artesunate, Bisantrene, Bryostatin 1, Carboplatin, Cisplatin, Defofa Bebeerine, Berberine, Chirata, Chlorguanide, Chloroquine, mide, Demecolcine, Diaziquone, Elformithine, Elliptinium Chlorproguanil, Cinchona, Cinchonidine, Cinchonine, Acetate, Etoglucid, Etoposide, Gallium Nitrate, Hydrox Cycloguanil, Gentiopicrin, Halofantrine, Hydroxychloro yurea, Interferon-C. Interferon-?3, Interferon-Y, Interleukine quine, Mefloquine Hydrochloride, 3-Methylarsacetin, Pam 2, Lentinan, Letrozole, Lonidamine, MitoguaZone, Mitox antrone, Mopidamol, Nitracrine, Pentostatin, Phenamet, aquine, Plasmocid, Primaquine, Pyrimethamine, Quinacrine, Pirarubicin, Podophyllinicc Acid, 2-Ethylhydrazide, Polyni Quinine, Quinine Bisulfate, Quinine Carbonate, Quinine trocubanes, Procarbazine, PSK7, Razoxane, Sizofuran, Dihydrobromide, Quinine Dihydrochloride, Quinine Ethyl Spirogermanium, Taxol. Teniposide, TenuaZonic Acid, Tri carbonate, Quinine Formate, Quinine Gluconate, Quinine aziquone, 2.2.2"-Trichlorotriethylamine, Urethan, Vinblas Hydriodide, Quinine Hydrochloride, Quinine Salicylate, tine, Vincristine, Vindesine and Vinorelbine; Quinine Sulfate, Quinine Tannate, Quinine Urea Hydrochlo 0302 Antineoplastic (hormonal) drugs, including: Andro ride, Quinocide, Quinoline and Sodium Arsenate Diabasic; gens such as Calusterone, Dromostanolone Propionate, Epi 0289 Antimigraine drugs such as Alpiropride, Dihydro tioStanol, Mepitiostane and Testolactone; ergotamine, Eletriptan, Ergocornine, Ergocorninine, Ergoc 0303 Antiadrenals such as Aminoglutethimide, Mitotane ryptine, Ergot, Ergotamine, Flumedroxone acetate, Fonazine, and Trilostane; Lisuride, Methysergid(e), Naratriptan, Oxetorone, Pizoty 0304 Antiandrogens such as Flutamide and Nilutamide: line, Rizatriptan and Sumatriptan; and 0290 Antinauseant drugs such as Acetylleucine Monoet 0305 Antiestrogens such as Tamoxifen and Toremifene: hanolamine, Alizapride, BenZquinamide, Bietanautine, Bro 0306 Antineoplastic adjuncts including folic acid replen mopride, Buclizine, Chlorpromazine, Clebopride, Cyclizine, ishers such as Frolinic Acid; Dimenhydrinate, Dipheniodol, Domperidone, Granisetron, 0307 Antiparkinsonian drugs such as Amantadine, Meclizine, Methalltal, Metoclopramide, Metopimazine, Benserazide, Bietanautine, Biperiden, Bromocriptine, Nabilone, Ondansteron, Oxypendyl, Pipamazine, Piprinhy Budipine, Cabergoline, Carbidopa, Deprenyl (a/k/a L-depre drinate, Prochlorperazine, Scopolamine, Tetrahydrocannab nyl, L-deprenil, L-deprenaline and selegiline), Dexetimide, inols. Thiethylperazine. Thioproperzaine and Trimethoben Diethazine, Diphenhydramine, Droxidopa, Ethopropazine, Ethylbenzhydramine, Levodopa, Naxagolide, Pergolide, Zamide; Piroheptine, Pramipexole, Pridinol, Prodipine, Quinpirole, 0291 Antineoplastic drugs, including: Alkylating agents, Remacemide, Ropinirole, Terguride, Tigloidine and Trihex Such as Alkyl Sulfonates such as BuSulfan, ImproSulfan and yphenidyl Hydrochloride; Piposulfan; 0308 Antipheochromocytoma drugs such as Metyrosine, 0292 AZiridines such as Benzodepa, Carboquone, Phenoxybenzamine and Phentolamine; Meturedepa and Uredepa; 0309 Antipneumocystis drugs such as Efformithine, Pen 0293 Ethylenimines and methylmelamines such as Altre tamidine and Sulfamethoxazole; tamine, Triethylenemelamine, Triethylenephosphoramide, 0310 Antiprostatic hypertrophydrugs such as Gestonor Triethylenethiophosphoramide and Trimethylolomelamine; one Caproate, Mepartricin, Oxendolone and Proscar7; 0294 Nitrogen mustards such as Chlorambucil, Chlor 0311 Antiprotozoal drugs (Leshmania) Such as Antimony naphazine, Chclophosphamide, Estramustine, Ifosfamide, Sodium Gluconate, Ethylstibamine, Hydroxy stilbamidine, Mechlorethamine, Mechlorethamine Oxide Hydrochloride, N-Methylglucamine, Pentamidine, Stilbamidine and Urea Melphalan, Novembichin, Phenesterine, Prednimustine, Tro Stibamine; fosfamide and Uracil Mustard; 0312 Antiprotozoal drugs (Trichomonas) such as Acetar Sone, Aminitrozole, Anisomycin, AZanidazole, Forminitra 0295 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; 0313 Antiprotozoal drugs (Trypanosma) such as Ben 0296 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, 0314 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 0297 Antimetabolites, including: Folic acid analogs such Trimeprazine; as Denopterin, Methotrexate, Pteropterin and Trimetrexate: 0315 Antipsoriatic drugs such as Acitretin, Ammonium 0298 Purine analogs such as Fludarabine, 6-Mercaptopu Salicylate, Anthralin, 6-AZauridine, Bergapten(e), Chrysar rine. Thiamiprine and Thioguanaine; and obin, Etretinate and Pyrogallol; US 2009/00982O7 A1 Apr. 16, 2009 24

0316 Antipsychotic drugs, including: Butyrophenones 0327 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; 0328 Phenols such as Acetomeroctol, Bithionol, Cad 0317 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; 0329 Quinolines such as Aminoquinuride, BenZOxiduine, Metofenazate, Oxaflumazine, Perazine, Pericyazine, Broxyquinoline, Chloroxine, Chlorquinaldol, Cloxyquin, Perimethazine, Perphenazine, Piperacetazine, Pipotiazine, Ethylhydrocupreine, Euprocin, Halquinol, Hydrastine, 8-Hy Prochlorperazine, Promazine, Sulforidazine. Thiopropazate, droxquinoline, 8-Hydroxquinoline Sulfate and Iodochlorhy Thioridazine, Trifluoperazine and Triflupromazine: droxyquin; and 0318. Thioxanthenes such as Chlorprothixene, Clo 0330 others such as Aluminum Acetate Solution, Alumi penthixol, Flupentixol and Thiothixene; num Subacetate Solution, Aluminum Sulfate, 3-Amino-4- 0319 other tricyclics such as Benzquinamide, hydroxybutyric Acid, Boric Acid, Chlorhexidine, Chloroazo Carpipramine, Clocapramine, Clomacran, Clothiapine, din, m-CresylAcetate, Cupric Sulfate, Dibromopropamidine, Clozapine, Opipramol, Prothipendyl, Tetrabenazine, and lchthammol, Negatol7, Noxytiolin, Ornidazole, B-Propiolac Zotepine; and tone, C-Terpineol; 0320 others such as Alizapride, Amisui pride, Buramate, 0331 Antispasmodic drugs such as Alibendol, Ambuceta Fluspirilene, Molindone, Penfluridol, Pimozide, Spirilene mide, Aminopromazine, Apoatropine, Bevonium Methyl and Sulpiride; Sulfate, Bietamiverine. Butaverine, Butropium Bromide, 0321) Antipyretics Such as Acetaminophen, Acetaminos N-Butylscopolammonium Bromide, Caroverine, alol, Acetanilide, Aconine, Aconite, Aconitine, Alclofenac, Cimetropium Bromide, Cinnamedrine, Clebopride, Coniine Aluminum Bis(acetylsalicylate), Aminochlorthenoxazin, Hydrobromide, Coniine Hydrochloride, Cyclonium Iodide, Aminopyrine, Aspirin, Benorylate, BenZydamine, Berberine, Difemerine, Diisopromine, Dioxaphetyl Butyrate, Diponium p-Bromoacetanilide, Bufexamac, Bumadizon, Calcium Bromide, Drofenine, Emepronium Bromide, Ethaverine, Acetysalicylate, Chlorthenoxazin(e), Choline Salicylate, Feclemine, Fenalamide, Fenoverine, Fenpiprane, Fenpiver Clidanac, Dihydroxyaluminum Acetylsalicylate, Dipyro inium Brcmide, Fentonium Bromide, Flavoxate, Flopropi cetyl, Dipyrone, Epirizole, Etersalate. Imidazole Salicylate, one, Gluconic Acid, Guaiactamine, Hydramitrazine, Hyme Indomethacin, IsofeZolac, p-Lactophenetide, Lysine Acetyl cromone, Leiopyrrole, Mebeverine, Moxaverine, Nafiverine, salicylate, Magnesium Acetylsalicylate, Meclofenamic Acid, Octamylamine, Octaverine, Pentapiperide, Phenamacide Morazone, Morpholine Salicylate, Naproxen, NifenaZone, Hydrochloride, Phloroglucinol, Pinaverium Bromide, Piperi 51-Nitro-2'-propoxyacetanilide, Phenacetin, Phenicarbazide, late, Pipoxolan Hydrochloride, Pramiverin, Prifinium Bro Phenocoll, Phenopyrazone, PhenylAcetylsalicylate, Phenyl mide, Properidine, Propivane, Propyromazine, Prozapine, Salicylate, PipebuZone, Propacetamol, Propyphenazone, Racefemine, Rociverine, Spasmolytol, Stilonium Iodide, Ramifenazone, Salacetamide, Salicylamide O-Acetic Acid, Sultroponium, Tiemonium Iodide, Tiquizium Bromide, Tiro Sodium Salicylate, Sulfamipyrine, Tetrandrine and Tinori pramide, Trepibutone, Tricromyl, Trifolium, Trimebutine, dine; N.N-1Trimethyl-3,3-diphenyl-propylamine, Tropenzile, Tro 0322 Antirickettsial drugs such as p-Aminobenzoic Acid, spium Chloride and Xenylropium Bromide: Chloramphenicol, Chloramphenicol Palmitate, Chloram 0332 Antithrombotic drugs such as Anagrelide, Arga troban, Cilostazol, Chrysoptin, Daltroban, Defibrotide, phenicol Pantothenate and Tetracycline; Enoxaparin, Fraxiparine7, Indobufen, Lamoparan, OZagrel, 0323 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; 0333 Antitussive drugs such as Allocamide, Amicibone, 0324 Antiseptics, including: Guanidines such as Alexi Benproperine, BenZonatate, Bibenzonium Bromide, Bromo dine, Ambazone, Chlorhexidine and Picloxydine: form, Butamirate. Butethamate, Caramiphen Ethanedisul 0325 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, lodic 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 0326 Mercurial compounds such as Hydragaphen, Mera nile, Levopropoxyphene, Morclofone, 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 0334 Antiulcerative drugs such as Aceglutamide Alumi Triiodomercurate (II) Solution. Thimerfonate Sodium and num Complex, 6-Acetamidocaproic Acid Zinc Salt, Acetox Thimerosal; olone, Arbaprostil, Benexate Hydrochloride, Bismuth Subci US 2009/00982O7 A1 Apr. 16, 2009

trate Sol (Dried), Carbenoxolone, Cetraxate, Cimetidine, 0348 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, 0349 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 0350 piperazine derivatives such as Cinnarizine, Flunar midine; isine and Lidoflazine; and others such as Bencyclane, 0335 Antiurolithic drugs such as Acetohydroxamic Acid, Etafenone and Perhexyline: Allopurinol, Potassium Citrate and Succinimide: 0351 Calcium regulators such as Calcifediol, Calcitonin, 0336 Antivenin drugs such as Lyovac7 Antivenin, Calcitriol, Clodronic Acid, Dihydrotachysterol, Elcatonin, 0337 Antiviral drugs, including: Purines and pyrimidino Etidronic Acid, Ipriflavone, Pamidronic Acid, Parathyroid nes such as Acyclovir, Cytarabine, Dideoxyadenosine, Hormone and Teriparatide Acetate; Dideoxycytidine, Dideoxyinosine, Edoxudine, Floxuridine, 0352 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, 0338 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, Ibopamine, 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; 0339. Anxiolytic drugs, including: Arylpiperazines such 0353 Chelating agents such as Deferozmine, Ditiocarb as Buspirone, Gepirone, Ipsapirone and TondoSpirone; Sodium, Edetate Calcium Disodium, Edetate Disodium, 0340 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 0354 Cholecystokinin antagonists such as Proglumide: Loflazepate, Etizolam, Fluidazepam, Flutazolam, Fluto 0355 Cholelitholytic agents such as Chenodiol, Methyl prazepam, Halazepam, Ketazolam, Lorazepam, Loxapine, tert-Butyl Ether, Monooctanoin and Ursodiol; Medazepam, Metaclazepam, Mexazolam, Nordazepam, 0356 Choleretics such as Alibendol, Anethole Trithion, Oxazepam, Oxazolam, Pinazepam, Prazepam and Tofiso AZintamide, Cholic Acid, Cicrotoic Acid, Clanobutin, pam, Cyclobutyrol, Cyclovalone, Cynarin(e), Dehydrocholic 0341 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 nyl)-2-phenylacrylic Acid, Metochalcone, Moquizone, 0342 others such as Alpidem, Benzoctamine, Captodi Osalmid, Ox Bile Extract, 4.4'-Oxydi-2-butanol, Piprozolin, amine, ChlormeZanone, Etifoxine, Flesinoxan, Fluoresone, Prozapine, 4-Salicyloylmorpholine, Sincalide, Taurocholic Glutamic Acid, Hydroxyzine, Lesopitron, Mecloralurea, Acid, Timonacic, Tocamphyl, Trepibutone and Vanitiolide; Mephenoxalone, Mirtazepine, Oxanamide, Phenaglycodol, 0357 Cholinergic agents such as Aceclidine, Acetylcho Suriclone and Zatosetron; line Bromide, Acetylcholide Chloride, Aclatonium Napadisi 0343 Benzodiazepine antagonists such as Flumazenil; late, BenZpyrinium Bromide, Bethanechol chloride, Carba 0344 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, 0358 Cholinesterase inhibitors such as Ambenonium Terbutaline and Tulobuterol; Chloride, Distigmine Bromide and Galanthamine: 0345 Quaternary ammonium compounds such as Bevo 0359 Cholinesterase reactivators such as Obidoximine nium Methyl Sulfate, Clutropium Bromide, Ipratropium Bro Chloride and Pralidoxime Chloride; mide and Oxitropium Bromide: 0360 Central nervous system stimulants and agents such 0346 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, Diethipropion, Theobromine, 1-Theobromineacetic Acid and Theophylline: N-Ethylamphetamine, Ethamivan, Etifelmin, Etryptamine, and Fencamfamine, Fenethylline, Fenosolone, Fluorothyl, Gal 0347 others such as Fenspiride, Medibazine, Monteku anthamine, Hexacyclonate Sodium, Homocamfin, Mazindol, last, Methoxyphenanime, Tretoquinol and Zafirkulast; Megexamide, Methamphetamine, Methylphenidate, US 2009/00982O7 A1 Apr. 16, 2009 26

Nikethamide, Pemoline, Pentylenetetrazole, Phenidimetra diol Benzoate, Estradiol 17B-Cypionate, Estriol, Estrone, zine, Phenmetrazine, Phentermine, Picrotoxin, Pipradrol, Ethinyl Estradiol, Mestranol, Moxestrol, Mytatrienediol, Prolintane and Pyrovalerone; Quinestradiol and Quinestrol; 0361 Decongestants such as Amidephrine, Cafaminol, 0381 Gastric secretion inhibitors such as Enterogastrone Cyclopentamine, Ephedrine, Epinephrine, Fenoxazoline, and Octreotide; Indanazoline, Metizoline, Naphazoline, Nordefrin Hydro 0382 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, 0362. 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 0363. Desensitizing Agents such as Potassium Nitrate and Acetate, Fluprednidene Acetate, Fluprednisolone, Flurandre Citrate Oxalate; nolide. Formocortal, Halcinonide, Halometasone, Halopre 0364 Depigmentors such as Hydroquinine, Hydro done Acetate, Hydrocortamate, Hydrocortisone, Hydrocorti quinone and Monobenzone; Sone Acetate, ydrocortisone Phosphate, Hydrocortisone 0365 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 0366 Pteridines such as Furterene and Triamterene: nisone Sodium Phosphate, Prednisolone Sodium Succinate, 0367 Purines such as Acefylline, 7-Morpholinomethylth Prednisolone Sodium 21-m-Sulfobenzoate, Prednisolone eophylline, Pamabrom, Protheobromine and Theobromine; 21-Stearoylglycolate, Prednisolone Tebutate, Prednisolone 0368 Steroids such as Canrenone, Oleandrin and 21-Trimethylacetate, Prednisone, Prednival, Prednylidene, Spironolactone; Prednylidene 21-Diethylaminoacetate, Tixocortal, Triamci 0369 Sulfonamide derivatives such as Acetazolamide, nolone, Triamcinolone Acetonide, Triamcinolone Bene Ambuside, AZosemide, Bumetanide, Butazolamide, tonide and Triamcinolone Hexacetonide; Chloraminophenamide, Clofenamide, Clopamide, Clorex 0383 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 0384 Gonadotropic hormones such as LH and PMSG: ide and Xipamide; 0385 Growth hormone inhibitors such as Octreotide and 0370 Uracils such as Aminometradine and Amisometra Somatostatin; dine; 0386 Growth hormone releasing factors such as Semore 0371 others such as Amanozine, Amiloride, Arbutin, Chlorazanil, Ethacrynic Acid, Etozolin, Hydracarbazine, lin; Isosorbide, Mannitol, Metochalcone, Muzolimine, Perhexy 0387 Growth stimulants such as Somatotropin: line, Ticrynafen and Urea; 0388. Hemolytic agents such as Phenylhydrazine and Phe 0372 Dopamine receptoragonists such as Bromocriptine, nylhydrazine Dopexamine, Fenoldopam, Ibopamine, Lisuride, Naxagolide (0389) Hydrochloride; and Pergolide; 0390 Heparin antagonists such as Hexadimethrine Bro 0373) Ectoparasiticides such as Amitraz, Benzyl Ben mide and Protamines; Zoate, Carbaryl, Crotamiton, DDT. Dixanthogen, Isobornyl 0391 Hepatoprotectants such as S-Adenosylmethionine, Thiocyanoacetate Technical, Lime Sulfurated Solution, Betaine, Catechin, Citolone, Malotilate, Orazamide, Phos Lindane, Malathion, Mercuric Oleate, Mesulphen and Sul phorylcholine, Protoporphyrin IX, Silymarin-Group. Thiotic phur-Pharmaceutical; Acid and Tiopronin; 0374 Enzymes, including: Digestive enzymes such as 0392 Immunomodulators such as Amiprilose, Bucil C.-Amylase (Swine Pancreas), Lipase, Pancrelipase, Pepsin lamine, Ditiocarb Sodium, Inosine Pranobex, Interferon-y, and Rennin; Interleukin-2, Lentinan, Muroctasin, Platonin, Procodazole, 0375 Mucolytic enzymes such as Lysozyme; Tetramisole, Thymomodulin, Thymopentin and Ubenimex: 0376 Penicillin inactivating enzymes such as Penicilli 0393 Immunosuppressants such as Azathioprine, nase; and Cyclosporins and Mizoribine; 0377 Proteolytic enzymes such as Collagenase, Chymo 0394 Ion exchange resins such as Carbacrylic Resins, papain, Chymotrypsins, Papain and Trypsin; Cholestyramine 0378 Enzyme inducers (hepatic) such as Flumecinol: 0395. Resin, Colestipol, Polidexide, Resodec and Sodium 0379 Estrogens, including: Nonsteroidal estrogens such Polystyrene Sulfonate; as Benzestrol, Broparoestrol, Chlorotrianisene, Dienestrol, 0396 Lactation stimulating hormone such as Prolactin; Diethylstilbestrol, Diethylstilbestrol Diproprionate, LH-RH agonists such as Buserelin, Goserelin, Leuprolide, Dimestrol, Fosfestrol, Hexestrol, Methallenestril and Meth Nafarelin, and Triptorelin; estrol; and 0397 Lipotropic agents such as N-Acetylmethionine, 0380 Steroidal estrogens such as Colpormon, Conjugated Choline Chloride, Choline Dehydrocholate, Choline Dihy Estrogenic Hormones, Equilenin, Equilin, Estradiol, Estra drogen Citrate, Inositol, Lecithin and Methionine: US 2009/00982O7 A1 Apr. 16, 2009 27

0398 Lupus erythematosus suppressants such as Bismuth 0415 Prostaglandins and prostaglandin analogs such as Sodium Triglycollamate, Bismuth Subsalicylate, Chloro Arbaprostil, Carboprost, Enprostil, Bemeprost, Limaprost, quine and Hydroxychloroquine; Misoprostol, Ornoprostil, Prostacyclin, Prostaglandin E, 0399 Mineralcorticoids such as Aldosterone, Deoxycor Prostaglandin E. Prostagland in E. Rioprostil, Rosapros ticosterone, Deoxycorticosterone Acetate and Fludrocorti tol, Sulprostone and Trimoprostil; Sone; 0416 Protease inhibitors such as Aprotinin, Camostat, 0400 Miotic drugs such as Carbachol, Physostigmine, Gabexate and Nafamo.stat; Pilocarpine and Pilocarpus; 0417 Respiratory stimulants such as Almitrine, Beme 0401 Monoamine oxidase inhibitors such as Deprenyl, gride, Carbon Dioxide, Cropropamide, Crotethamide, Dime Iproclozide, Iproniazid, Isocarboxazid, Moclobemide, Octo fline, Dimorpholamine, Doxapram, Ethamivan, Fominoben, moxin, Pargyline, Pheneizine, Phenoxypropazine, Pivalyl Lobeline, Mepixanox, Metamivam, Nikethamide, Picro benzhydrazine, Prodipine, Toloxatone and Tranylcypromine; toxin, Pimeclone, Pyridofylline, Sodium Succinate and 0402 Mucolytic agents such as Acetylcysteine, Bromhex Tacrine; ine, Carbocysteine, Domiodol, Letosteine, Lysozyme, 0418 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, 0403 Muscle relaxants (skeletal) such as Afloqualone, Sodium Tetradecyl Sulfate and Tribenoside; Alcuronium, Atracurium Besylate, Baclofen, BenZoctamine, 0419 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, 0420 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 0421 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, 0422 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, 0404 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 04.05 Neuroprotective agents such as Dizocilpine; Vinylbital; 0406 Nootropic agents such as Aceglutamide, Acetylcar 0423 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; 0407 Ophthalmic agents such as 15-ketoprostaglandins: 0424 Bromides such as Ammonium Bromide, Calcium Bromide, Calcium Bromolactobionate, Lithium Bromide, 0408 Ovarian hormone such as Relaxin; Magnesium Bromide, Potassium Bromide and Sodium Bro 04.09 Oxytocic drugs such as Carboprost, Cargutocin, mide; Deaminooxytocin, Ergonovine, Gemeprost, Methylergono 0425 Carbamates such as Amyl Carbamate Tertiary, vine, Oxytocin, Pituitary (Posterior), Prostaglandin E. Pros Ethinamate, Hexaprpymate, Meparfynol Carbamate, taglandin F, and Sparteine; Novonal and Tricholorourethan; 0410 Pepsin inhibitors such as Sodium Amylosulfate; 0426 Chloral derivatives such as Carbocloral, Chloral 0411 Peristaltic stimulants such as Cisapride; Betaine, Chloral Formamide, Chloral Hydrate, Chloralan 0412 Progestogens Such as Allylestrenol, Anagestone, tipyrine, Dichloralphenazone, Pentaerythritol Chloral and Chlormadinone Triclofos; 0413 Acetate, Delmadinone Acetate, Demegestone, 0427 Piperidinediones such as Glutehimide, Methypry Desogestrel, Dimethisterone, Dydrogesterone, Ethisterone, lon, Piperidione, Pyrithyidione, Taglutimide and Thalido Ethynodiol, Fluorogestone Acetate, Gestodene, Gestonorone mide; Caproate, Haloprogesterone, 17-Hydroxy-16-methylene— 0428 Quinazolone derivatives such as Etaqualone, progesterone, 17C.-Hydroxyprogesterone, 17C-Hydroxyges Mecloqualone and Methaqualone; and others such as Acetal, terone Caproate, Lynestrenol, Medrogestone, MedroX Acetophenone, Aldol, Ammonium Valerate, Amphenidone, yprogesterone, Megestrol Acetate, Melengestrol, d-Bornyl C-Bromoisovalerate, d-Bornyl Isovalerate, Bromo Norethindrone, Norethynodrel, Norgesterone, Norgestimate, form, Calcium 2-Ethylbutanoate, Carfinate, C.-Chlorolose, Norgestrel, Norgestrienone, Norvinisterone, Pentagestrone, Clomethiazole, Cypripedium, Doxylamine, Etodroxizine, Progesterone, Promegestone, Quingestrone and Trengestone; Etomidate, Fenadiazole, Homofenazine, Hydrobromic Acid, 0414 Prolactin inhibitors such as Metergoline; Mecloxamine, Menthyl Valerate, Opium, Paraldehyde, Per US 2009/00982O7 A1 Apr. 16, 2009 28 lapine, Propiomazine, Rilmazafone, Sodium Oxybate, Sul Methotrexate and Tioguanine (formerly Thioguanine); Pep fonethylmethane and Sulfonmethane: tide antibiotics Such as Bleomycin; Platinum-based agents 0429 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 0430 Thyrotropic hormones such as TRH and TSH: Vinorelbine. 0431 Uricosurics such as Benzbromarone, Ethebenecid, 0443) 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 0432 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, 0444 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. 0433) 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 0445. 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%, 0434 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, 0446. 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, 0435 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 0436 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, 0437 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, 0438 Anticoagulants such as heparin; 87:9188-9192 (1990)), human serum albumin, human growth 0439 Miscellaneous such as Erythropoietin (Hematinic), hormone, parathyroid hormone and calcitonin. In some Filgrastim, Finasteride (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 0440 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 0441 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 0442 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 0447 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 US 2009/00982O7 A1 Apr. 16, 2009 29 other small molecule of interest. For example, in one embodi also can be used. Compounds that can be used as anionic ment, the Small molecule is tetracycline, the counterion is counterions also include, but are not limited to, the following: 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 0448. 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 0453 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 0449 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 0454 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. 0455 The counterions can be used as acids (e.g. sulfuric 0450 Nature and Concentration of Counterion acid) or bases (e.g. imidazole) or their salts (e.g. sodium 0451. 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, pI, 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 0456. 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 0452. 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 (p (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 US 2009/00982O7 A1 Apr. 16, 2009 30 counterion should be decreased, while failure to form micro and excipients). The solvent and antisolvent can be a combi spheres (broken glass-like crystals or flakes) or formation of nation of an aqueous liquid and a non-aqueous and/or organic microspheres below the desired size indicates that the con liquid, or both can be non-aqueous and/or organic liquids. centration of counterion should be increased. 0463 Nature and Concentration of Solvent 0457 Counterions that produce Microspheres in the 0464 A number of macromolecules and small molecules, Absence of added Compound among the microparticle-forming compounds of interest, are 0458 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 0459. The above types of counterions can be useful as a vents for water-insoluble compounds. tool for catalyzing microsphere formation in molecules that 0465. Organic solvents that can be used in the methods otherwise might not form microparticles. provided herein include alcohols and others listed as Class 3 0460 Solvent/Antisolvent System and 2 solvents in International Conference on Harmonisation 0461) 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 0466 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 0462. 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 0467 Nature and Concentration of Antisolvent cible, or partially miscible, at the temperatures used for dis 0468. 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, US 2009/00982O7 A1 Apr. 16, 2009 alcohols (methanol, ethanol. 1-propanol, isopropanol, example, empirical determinations including changing the 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 pI of the protein provides optimal microsphere formation. (DMF), dimethyl sulfoxide (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. 0469. In some embodiments, the organic solvent can be 0475. Ionic Strength volatile. In other embodiments, when incorporation of the 0476. 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. 0477 Cooling Ramp 0470 Organic solvents that can be used in the methods 0478. 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 0471 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 0472 pH and other characteristics such as size of the microspheres. In 0473. 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 0479. 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 0474. 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° US 2009/00982O7 A1 Apr. 16, 2009 32

C./min to about or at 10° C./min or about or at 15° C./min, neously. In some embodiments, a number of pH values in 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 0480. 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. 0481 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 0482 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 0486 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 0487. 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. 0488. The large scale manufacture by a batch process or by 0483 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 0489 Precipitation of the molecule into microspheres. 0484 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 0485 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 US 2009/00982O7 A1 Apr. 16, 2009 33

the vessel is placed onto a cold Surface or a cooling coil ery. Vaccine 2001; 19(17-19):2629-2636; MaaY F. Prestrel 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: 1 pump. Alternatively, the microspheres can be formed by (3):283-302: MaaYF, Nguyen PA, Hsu SW.Spray-drying of precipitation in a continuous mode, by passing the cock air-liquid interface sensitive recombinant human growth hor tail solution through a heat exchanger(s) once using a mone. J Pharm Sci 1998: 87(2):152-159; Vanbever R, Mint peristaltic pump. Zes J. D. Wang Jet al. Formulation and physical characteriza 0490 Removal of bulk liquid. The suspension of the tion of large porous particles for inhalation. Pharm Res 1999; microspheres can be concentrated using standard cen 16(11):1735-1742: Bot AI, Tarara T E. Smith DJ, Bot SR, trifugation, continuous flow centrifugation (e.g., CARR Woods C M. Weers J. G. Novel lipid-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 0491 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 0492. 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. Eur J 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 0493. 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, 0494 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 US 2009/00982O7 A1 Apr. 16, 2009 34 diagnostic agents, processed foods, dietary Supplements, include but are not limited to polysaccharides, glycans, pro 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 0499 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 0495. 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 0500. 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 0496 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 0501) 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 0497. 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 0498. 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 0502. The hydrogel microspheres can be crosslinked to molecules useful for preparation of Such microspheres decrease their solubility/erosion and to provide a more Sus US 2009/00982O7 A1 Apr. 16, 2009

tained release. Cross-linking can be performed using a variety 30 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65mg, 70 mg, 75 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 0508. 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 0503. 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 0504. 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. 0509. In other embodiments, the molecule is a macromol 0505. 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, 0506. 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 0507. 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 0510. 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 0511. 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), a 1-antitrypsin, Ecotin, eglin c. US 2009/00982O7 A1 Apr. 16, 2009 36 serpin, Pulmozyme (rhDNase), BetaxololTM, DiclofenacTM, ments, the molecule or compound also can serve as the coun doxorubicin, acetyl cysteine, leuprolide acetate, luteinizing terion and initiate and/or facilitate the formation of micro hormone releasing hormone (LHRH), (D-Tryp6)-LHRH, spheres. nafarelin acetate, insulin, Sodium insulin, Zinc insulin, prota 0516. When preparing microspheres containing a protein, mine, lysozyme, alpha-lactalbumin, basic fibroblast growth a proteinstabilizer Such as glycerol, fatty acids, Sugars such as factor (bFGF), beta-lactoglobulin, Trypsin, calcitonin, par Sucrose, ions such as Zinc, Sodium chloride, or any other athyroid hormone, carbonic anhydrase, ovalbumin, bovine protein stabilizers known to those skilled in the art can be serum albumin (BSA), human serum albumin (HSA), phos added prior to cooling the cocktail during microsphere for phorylase b, alkaline phosphatase, beta-galactosidase, IgG, mation, to minimize protein denaturation. Such stabilizers fibrinogen, poly-L-lysine, IgM, DNA, desmopressin acetate, also can be added to microspheres formulated from other macromolecules or Small molecules. growth hormone releasing factor (GHRF), Somatostatin, leu 0517. In some embodiments the microspheres can further prolide, antide, Factor VIII, G-CSF/GM-CSF, human growth be coated on the surface with suitable molecules and/or coat hormone (hGH), beta interferon, antithrombin III, alpha ing agents. Such as those that lend resistance to acids, such as interferon, alpha interferon 2b. digestive acids, or proteases. In other embodiments, the 0512. The term “macromolecule' or “small molecule' microspheres can be non-covalently coated with compounds also can include a plurality of different macromolecules and/ Such as fatty acids or lipids. The coating can be applied to the or Small molecules and includes combinations such as a com microspheres by immersion in the solubilized coating Sub bination of a pharmaceutical compound and an affinity mol stance, then spraying the microspheres with the Substance, or ecule for targeting the pharmaceutical compound to a tissue, by using other methods known to those of skill in the art. In organ or tumor requiring treatment. An affinity molecule can some embodiments, the fatty acids or lipids are added directly be, for example, a ligand or a receptor. Examples of ligands to the microsphere-forming cocktail solution. can include viruses, bacteria, polysaccharides, or toxins that 0518. Formation of the microspheres by decreasing tem can act as antigens to generate an immune response when perature can be performed by a multitude of conventional administered to an animal and cause the production of anti methods in batch or continuous modes. Microsphere forma bodies. The microspheres provided herein also can be pre tion can further be triggered by other methods including, but pared from combinations or mixtures of macromolecules and not limited to, modulating atmospheric pressure, g-force or Small molecules Surface expansion, including seeding. Microsphereformation 0513. An inhaler device can be used to deliver a therapeu can occur immediately upon exposure to these conditions or tic compound or diagnostic compound, such as those listed can require an extended period of time as provided herein. above, to the respiratory airways and lungs of a Subject. For example, protein microspheres, or antibiotic microspheres, D. Exemplary Compounds Such as Vancomycin microspheres, can be prepared, for 0519 A. Peptides example by contacting an aqueous solution of the protein or 0520 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 0521. Somatostatins panol, and cooling the Solution to form the microspheres. The 0522. 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 0514 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 0523 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 0515. In some embodiments, a molecule or compound that pancreas. does not produce microspheres of desirable characteristics, 0524. 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 US 2009/00982O7 A1 Apr. 16, 2009 37 active intestinal peptide (VIP), Gastric inhibitory polypeptide 0537 C. Chemotherapeutic Agents (GIP), Enteroglucagon (GIP); and pancreatic hormones, glu 0538 Pharmacologic agents that are useful in the treat cagon and insulin. ment of cancer fall under the general umbrella of chemothera 0525) Leuprolide peutic agents, and are contemplated for preparation in the 0526 Leuprorelin (INN) or leuprolide acetate (USAN) is form of microspheres according to the methods provided a gonadotropin-releasing hormone agonist (GnRH agonist). herein. An exemplary chemotherapeutic agent is paclitaxel. By causing constant stimulation of the pituitary GnRH recep 0539 Paclitaxel tors, it initially causes stimulation (flare), but thereafter 0540 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: 0541. D. Nucleic Acids 22. 0542) Nucleic acids, including those of therapeutic sig 0527 B. Antibiotics nificance, are contemplated for the preparation of micro 0528. 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 (0543 siRNA to form microparticles by the methods provided herein are 0544 Small interfering RNA (siRNA), sometimes known described below. as short interfering RNA or silencing RNA, are a class of 0529) Aminoglycosides 20-25 nucleotide-long double-stranded RNA molecules that 0530 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 0531 Glycopeptides screens that are designed to identify the important genes in 0532 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. Sci. 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 0533 Penicillins sequences of these siRNAs (sense and antisense Strands) are 0534 Penicillins are a class of B-lactam antibiotics that set forth in SEQID NOS: 23-26. include compounds such as Ampicillin, AZiocillin, Carbeni (0545 E. Prostaglandins cillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Meziocillin, Nafcillin, Penicillin, Piperacillin and Ticarcillin. B-lactam 0546 A prostaglandin is any member of a group of lipid antibiotics work by inhibiting the formation of peptidoglycan compounds that are derived enzymatically from fatty acids, cross links in the bacterial cell wall. The B-lactam moiety of are hormone or hormone-like, and have important and pleio penicillin binds to the enzyme (transpeptidase) that links the tropic functions and effects in the animal body. Every pros peptidoglycan molecules in bacteria, and this weakens the taglandin contains 20 carbon atoms, including a 5-carbon cell wall of the bacterium (in other words, the antibiotic ring. They are mediators and have a variety of physiological causes cytolysis or death). effects. The prostaglandins together with the thromboxanes 0535 Tetracyclines and prostacyclins form the prostanoid class of fatty acid 0536 Tetracyclines are a class of natural and synthetic derivatives; the prostanoid class is a Subclass of eicosanoids. broad-spectrum antibiotics whose members include, for 0547. Due to their pleiotropic effects, prostaglandins have example, Tetracycline, Chlortetracycline, Oxytetracycline, a variety of clinical applications, including: Demeclocycline, Semi-synthetic Doxycycline, Lymecycline, (0548. To induce childbirth, parturition or abortion (PGE Meclocycline, Methacycline, Minocycline and Rolitetracy or PGF2, with or without mifepristone, a progesterone cline. Tetracyclines inhibit cell growth by inhibiting transla antagonist); tion. The tetracyclines bind to the 16S part of the 30S ribo 0549. To prevent closure of patent ductus arteriosus in somal subunit and prevent the amino-acyl tRNA from binding newborns with particular cyanotic heart defects (PGE): to the A site of the ribosome. 0550. To prevent and treat peptic ulcers (PGE): US 2009/00982O7 A1 Apr. 16, 2009

0551 As a vasodilator in severe Raynaud's phenomenon influenza viruses and used as the receptor through which the or ischemia of a limb; virus binds and initiates infection. Human influenza viruses, 0552. In pulmonary hypertension; however, seem to prefer Neu5Ac O(2.6)-Gal, while avian and 0553. In treatment of glaucoma; and equine influenza viruses predominantly recognize Neu5Ac 0554. 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 0555 F. Viruses of sialic acids, but C.(2,6)-linked sialic acid is more abundant 0556 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 0557 Tobacco Mosaic Virus (TMV) infection process including, but not limited to, M. pneumo 0558 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 0564 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 0559. 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 0560 G. Proteins Substrate molecule. Many sialidase proteins have been puri 0561 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 0562 Sialidases sialic acid residues than can serve as receptors for pathogenic 0563 Sialidases, also referred to as neuraminidases and microorganisms. For example, among the large bacterial N-acylineuraminosylglycohydrolases, 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 0565 Sialidase-GAG. Fusion Proteins Sion, and the interaction between inflammatory cells and 0566 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 0567 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 US 2009/00982O7 A1 Apr. 16, 2009 39 cell membranes, including the Surface of respiratory epithe structural homology (up to 68% amino acid identity) (Spre lium. Many proteins specifically bind to heparin/heparan Sul cher et al. (1995) J Biol Chem 270:29854-29861). PI-8 has fate, and the GAG-binding sequences in these proteins have been shown to inhibit trypsin, thrombin, factor Xa, subtilisin been identified. For example, the GAG-binding sequences of A furin, and also chymotrypsin in vitro. It is released by 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 0572. 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 ber of serine protease inhibitors have been shown to reduce 400; Weisgraber et al. (1986) J Bio Chem 261:2068-2076). influenza virus activation in cultured cells, chicken embryos The GAG-binding sequences of these proteins are distinct and in the lungs of infected mice. The serpins bind to hemag from their receptor-binding sequences, so they do not induce glutinin (HA) molecules on the surface of the influenza virus the biological activities associated with the full-length pro and inhibit its activity, thus reducing the infectivity of the teins or the receptor-binding domains. These sequences, or virus. For example trypsin inhibitors, such as: aprotinin (Zhi other sequences that can bind heparin/heparan Sulfate, can be mov et al. (2002) JVirol 76:8682-8689), leupeptin (Zhimov et used as epithelium-anchoring-domains in sialidase-GAG al. (2002) J Virol 76:8682-8689: Tashiro et al. (1987) J Gen fusion proteins. Virol 68:2039–2043), soybean protease inhibitor (Barbey 0568. 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 Morel et al. (1987) J Infect Dis 155:667-672) have all been tein from A. viscosus set forth in SEQID NO:1. In another shown to inhibit influenza virus infection, and are candidate example, the sialidase-GAG fusion protein can contain the therapeutic agents for use in the treatment of influenza virus 394 amino acid catalytically active portion of a sialidase infection. Thus, as a related trypsin inhibitor, PI8 also can be protein from A. viscosus set forth in SEQ ID NO:2. The used as a therapeutic agent in the treatment of influenza virus GAG-binding sequence can be linked to the sialidase by infection. recombinant methods. In some examples, the fusion protein (0573 Surface Active Agents can include an amino acid linker, Such as four glycine resi 0574. The compositions provided herein can contain one dues. Furthermore, linkage can be via the N- or C-terminus of or more surface active agents that are added in an amount the GAG-binding sequence, or the N- or C-terminus of the sufficient to stabilize the cocktail solutions and/or the micro sialidase. Exemplary examples of Sialidase-GAG fusion pro spheres. The selection of an appropriate amount of Surface teins include those polypeptides set forth in SEQ ID NOS: active agent is a function of the nature of the compound, 9-13, and 17. In a further example, the sialidase and GAG Solvent and antisolvent. binding sequence components can be linked using chemical 0575. In certain embodiments, the surface active agent can or peptide linkers, by any method known in the art. be selected from sodium lauryl sulfate; sorbitan laurate, sor 0569 Proteinase Inhibitor 8 bitan palmitate, sorbitan stearate (available under the trade 0570 Proteinase inhibitor 8 (PI8), also known as Serpin name SpanR 20-40-60 etc.); polysorbates such as polyoxy B8, is a serine protease inhibitor (Serpin) Serpins are a large ethylene (20) sorbitan monolaurate, polyoxyethylene (20) Superfamily of structurally related proteins that are expressed Sorbitan monopalmitate, polyoxyethylene (20) Sorbitan in viruses, insects, plants and higher organisms, but not in monostearate (available under the tradename TWEENS(R) bacteria or yeast. Serpins regulate the activity of proteases 20-40-60 etc.); benzalkonium chloride, mixed chain phos involved in many biological process, including coagulation, pholipids, cationic lipids, oligolipids, phospholipids, car fibrinolysis, inflammation, cell migration, and tumorigenesis. nitines, sphingosines, sphingomyelins, ceramides, glycolip They contain a surface-exposed reactive site loop (RSL), ids, lipoproteins, apoproteins, amphiphilic proteins, which acts as a "bait for proteases by mimicking a protease amphiphilic peptides, amphiphilic synthetic polymers, and Substrate sequence. On binding of the target protease to the combinations thereof. Other exemplary Surface active agents serpin, the RSL is cleaved, after which the protease is for use herein include, but are not limited to covalently linked to the serpin. The protease in the newly 0576 i) Natural lipids, i.e. Cholesterol, Sphingosine and formed serpin-protease complex is inactive (Huntington et al. Derivatives, Gangliosides, Sphingosine derivatives (Soy (2000) Nature 407:923-926). Bean), Phytosphingosine and derivatives (Yeast), Choline 0571 PI8 is a member of a subfamily of serpins of which (Phosphatidylcholine), Ethanolamine (Phosphatidylethano chicken ovalbumin is the archtype. Like other serpins that lamine), Glycerol (Phosphatidyl-DL-glycerol), Inositol belong to this family, PI8 lacks a typical cleavable N-terminal (Phosphatidylinositol), Serine (Phosphatidylserine (Sodium signal sequence, resulting in a 374 amino acid protein (SEQ Salt)), Cardiolipin, Phosphatidic Acid, Egg Derived, Lyso IDNO:14) that resides mainly intracellularly. Other members (Mono Acyl) Derivatives (Lysophosphatides), Hydrogenated of this human ovalbumin-like Subfamily include plasminogen Phospholipids, Lipid Tissue Extracts, activator inhibitor type 2 (PAI-2), monocyte neutrophil 0577 ii) Synthetic lipids, i.e. Asymmetric Fatty Acid, elastase inhibitor (MNEI), squamous cell carcinoma antigen Symmetric Fatty Acid Saturated Series, Symmetric Fatty (SCCA)-1, leupin (SCCA-2) maspin (PI5), protease inhibitor Acid Unsaturated Series, Acyl Coenzyme A (Acetoyl 6 (PI6), protease inhibitor (PI9) and bomapin (PI10). Within Coenzyme A. Butanoyl Coenzyme A, Crotanoyl Coenzyme this family the serpins PI6, PI8, and PI9 show the highest A, Hexanoyl Coenzyme A, Octanoyl Coenzyme A, Decanoyl US 2009/00982O7 A1 Apr. 16, 2009 40

Coenzyme A. Lauroyl Coenzyme A, Myristoyl Coenzyme A, Ethylphosphocholine), Synthetic PAF & Derivatives Palmitoyl Coenzyme A, Stearoyl Coenzyme A, Oleoyl Coen (1-Alkyl-2-Acyl-Glycero-3-Phosphocholine & Derivatives), Zyme A, Arachidoyl Coenzyme A, Arachidonoyl Coenzyme 0582 vii) Fluorescent lipids, i.e.: Glycerol Based (Phos A, Behenoyl Coenzyme A, Tricosanoyl Coenzyme A. Ligno phatidylcholine (NBD), Phosphatidic Acid (NBD), Phos ceroyl Coenzyme A, Nervonoyl Coenzyme A, Hexacosanoyl phatidylethanolamine (NBD), Phosphatidylglycerol (NBD), Coenzyme A, Phosphatidylserine (NBD)), Sphingosine Based (Ceramide 0578 iii) Sphingolipids, i.e. D-erythro (C-18) Derivatives (NBD), Sphingomyelin (NBD), Phytosphingosine (NBD), (Sphingosine, Such as: D-erythro Sphingosine (synthetic), 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), 0583 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 0584) 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, B name SpanR 20-40-60 etc.); polysorbates such as D-Glucosyl, Sphingosine, B D-Galactosyl, Sphingosine.f3D polyoxyethylene (20) Sorbitan monolaurate, polyoxyethyl Lactosyl), Glycosylated Ceramide (D-Glucosyl-B1-1 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- 0585. 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 0586. 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 0587. The amount of surface active agent can be empiri (Soy Bean) (Glucosylceramide), Phytosphingosine Deriva cally determined and is a function of the agent selected, and tives (Yeast) (Phytosphingosine, D-ribo-Phytosphingosine the desired form of the resulting microsphere composition. 1-Phosphate, N-Acyl Phytosphingosine C2, N-Acyl The amount included can be from less than 0.1% by weight up Phytosphingosine C8, N-Acyl Phytosphingosine C18, to 35% or more. In certain embodiments, the surface active 0579 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 0580 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 0581 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% US 2009/00982O7 A1 Apr. 16, 2009 of the total weight of the composition. In other embodiments, diagnostics, diuretics, electrolytes, enzymes, hypnotics, hor the Surface active agent is presentata concentration of about mones, hypoglycemics, hyperglycemics, muscle contracta 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 0592. 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 0588 Optional Additional Agents about 0.1% up to about 7%, from about 0.1% up to about 6%, 0589. 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 0590 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 0593 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 0591 Agents can be selected from inorganic and organic drugs including, but not limited to drugs that act on the 0594. 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 0595. 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 0596. The microparticles provided herein can be used to diovascular drugs, calcium channel inhibitors, depressants, treat Infectious diseases, such as arboviral infections, botu US 2009/00982O7 A1 Apr. 16, 2009 42 lism, brucellosis, candidiasis, campylobacteriosis, chicken 0601 The articles of manufacture provided herein can pox, chlamydia, cholera, coronovirus infections, staphyllo contain packaging materials. Packaging materials for use in coccus infections, coxsackie virus infections, Creutzfeldt packaging pharmaceutical products are well known to those Jakob disease, cryptosporidiosis, cyclospora infection, of skill in the art. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052, cytomegalovirus infections, Epstein-Barr virus infection, 558 and 5,033,252. Examples of pharmaceutical packaging dengue fever, diphtheria, ear infections, encephalitis, influ materials include, but are not limited to, blister packs, bottles, enza virus infections, parainfluenza virus infections giardia tubes, inhalers, pumps, bags, vials, containers, bottles, and sis, gonorrhea, Haemophilus influenzae infections, hantavi any packaging material Suitable for a selected formulation rus infections, viral hepatitis, herpes simplex virus infections, and intended mode of administration and treatment. HIV/AIDS, helicobacter infection, human papillomavirus 0602. The following examples are included for illustrative (HPV) infections, infectious mononucleosis, legionellosis, purposes only and are not intended to limit the scope of the leprosy, leptospirosis, listeriosis, lyme disease, lymphocytic invention. choriomeningitis, malaria, measles, marburg hemorrhagic fever, meningitis, monkeypox, mumps, mycobacteria infec tion, mycoplasma infection, norwalk virus infection, pertus EXAMPLE 1. sis, pinworm infection, pneumococcal disease, Streptococcus pneumonia infection, Mycoplasma pneumoniae infection, Preparation of Microspheres of the Sialidase Fusion Moraxella catarrhalis infection, Pseudomonas aeruginosa Protein, DAS181 infection, rotavirus infection, psittacosis, rabies, respiratory syncytial virus infection, (RSV), ringworm, rocky mountain 0603 A. Purification of DAS181 spotted fever, rubella, salmonellosis, SARS, scabies, sexually 0604 DAS181 is a fusion protein containing the heparin transmitted diseases, shigellosis, shingles, sporotrichosis, (glycosaminoglycan, or GAG) binding domain from human streptococcal infections, syphilis, tetanus, trichinosis, tuber amphiregulin fused via its N-terminus to the C-terminus of a culosis, tularemia, typhoid fever, viral meningitis, bacterial catalytic domain of Actinomyces Viscosus (sequence of meningitis, west nile virus infection, yellow fever, adenovi amino acids set forth in SEQIDNO:17). The DAS181 protein rus-mediated infections and diseases, retrovirus-mediated was purified as described in Malakhov et al., Antimicrob. infectious diseases, yersiniosis Zoonoses, and any otherinfec Agents Chemother, 1470–1479, 2006, which is incorporated tious respiratory, pulmonary, dermatological, gastrointestinal in its entirety by reference herein. Briefly, the DNA fragment and urinary tract diseases. coding for DAS181 was cloned into the plasmid vector 0597 Other diseases and conditions, including arthritis, pTrc99a (Pharmacia: SEQID NO:16) under the control of a asthma, allergic conditions, Alzheimer's disease, cancers, IPTG (isopropyl-B-D-thiogalactopyranoside)-inducible pro cardiovascular disease, multiple sclerosis (MS), Parkinson's moter. The resulting construct was expressed in the BL21 disease, cystic fibrosis (CF), diabetes, non-viral hepatitis, strain of Escherichia Coli (E. Coli). hemophilia, bleeding disorders, blood disorders, genetic dis 0605. The E. Coli cells containing the expressed construct orders, hormonal disorders, kidney disease, liver disease, were lysed by sonication in 50 mM phosphate buffer, pH 8.0: neurological disorders, metabolic diseases, skin conditions, 0.3 MNaCl and 10% glycerol. The clarified lysate was passed thyroid disease, osteoporosis, obesity, stroke, anemia, through an SP-Sepharose column. Proteins were eluted from inflammatory diseases and autoimmune diseases. the column with lysis buffer that contained 0.8 MNaCl. The fraction eluted from SP-Sepharose was adjusted to 1.9 M ammonium sulfate ((NH4)2SO4), clarified by centrifugation, F. Combinations, Kits, Articles of Manufacture and loaded onto abutyl-Sepharose column. The column was 0598 Combinations and kits containing the combinations washed with two volumes of 1.3 M (NH)SO, and the provided herein, including microparticles or ingredients for DAS181 fusion protein was eluted with 0.65 M (NH)SO. forming the microparticles Such as a small molecule or a 0606 For the final step, size exclusion chromatography macromolecule of interest, counterions, solvents, buffers, or was performed on Sephacryl S-200 equilibrated with phos salts and optionally including instructions for administration phate-buffered saline (PBS). The protein purity was deter are provided. The combinations include, for example, the mined to be greater than 98% as assessed by sodium dodecyl compositions as provided herein and reagents or Solutions for Sulfate-polyacrylamide gel electrophoresis, reversed-phase diluting the compositions to a desired concentration for high-pressure liquid chromatography, and enzyme-linked administration to a host Subject, including human beings. The immunosorbent assay with antibodies generated against E. combinations also can include the compositions as provided Coli cell proteins. The purified DAS181, molecular weight herein and additional nutritional and/or therapeutic agents, 44,800 Da, was dialyzed against 2 mM sodium acetate buffer, including drugs, as provided herein. pH 5.0. 0599 Additionally provided herein are kits containing the 0607 B. Activity of DAS181 above-described combinations and optionally instructions for 0608. The sialidase activity of DAS181 was measured administration by oral, Subcutaneous, transdermal, intrave using the fluorogenic substrate 4-methylumbelliferyl-N- nous, intramuscular, ophthalmic or other routes, depending acetyl-C-D-neuraminic acid (4-MU-NANA; Sigma). One on the protein and optional additional agent(s) to be delivered. unit of Sialidase is defined as the amount of enzyme that 0600 The compositions provided herein can be packaged releases 10 nmol of MU from 4-MU-NANA in 10 minutes at as articles of manufacture containing packaging material, a 37° C. (50 mM CHCOOH NaOH buffer, pH 5.5) in a composition provided herein, and a label that indicates that reaction that contains 20 nmol of 4-MU-NANA in a 0.2 ml the composition, e.g., a DAS181 formulation or a Vancomy volume (Potieret al., Anal. Biochem.,94:287-296, 1979). The cin formulation, is formulated for oral, pulmonary or other specific activity of DAS181 was determined to be 1,300 U/mg delivery. protein (0.77 ug DAS181 protein per unit of activity). US 2009/00982O7 A1 Apr. 16, 2009

0609 C. Preparation of Microspheres Using Purified counterion (sodium citrate, 5 mM) and isopropanol organic DAS181 solvent (10%, 20% or 30%). The resulting cocktail solutions 0610 DAS181 (10 mg/ml), purified and prepared as were cooled from ambient temperature (about 25°C.) to 4° described under Section A above, was used to form 200 ul C., followed by cooling to -20°C., followed by freezing to cocktails as shown below. The cocktails contained either gly -80°C., as described in Example 1. Upon freezing to -80°C., cine or citrate as counterions, and isopropanol as organic the tubes are placed in a lyophilizer and the volatiles (water solvent, as follows: and isopropanol) were removed by Sublimation, leaving the 0611) 1) DAS181+5 mM glycine, pH 5.0; dry powder containing microspheres. 0612. 2) DAS181+5 mM glycine, pH 5.0+10% isopro 0624 Results: Microsphere formation was observed with panol: all three concentrations: 10%, 20%, or 30%, of the organic 0613. 3) DAS181+5 mM sodium citrate, pH 5.0; Solvent isopropanol. The dimensions of the microspheres 0614 4) DAS181+5 mM sodium citrate, pH 5.0+10% iso however varied, depending on the concentration of the propanol: organic solvent. The sizes of the microspheres as determined 0615 Plastic microcentrifuge tubes containing the cock by comparing the particles to a grid on a hemocytometer were tails with ingredients as described in 1)-4) above were gradu estimated to be 2 microns using 10% isopropanol, 4 microns ally cooled from: using 20% isopropanol, and 5-6 microns using 30% isopro 0616 (a) ambient temperature (about 25°C.) to 4°C. by panol. These results demonstrate that the size of the micro placing the cocktails in a refrigerator, followed by: particles can be engineered as desired using an appropriate 0617 (b) cooling to -20°C. by placing the resulting cock concentration of organic solvent. tail from (a) in a freezer, followed by: 0618 (c) freezing to -80° C. by placing the resulting cock EXAMPLE 3 tail from b) in a freezer. 0619 Under optimal conditions, microspheres would be Size of DAS181 Microspheres as a Function of Pro expected to form between about 4°C. to about -20°C. (gen tein Concentration erally in the range of about -2°C. to about -15°C.). Freezing to -80° C. is carried out to remove ingredients from the 0625 DAS181 was purified and used to prepare micro cocktail other than the microspheres (e.g., solvent, etc.) by spheres as described above in Example 1 (see cocktail 4)), freeze-drying. Cocktail 4) was prepared in triplicate, two using a combination of DAS181 protein (5 mg/ml or 10 aliquots in plastic tubes and one in a glass tube. One aliquot mg/ml), citrate counterion (sodium citrate, 5 mM) and iso (in a plastic tube) was cooled as described above, while the propanol (5% or 20%). The resulting cocktail solutions were two other aliquots (one in a plastic tube and one in a glass cooled from ambient temperature (about 25°C.) to 4°C., tube) were subjected to Snap cooling/freezing by dipping the followed by cooling to -20°C., followed by freezing to -80° tubes into liquid nitrogen. C., as described in Example 1. Upon freezing to -80°C., the 0620. Upon freezing, all tubes were placed into the lyo tubes were placed in a lyophilizer and the volatiles (water and philizer and the volatiles (water and isopropanol) were isopropanol) were removed by Sublimation, leaving the dry removed by sublimation, leaving the dry pellets. powder containing microspheres. 0621 Results: The dry pellets recovered from the cock 0626. Results: Microsphere formation was observed with tails treated as described above, were tested for the presence both concentrations of protein (5 mg/ml and 10 mg/ml), and of microspheres. Of the above samples, microspheres with both concentrations of organic solvent (5% or 20%). The good dispersivity characteristics, about 2 microns (Lm) in dimensions of the microspheres however varied. Cocktails size, were observed only with cocktail 4) containing citrate containing 5 mg/ml or 10 mg/ml protein and 5% isopropanol counterion and isopropanol and Subjected to gradual cooling. produced microspheres estimated to be about 1.5 micron in The counterion glycine did not prove to be optimal for the size. The cocktail containing 5 mg/ml protein and 20% iso DAS181 protein (cocktail2)), showing a mixture of glass-like propanol produced microspheres of an estimated size of crystals and agglomerates with only a few microspheres. about 3 microns, while the cocktail containing 10 mg/ml When no organic solvent was present, a glass-like mass of protein and 20% isopropanol produced microspheres of an lyophilized DAS181 protein was obtained and no micro estimated size of about 4 microns. These results demonstrate spheres were observed (cocktails 1) and 3)). Snap-freezing of that the size of the microparticles can be engineered as desired cocktail 4) in a glass tube produced glass-like crystals and no using an appropriate concentration of protein, or an appropri microspheres, while Snap-freezing of cocktail 4) in a plastic ate combination of concentration of organic solvent and con tube (cooling rate is slightly slower due to slower diffusion of centration of protein. heat through plastic than through glass) produced agglomer ated microspheres. EXAMPLE 4 0622. This example demonstrates that microspheres with Size of DAS181 Microspheres as a Function of narrow size distribution and good dispersivity (minimal Counterion Concentration agglomeration) can be produced by a combination of appro priate protein, counterion, organic solvent and gradual cool 0627 DAS181 was purified and used to prepare micro ing, using the methods provided herein. spheres as described above in Example 1 (see cocktail 4)), using a combination of DAS181 protein (10 mg/ml), citrate EXAMPLE 2 counterion (sodium citrate; 2 mM, 3 mM or 6 mM) and Size of DAS181 Microspheres as a Function of isopropanol (20%). The cocktail solutions were mixed in Organic Solvent Concentration glass vials and cooled from +20° C. to -40° C. at a freeze 0623 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 US 2009/00982O7 A1 Apr. 16, 2009 44 and secondary drying at 30° C. for 3 hours, leaving the dry Efficient microsphere formation was observed in all cases. powder containing microspheres. When the samples containing Surfactant were compared to 0628 Results: Microsphere formation was observed at all the sample containing all the remaining ingredients but no three tested concentrations of citrate counterion. The size of added surfactant, it was noted that the microspheresformed in the microspheres increased from 1 micron at 2 mM citrate, to the presence of Surfactant had improved dispersivity (lesser 3 microns at 3 mM citrate, to 5 microns at 6 mM citrate. agglomeration or aggregation). Addition of 1 mM sodium acetate or 1 mM sodium chloride to the cocktail containing 2 mM citrate did not affect formation EXAMPLE 6 of the microspherestriggered by the citrate counterion. These Preparation of Microspheres of Bovine Serum Albu results demonstrate that the size of the microparticles can be engineered as desired using an appropriate concentration of min (BSA) by Selection of Suitable Types and Con counterion. centrations of Organic Solvents and Counterions 0632. As described herein, the methods provided herein EXAMPLE 5 can empirically be optimized in high-throughput format to obtain microspheres having desired characteristics including DAS181 Microspheres Formed in the Presence of size, flowability and dispersivity. The purpose of this experi Surfactants ment was to demonstrate that by varying types and concen 0629. The addition of surfactants to macromolecular (e.g., trations of organic solvents and counterions, as well as pH of protein) microspheres often can improve characteristics of the cocktail, size and quality of microspheres of a protein of the microspheres that render them suitable for administration interest, in this case bovine serum albumin (BSA), can be to a subject, Such as flowability, dispersivity and disposition adjusted. for a particular route of administration, Such as intranasal or 0633 Cocktail solutions containing 5 mg/ml of BSA and oral inhalation. To test whether Surfactants can be incorpo various organic solvents and counterions at indicated pH and rated into the methods of manufacturing microspheres as concentrations (see Table 1) were placed in a microtiter plate provided herein, the production of DAS181 microspheres (final volume per well of 0.1 ml). Cocktails were cooled from was undertaken as described in Example 1 above, except that +20° C. to -40°C. at a freeze ramp of 1° C. per minute in a in addition, a Surfactant was added to the Solution. Millrock Lab Series lyophilizer. Volatiles were removed by 0630. To a cocktail solution containing 5 mg/ml DAS181, sublimation at 100 mTorr, with a primary drying at -30°C. 5 mM sodium citrate, and 20% isopropanol, was added a for 12 hours and secondary drying at 30° C. for 3 hours. surfactant (3.5% w/w lecithin, 0.7% w/w Span-85.R. (sorbitan 0634. Results: The results are shown in Table 1 below. For trioleate), or 3.5% w/w oleic acid). The microspheres were the BSA protein, combinations (of counterion and organic formed by cooling the solutions to 4°C., followed by cooling Solvent, respectively) that produced the most uniform micro to -20°C., followed by freezing to -80°C. for lyophilization spheres with minimal crystallization or aggregation include: as described above in Example 1. Upon freezing, the tubes 0635 (1) citrate--isopropanol were placed into a lyophilizer and the volatiles (water and 0636 (2) citrate+acetone isopropanol) were removed by Sublimation, leaving the dry 0637 (3) itaconic acid+1-propanol powder containing microspheres. 0638 (4) glycine--dioxane 0631 Results: The microspheres resulting from treatment 0639 (5) glycine--1-propanol of each of the above cocktails as described above were spread 0640 (6) rubidium+1-propanol on glass slides using cover slips rubbed in a circular motion. 0641 (7) perchlorate+1-propanol

TABLE 1. High-throughput screening of BSA microspheres formed under different conditions Counterion pH Organic Solvent Product description 5 mM pivalic acid 4.0 5% Cyclohexanol 0.5-1 micron microspheres with occasional crystals 5 mM pivalic acid 4.0 5% 1-propanol 0.5-1 micron microspheres with Some aggregates 5 mM pivalic acid 4.0 5% butyl alcohol Aggregated microspheres 5 mM pivalic acid 4.0 5% p-Dioxane Aggregated microspheres 5 mM rubidium 9.0 5% Cyclohexanol 0.5-1 micron microspheres. chloride Aggregates and occasional crystals 5 mM rubidium 9.0 5% 1-propanol 0.5-1 micron microspheres chloride 5 mM rubidium 9.0 5% butyl alcohol Few microspheres (0.5-1 chloride micron). Mostly aggregates and crystals 5 mM rubidium 9.0 5% p-Dioxane 1-2 microns microspheres with chloride Some aggregates 5 mM sodium 4.0 5% Cyclohexanol 1-2 microns microspheres with bromide Some aggregates 5 mM sodium 4.0 5% 1-propanol Few microspheres (0.5-2 bromide micron). Mostly aggregates and crystals 5 mM sodium 4.0 5% butyl alcohol Few microspheres (0.5-1 bromide micron). Mostly aggregates and crystals US 2009/00982O7 A1 Apr. 16, 2009 45

TABLE 1-continued

High-throughput screening of BSA microspheres formed under different conditions Counterion pH Organic Solvent Product description 5 mM sodium 4.0 5% p-Dioxane 1-2 microns microspheres with bromide Some aggregates 5 mM sodium 4.0 5% Cyclohexanol 0.5-2 microns microspheres with perchlorate Some crystals and aggregates 5 mM sodium 4.0 5% 1-propanol 0.5-1 micron microspheres perchlorate 5 mM sodium 4.0 5% butyl alcohol Few 1-2 microns microspheres. perchlorate Mostly crystals and aggregates 5 mM sodium 4.0 5% p-Dioxane Aggregated microspheres perchlorate 5 mM calcium 4.0 5% Cyclohexanol Few 1-2 microns microspheres, phosphate mostly aggregates 5 mM calcium 4.0 5% 1-propanol 1-2 microns microspheres with phosphate Some aggregates 5 mM calcium 4.0 5% butyl alcohol Few 1-2 micron microspheres. phosphate Mostly crystals and aggregates 5 mM calcium 4.0 5% p-Dioxane Aggregated microspheres phosphate 5 mM triethylamine 9.0 5% Cyclohexanol 0.5-1 micron microspheres with Some crysta S and aggregates 5 mM triethylamine 9.0 5% 1-propanol 1-2 micron microspheres with Some aggregates 5 mM triethylamine 9.0 5% butyl alcohol Few 1-2 micron microspheres. Mostly crystals and aggregates 5 mM triethylamine 9.0 5% p-Dioxane Aggregated microspheres 5 mM glycine 9.0 5% Cyclohexanol 0.5-1 micron microspheres with Some crysta S and aggregates 5 mM glycine 9.0 5% 1-propanol 0.5-2 micron microspheres with occasional aggregates 5 mM glycine 9.0 5% butyl alcohol Few 1-2 micron microspheres. Mostly crys als and aggregates 5 mM glycine 9.0 5% p Dioxane 1-2 micron microspheres 5 mM sodium 4.0 15% isopropanol 1-2 micron microspheres citrate 5 mM sodium 4.015% acetone 0.5-1 micron microspheres citrate 5 mMitaconic acid 4.0 15% 1-propanol 1-2 micron microspheres

0642. These results demonstrate that, for each protein, 0644 DNase I, trypsin and hemoglobin were purchased multiple formulations can readily be screened for the best from Worthington. Lysozyme, ovalbumin, and RNAse A microsphere formation (desired dimensions, uniformity, dis were purchased from Sigma. Purification of 6xHis tagged persivity, minimal aggregation and crystal formation, etc.) in PI8, GFP and RFP: 6xHis tagged PI8, GFP and RFP were expressed and purified essentially as described for DAS181 in high-throughput format. The combinations of reagents and Example 1 above, with the following modifications conditions (counterion, organic solvent, pH, concentrations) 0645 Purification of 6xHis tagged GFP and 6xHis tagged selected from the initial screen can then further be fine-tuned RFP: Constructs encoding Red Fluorescent protein and as desired. Green Fluorescent protein with N-terminal His tags were expressed in E. Colias 6xHis-tagged proteins. Expression of EXAMPLE 7 Red Fluorescent protein was allowed to proceed overnight in LB medium with 1 mMIPTG. Green Fluorescent protein was Preparation of Microspheres Using a Variety of Pro induced for 3 hour in TB medium with 1 mM IPTG. Cell teins lysates from 4 liters of induced cultures were clarified by centrifugation and the proteins were purified by metal chelate 0643. The methods provided herein can be used to prepare affinity chromatography on Fast-Flow Chelating resin (GE microspheres using a variety of proteins. In addition to Healthcare) charged with Nickel and packed into C-10 col DAS181 and BSA exemplified above, the methods were used umns (GE Healthcare). to prepare microspheres from trypsin, hemoglobin, DNase I. 0646. The proteins were further purified by Gel Filtration lysozyme, ovalbumin, RNASe A, hexahistidine-tagged Chromatography on a 0.5 cmx70 cm Sephacryl 200 column human proteinase inhibitor 8 (PI8, having the sequence of equilibrated with phosphate buffered saline. The proteins amino acids set forth in SEQ ID NO:15), red fluorescent were dialyzed against 2 mM sodium acetate buffer, pH 5.0, protein (RFP) and green fluorescent protein (GFP). and concentrated on a Centriprep (Amicon). US 2009/00982O7 A1 Apr. 16, 2009 46

0647. Purification of 6xHis tagged PI8: A construct (a) A DAS181 aqueous solution containing 14 mg/ml encoding PI8 with an N-terminal His tag was expressed in E. DAS181, 5 mM sodium citrate, pH 5.0 was spray dried into an Coli as 6xHis-tagged PI8. Purification was performed as air stream at 55° C., to produce microspheres. described for 6xHis RFP and 6xHis GFP above, with the (b) Alternately, DAS181 microspheres were produced exception that all buffers used in the various chromatographic purification steps contained 1 mM TCEP (Tris(2-carboxy according to the methods provided herein. To a DAS181 ethyl)phosphine hydrochloride). aqueous solution containing 14 mg/ml DAS181, 5 mM Sodium citrate, pH 5.0, was added 5% isopropanol as organic 0648 Preparation of microspheres: Cocktail solutions solvent. The resulting solution was cooled from +20° C. to containing 5 mg/ml of protein and various counterions, -40°C. at a freeze ramp of 1° C. per minute in a Millrock Lab organic solvents and pH as listed below were prepared in a Series lyophilizer. Volatiles (water and isopropanol) were microtiter plate as described above in Example 6. removed by sublimation at 100 mTorr with primary drying at -30° C. for 12 hours and secondary drying at 30° C. for 3 TABLE 2 hours, leaving the dry powder containing microspheres. Combinations Used to Produce Microspheres of Different Proteins 0654 B. Aerodynamic Particle Size Distribution of Microsphere Microspheres Organic Size 0655 The microspheres prepared as described in Example Protein Counterion pH Solvent (microns) 8A were tested by Andersen Cascade Impaction. The depo Trypsin 5 mM 8.0 5% isopropanol OS-1 sition of pharmaceuticals in the respiratory tract can be pre arginine dicted by the aerodynamic behavior of particles (micro Lysozyme 5 mM citrate 8.0 5% isopropanol 4-5 PIN 168 (PI8) 5 mM citrate 5.0 7% isopropanol 2-5 spheres) on the stages/collection plates of the cascade DNase I 5 mM citrate 4.0 5% isopropanol 0.4-1 impactor. RNaseA 5 mM citrate 4.0 5% isopropanol 0.4-1 Hemoglobin 5 mM 5.0 10% isopropanol O4-O-7 0656. The cascade impaction experiment was performed glycine using DAS181 microspheres prepared by one of the two Ovalbumin 5 mM pivalic 4.0 10% isopropanol OS-1 alternate methods described in section A above, i.e., either by acid Red fluorescent 5 mM pivalic 7.0 10% 1-propanol 1-4 (occasional spray-drying or by the methods provided herein. The micro protein acid aggregates) spheres (10 mg) were loaded into gelatin capsules. The gela Green 5 mM pivalic 7.0 10% 1-propanol O.S.-1.5 tin capsules were placed into a CycloHaler (PharmaChemie) luorescent acid dry powder inhaler and Subjected to cascade impaction. An protein 8-stage, non-viable Andersen Cascade Impactor (Thermo Electron, Boston) modified for use at 90 liters per minute of 0649. The microtiter plate was cooled from +20° C. to air flow and equipped with a USP throat, induction cone and -40°C. at a freeze ramp of 1° C. per minute in a Millrock Lab no preseparator, was used. The collection plates of the impac Series lyophilizer. Volatiles (water and isopropanol) were tor representing various areas/stages of deposition post-inha removed by sublimation at 100 mTorr with primary drying at lation (trachea, primary and secondary bronchi, terminal -30° C. for 12 hours and secondary drying at 30° C. for 3 bronchi, alveoli, etc.) were coated with silicon spray to pre hours, leaving the dry powder containing microspheres. vent bouncing of the microspheres. The microspheres from 0650. The dry powders were spread on glass slides and the stages and collection plates were recovered into a phos microphotography was performed through either 32x or 100x phate buffered saline containing 0.1%Tween, and the amount objective. All the combinations listed in Table 2 above pro of deposited DAS181 recovered from each stage and collec duced microspheres of good quality (uniform size distribu tion plate was quantified by measuring absorbance at 280 nm. tion, dispersivity, with few aggregates and/or crystals). The 0657 Results: The geometric size of microspheres pro microspheres varied in size from about 0.4-1 micron (RNAse duced by the two methods was assessed by light microscopy A, DNAse I) to about 2-5 microns (6xHis PI8, lysozyme), and found to be essentially identical (range of 1.5-3.0 depending on the protein. This example demonstrates that the microns) for both methods. As shown in Table 3 below, how methods provided herein can be used to produce micro ever, the aerodynamic particle size distribution of the two spheres from a wide variety of proteins. preparations differs significantly between the two methods. For the microspheres produced according to a method as EXAMPLE 8 provided herein (i.e., method (b) as set forth in section A above), less than 25% remained trapped in the mouth (throat/ Aerodynamic Particle Size Distribution of DAS181 cone of the impactor assembly), while greater than 70% of the Microspheres for Inhalation: A Comparison of the microspheres were delivered to the trachea and lungs (with Method Provided Herein with Spray-Drying greater than 40% in the terminal bronchi and alveoli). In comparison, less than 50% of the DAS181 microspheres 0651. As described herein, the methods provided herein formed by spray-drying (method (a) as set forth in section A can be used to produce microspheres in any desired size above) was delivered to the trachea and lungs (less than 20% range, including a range of about 0.5 micron to about 6-8 in the terminal bronchi and alveoli). The results demonstrate microns for delivery via inhalation. that methods provided herein can produce microspheres for 0652 A. Preparation of Microspheres delivery into deep lungs, and that the microspheres produced 0653) To test the aerodynamic particle size distribution of by methods provided herein have Superior disagglomeration DAS181 dry powder (microspheres) formulated for delivery and flowability properties (provide a higher delivered dose) by inhalation, DAS181 microspheres were prepared using compared to microspheres produced by a spray-drying two methods as follows: method. US 2009/00982O7 A1 Apr. 16, 2009 47

TABLE 3 Results of Cascade Impaction Analyses of DAS181 Microspheres Percent Deposition of DAS181 Component Microspheres of the Expected Produced by Andersen Corresponding Deposition in Method (a) Microspheres Cascade Size Cut-Off Respiratory (i.e., Spray Produced by Impactor (microns) Airways 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 1 3.5-5.2 trachea bronchi 12.5 9.3 2 26-35 Secondary 11.6 12.6 bronchi 3 1.7-2.6 terminal bronchi 11.0 24.O 4 1.0-1.7 alveoli 4.5 19.2 5 O43-10 Alveoli 1.4 3.5

EXAMPLE 9 tration cassettes (TFF cassettes. Such as those manufac tured by Millipore or Sartorius)). The removal of bulk Large Scale Manufacture of Microspheres liquid (50% or greater) can result in a faster drying cycle 0658. This example demonstrates that the methods pro and higher efficiency and throughput. vided herein can be scaled for the manufacture of large quan 0662 Drying the microspheres. The recovered micro tities of DAS181. The Batch Process described herein is spheres formed by any mode, can be dried by conven suitable for the manufacture of high quality dry powder tional lyophilization. Alternatively, the microspheres microspheres in an amount ranging from, for example, mil can be dried under ambient temperature and atmo ligrams to about a kilogram and is limited by the capacity of spheric pressure, eliminating the use of lyophilizer. the mixing tank and/or lyophilizer shelf space. An alternative 0663 Results: DAS181 protein was successfully pro “continuous” process described herein can be used to manu cessed into dry powder (microspheres) by a continuous mode facture amounts ranging from, for example, hundreds of as described herein. Cocktail containing 10 mg/ml DAS181, grams to hundred or more kilograms (100 grams to 100 kg 20% isopropanol. 2 mM sodium sulfate was passed through and above). Additional advantage of continuous process is a 35 SERIES heat exchanger (Exergy, Garden City, N.Y.) better control over the chilling of the cocktail. coupled with a NESLAB circulating cryostatusing a peristal 0659. The large scale manufacture by a batch process or by tic pump so that during the passage the cocktail was cooled a continuous process can follow, for example, one or more of from about 25° C. to about -12°C. the steps described below in any combination of steps or 0664. The resulting Suspension of microspheres exiting specific alternative methods: the heat exchanger was pumped into a prechilled lyophiliza 0660 Precipitation of protein into microspheres. This tion tray (-40°C.), frozen and lyophilized or, alternatively, step can be performed in a batch mode by placing the pumped directly into liquid nitrogen and then lyophilized. cocktail solution containing the desired concentration of The resulting microspheres, which were analyzed by micros protein, organic solvent and counterion in lyophilization copy and cascade impaction, showed uniform microspheres tray(s) and placing the tray(s) onto lyophilizer shelves. with minimal aggregation and good dispersivity and were Alternatively, trays can be chilled and frozen on a chilled similar in dimensions and aerodynamic particle size distribu platform or other type of equipment (e.g., a freezer) and tion to the microspheres produced by batch mode. When the stored for a period of time frozen and lyophilized later. formulated DAS181 cocktail solution was not chilled (not Alternatively, the microspheres can be formed by pre passed through heat exchanger, thus no precipitation of cipitation in a vessel with stirring, wherein the vessel is placed onto a cold Surface or a cooling coil is immersed microspheres was induced) and poured directly into liquid into liquid or while the cocktail is being recirculated nitrogen, no microspheres were observed and, instead, glass through a heat exchanger using a peristaltic pump. Alter like crystals were observed after lyophilization. natively, the microspheres can be formed by precipita tion in a continuous mode, by passing the cocktail solu EXAMPLE 10 tion through a heat exchanger(s) once using a peristaltic Batch Mode Process and Formulation of DAS181 pump. Microspheres for Delivery to Upper and Central Res 0661 Removal of bulk liquid. The suspension of the piratory Airways microspheres can be concentrated using standard cen trifugation, continuous flow centrifugation (e.g., CARR 0665. 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. US 2009/00982O7 A1 Apr. 16, 2009

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, 12 g/L Active ingredient Sodium acetate’ 1.12 mM API O688 mM pH buffer Acetic acid O.63 mM Solution O.O387 nM pH buffer Sodium Sulfate 500 mM O.O21.5 L 2 mM Microparticle formation agent (counterion) Isopropanol 100% viv O.269 L. S90 wiv Microparticle formation agent Calcium chloride 500 mM O.OO28 L. O.268 mM Stability enhancing agent Water for irrigation neat 179L NA Diluent '''Batch size: final volume of formulated cocktail 5.38 L. Theoretical yield 74 g of bulk DAS181 Dry Powder. °Components of the DAS181 protein (API) stock solution.

0666 A. Production of Bulk Drug Substance fied homogenate was then concentrated by allowing the lysate 0667 The terms Drug Substance, Active Pharmaceutical to recirculate without fresh buffer feed. Next, DAS181 pro Ingredient, and API are used interchangeably in this example tein was captured from the clarified homogenate on a Toyo pearl SP-550C resin which was washed in a series of buffers and refer to the DAS181 protein. Production of DAS181 (see Table 5) before the DAS181 protein was eluted from the protein in bulk was conducted as follows. First, bulk amounts resin. The sodium chloride concentration of the eluate was of DAS181 were expressed in E. coli (BL21 strain) essen adjusted to 1.0 M in a final buffer of 50 mM phosphate at pH tially as described in Example 1. The E. coli cells expressing 8.0. The DAS181-containing eluate was then passed through the DAS181 protein were washed by diafiltration in a fermen a Toyopearl Hexyl-650C resin for further purification using a tation harvest wash step using Toyopearl buffer 1, UFP-500 Toyopearl Buffer 4. The resin eluate containing DAS181 E55 hollow fiber cartridge (GE Healthcare) and a Watson protein was then buffer-exchanged into 5 mM sodium acetate Marlow peristaltic pump. in a diafiltration step (see step 8 in Table 5). The concentrated 0668. The recombinant DAS181 protein was then purified protein was next passed through a Sartorius Q SingleSep in bulk from the cells. The detailed specifications of the Filter in order to remove DNA in a flow-through mode. Iso components and buffers used in the bulk purification of propanol was added to the Q SingleSep filtrate to a final DAS181 are provided in Tables 5 and 6 below. The harvested concentration of 20% v/v. The DAS181 protein in the buffer and washed cells were lysed in a homogenization step by was passed through an Amberchrome CG300M resin equili passing the cells twice through using Niro-Soave Panda cell brated with an Amberchrom buffer (see step 11 in Table 5). disrupter. The homogenate thus obtained was clarified by The purified bulk DAS181 protein was then buffer-ex microfiltration using the Toyopearl buffer 1, Hydrosart 0.2 changed into formulation buffer and concentrated by diafil micron TFF cassette and a Watson Marlow pump. The clari tration (see step 12 of Table 5).

TABLE 5 Purification of bulk DAS181 drug substance 1 Purpose Fermentation Harvest Wash Cartridge GEUFP-500-E55 Specifications Activity Buffer Name Inlet PSI Diafiltration Toyopearl Buffer 1 25-35 2 Purpose Homogenization Activity Step Buffer Name Equilibration Equilibration Harvest Buffer Homogenization 1st Pass Sample Load Homogenization 2nd Pass Sample Load 3 Purpose Homogenate Clarification (Diafiltration) TFF Cartridge HydroSart 10K 0.6m* Specifications Activity Buffer Name Inlet PSI Recirculation Sample Load 40 Diafiltration Toyopearl Buffer 1 SO 4 Purpose Permeate Concentration TFF Cartridge HydroSart 10K 0.6 m? Specifications Activity Buffer Name Inlet PSI Recirculation Sample Load NS Concentration Sample Load SO US 2009/00982O7 A1 Apr. 16, 2009 49

TABLE 5-continued Purification of bulk DAS181 drug substance 5 Purpose DAS181 capture performed in bind and elute mode Resin Toyopearl SP-550C Activity Step Buffer Name Loading Sample Load Clar. Homogenate Wash SP Wash 1 Toyopearl Buffer 1 SP Wash2 Toyopearl Buffer 2 SP Wash 3 Toyopearl Buffer 3 SP Wash4 Toyopearl Buffer 2 SP Was S Toyopearl Buffer 1 Elution Elution Toyopearl Buffer 4 6 Purpose Adjust NaCl Concentration Method Add NaCl to 1.0 M Final Buffer 50 mM phosphate, 1.0 M NaCl, pH 8.0 7 Purpose DAS181 purification in flow-through mode Resin Toyopearl Hexyl-650C Activity Step Buffer Name Loading Sample Load Cond. Hexyl Load 8 Purpose Concentration &Diafiltration TFF Cartridge HydroSart 10K 0.6 m Specifications Activity Buffer Name Recirc. Limin Recirculation Toyopearl Buffer 6 15-16 Concentration Hexyl Product Pool 15-16 Diafiltration Toyopearl Buffer 6 15-16 Recirculation Toyopearl Buffer 6 NS 9 Purpose Remove DNA in flow-through mode Resin Sartorius Q SingleSep Filter Activity Step Buffer Name Loading Sample Load 10 Purpose Buffer Adjustment Method Add Isopropanol to 20% Final Buffer 5 mM Acetate, 20% Isopropanol, pH 5.0 11 Purpose DAS181 polishing in flow-through mode Resin Amberchrome CG300M Activity Step Buffer Name Loading Sample Load Amberchrom Load 12 Purpose Concentration &Diafiltration TFF Cartridge HydroSart 10K 0.6 m Specifications Activity Buffer Name Recirc. Limin Recirculation Formulation Buffer 15-16 Concentration Amberchrom Product Pool 15-16 Diafiltration Formulation Buffer 15-16 *Volumes in liters, except 4x denotes multiples of the retentate volume CV = Column Volumes NR = Not Recorded NS = Not Specified

TABLE 6 TABLE 6-continued Buffers used during the DAS181 purification process Buffers used during the DAS181 purification process Buffer Name Buffer Composition Buffer Name Buffer Composition Toyopearl Buffer 1 50 mM potassium phosphate, 0.3 MNaCl, pH 8.0 Toyopearl Buffer 2 1.1 mM potassium phosphate, 2.9 mM sodium O.SNNaOH O.SNNaOH phosphate, 154 mM NaCl, pH 7.4 O.1 NNaOH O.1 NNaOH Toyopearl Buffer 3 1.1 mM potassium phosphate, 2.9 mM sodium 70% Isopropyl 70% isopropanol phosphate, 154 mM NaCl, 1% Triton X-100, Alcohol 0.1% SDS, 0.5% sodium deoxycholate, 20% EtOH 20% ethyl alcohol pH 7.4 Toyopearl Buffer 4 50 mM potassium phosphate, 1.0 M NaCl, pH 8.0 Toyopearl Buffer 5 50 mM potassium phosphate, 0.5 M NaCl, pH 8.0 Toyopearl Buffer 6 5 mM sodium acetate, pH 5.0 0669 B. Batch Manufacturing Process Toyopearl Buffer 7 5 mM sodium acetate, 60% isopropanol, pH 5.0 0670. The ingredients set forth in Table 4 above were Formulation Buffer 1.75 mM sodium acetate, pH 5.0 combined to form DAS181 microspheres in a large scale 3% Isoproyl Alcohol 3% isopropanol Amberchrom Buffer 5 mM sodium acetate, 20% isopropanol, pH 5.0 batch process as described below. adjusted with acetic acid Step I: Thawing of bulk Drug Substance 1.ONNaOH.3% 1.0 N NaOH, 3% isopropanol Isopropanol 0671 Frozen 0.2 um-filtered bulk Drug Substance in plas 1.ONNaOH 1.ONNaOH tic bottles was thawed overnight at ambient temperature (25+3° C.). US 2009/00982O7 A1 Apr. 16, 2009 50

Step II: Weighing of the excipients and preparation of solu in a wide range (2-30%, V/v) of concentrations, and (3) it has tions a relatively high freezing point So its vapors can efficiently be 0672 35.51 g of Sodium Sulfate anhydrous powder was trapped during lyophilization. weighed and Q.S. to 500 mL with Water For Irrigation, then 0685. The protein concentration in the final formulation stirred to obtain a clear solution. 18.38g of Calcium Chloride could be varied (10-14 mg/ml), as could the concentration of counterion (1-5 mM) and isopropanol (2-30% V/v), without dihydrate powder was weighed and Q.S. to 250 mL with Substantial impact on the physical properties of the micro Water For Irrigation, then stirred to obtain a clear solution. spheres or the activity of the DAS181 protein in the micro Step III: Preparation of the DAS181 cocktail solution spheres. At higher concentrations of isopropanol (15-30%), 0673. To 3.3 L of concentrated Drug Substance (19.55 the microspheres formed while the cocktail was still fully g/L), 1.79 L of Water For Irrigation was added slowly with liquid. At lower concentrations (2-15%), ice crystals began to stirring, followed by 0.0215 L of Sodium Sulfate solution, form first, followed by precipitation to form microspheres. 0.0028 L of Calcium Chloride solution and 0.269 L of iso 0686 C. Yield of DAS181 in the Microspheres propanol. The Solution was stirred to ensure complete mixing 0687. The theoretical yield of DAS181 in the dry micro of components. spheres is calculated according to the following formula: Step IV: Filtration of Formulated Cocktail Solution Through Theoretical yield=DAS181 protein, g-protein fraction 0.2 um Filter in Dry Powder (microspheres) 0688. The protein fraction value (0.866) was established 0674. The formulated cocktail solution of Step III was empirically by analysis of several manufactured batches of filtered through a 0.2 um filter into sterile media bags to DAS181 microspheres. The theoretical yield for the amounts control particulates and bioburden. as set forth in Table 2 is 64.56 g--0.866–74.55g. The actual Step V: Filling into Lyophilization Trays yield of DAS181 Dry Powder was found to be 64 g. 0675. The formulated filtered solution was dispensed into 0689 Results: The suitability of the microspheres pre autoclaved Lyoguard lyophilization trays. To ensure even pared as described in section B above for administration by cooling of the Solution and formation of high quality micro oral inhalation was tested by Andersen Cascade Impaction. spheres, 6 trays were each filled with 0.9L or less of cocktail The results are summarized in Table 7 below. The deposition Solution. of pharmaceuticals in the respiratory tract can be predicted by deposition of particles (microspheres) on the stages/collec Step VI: Freezing and Lyophilization tion plates of the cascade impactor. For a pharmaceutical, e.g., DAS181 microspheres, that is administered to prevent or 0676. The trays were placed onto lyophilizer (Hull treat viral infections that initiate in the respiratory tract, such 120FSX200) shelves pre-chilled to -45+5° C. and the solu as influenza, it is desirable to deposit the pharmaceutical in tion was allowed to chill and freeze. Formation of micro the throat, trachea, bronchi (upper and central respiratory spheres occurred while the solution was being frozen. The airways). The DAS181 fusion protein delivered to upper and freezing is allowed to proceed for 1-2 h to ensure complete central respiratory airways cleaves off the receptor Sialic solidification. The product temperature was verified by read acids from mucous membranes, thus preventing viral binding ing the thermocouples attached to two of the six trays. and infection at these sites. For optimal delivery of the 0677. The lyophilization cycle steps are as follows: DAS181 microspheres to sites where respiratory viral infec 0678 a) Set vacuum to 160 microns and allow to evacu tion can be initiated, i.e., in the throat, trachea or bronchi, the ate to 100-200 microns; microspheres must not be (a) So big that they are trapped at the 0679 b) Ramp shelf temperature to +10° C. over 3 h; front end in the mouth (i.e., microspheres are too big, about 8 0680 c) Hold shelf temperature at +10° C. for 36 h microns or greater); or (b) so Small that they are deposited in (primary drying); deep lungs and absorbed systemically into the blood stream 0681 d) Thermocouple traces examined to verify that (i.e., 0.5 microns or smaller). For delivery of the DAS181 primary drying phase is completed and the product tem microspheres to the throat, trachea and bronchi, a size range perature has stabilized at +10° C.t5° C. for 15-30 h. of about 1 micronto about 5.5-6 microns generally is suitable. 0682 e) Ramp shelf temperature to +30°C. over 1 hand 0690 DAS181 microspheres manufactured as described hold for 3-5 h (secondary drying). above were characterized by Andersen cascade impaction Step VII: Transfer of Bulk DAS181 Microspheres into Con and found to be suitable for delivery to upper and central tainer and Mixing respiratory airways with sufficiently low percentage (<5%) 0683. A section on the bottom film of each Lyoguard lyo deposited in the alveoli. philization tray was cleaned using sanitizing wipes and a 3x3 cm opening was made with a scalpel. The dry microspheres TABLE 7 were transferred into a plastic bottle. The bottle was capped Aerodynamic Particle Size Distribution of DAS181 dry powder and tumbled forty times, changing directions with each inver at 60 liters per minute. sion. The tumbling was to ensure uniformity of bottle content. Samples for analytical testing were taken and the bottle was Corre- Percent of Component of sponding Expected DAS181 total recapped and sealed into plastic bags for storage. Andersen size deposition in protein DAS181 0684. In the DAS181 microsphere bulk manufacturing Cascade cut-off, respiratory deposited protein process as described above, Sulfate was demonstrated to be a Impactor microns airways (in mg) recovered safe Substance for use as a counterion, and reproducibly pro Inhaler 1.57 0.11 20.13% duced microspheres with a narrow size distribution. Further, (Cyclohaler) the organic solvent isopropanol was a good solvent of choice Throat? Cone >10 Oral cavity O.93 + 0.19 11.92% because (1) a class 3 solvent, (2) it can produce microspheres US 2009/00982O7 A1 Apr. 16, 2009 51

inhaler (as demonstrated by Andersen Cascade Impaction or TABLE 7-continued laser diffraction). Light microscopy of microspheres dis persed in a liquid surfactant (e.g. Triton X-100 or Tween 20) Aerodynamic Particle Size Distribution of DAS181 dry powder or non-polar solvent (e.g., alcohol, acetone, or acetonitrile) at 60 liters per minute. that does not dissolve the microspheres, confirmed that aggre Corre- Percent of gates are easily dissipated into individual uniform micro Component of sponding Expected DAS181 total spheres. Andersen size deposition in protein DAS181 Cascade cut-off, respiratory deposited protein EXAMPLE 11 Impactor microns airways (in mg) recovered Preparation of DAS181 Microspheres Using Sulfates -1 (Stage + Plate) 8.6-10 Oral cavity OSO 0.10 6.41% Other than the Sodium Salt -0 (Stage + Plate) 6.5-8.6 oropharynx O4O O.O3 S.13% 1 (Stage + Plate) 44-6.5 pharynx O-580.03 7.44% 2 (Stage + Plate) 3.3-4.4 trachea bronchi 0.83 + 0.07 10.64% 0692 Studies have shown that in certain instances, e.g., in 3 (Stage + Plate) 2.0-3.3 Secondary 18O, O.O9 23.08% Some asthmatics, the presence of sodium in formulations for bronchi pulmonary administration could carry a risk of inducing air 4 (Stage + Plate) 1.1-2.0 Terminal O.82 0.08 10.51% way hyperresponsiveness (Agrawal et al., Lung, 183:375-387 bronchi (2005)). This example therefore tested alternate salts, such as 5 (Stage + Plate) 0.54-1.1 alveoli O.23 O.O3 2.95% 6 (Stage + Plate) 0.25-0.54 alveoli O.14 - 0.03 1.79% salts of other metals such as potassium, magnesium and cal XACI (Emitted) 6.24 O. 10 80.00% cium. 0693 DAS181 microspheres were manufactured as 10+ 1.0 mg of DAS181 Dry Powder (8.5 mg + 10% DAS181 protein) was described above in Example 1. Cocktail solutions containing filled into HPMC capsule XACI (Emitted) fraction is the sum of all material recovered from USP 12 mg/mL DAS181 and 5% (v/v) isopropanol contained as Throat, Induction Cone and stages -1 to 6. counterions the indicated Sulfates at 2 mM concentration, pH 4.5-5.0. The microspheres were formed by cooling the solu (0691 DAS181 microspheres were further characterized tions from +25° C. to -45° C. Upon freezing, the volatiles by laser diffraction, which demonstrated, consistent with the (water and isopropanol) were removed by Sublimation, leav cascade impaction results, that the majority of the micro ing the dry powder containing microspheres. spheres produced by the method described in this Example 0694. The aerodynamic particle size distribution of the dry are within a size range of between 1 micron and 5 microns in powder was assessed by Andersen Cascade Impaction, and size. Scanning Electron Microscopy (FEI Quanta 200 Scan the amount of DAS181 per stage was determined by UV ning Electron Microscope. Everhart Thornley (ET) detector) measurement at 226 nm (A). The results are shown below of the DAS181 microspheres prepared according to the in Table8. The results demonstrate that sulfate salts other than method described in this Example revealed that the micro the sodium salt can be used as counterion to obtain DAS181 spheres are present as agglomerates of hundreds and thou microspheres of a size range Such that the majority are deliv sands of individual particles approximately 0.5-3 micron in ered to the throat, trachea and bronchi, in an amount that is size. The agglomerates however are easily dissipated by air comparable to the amount delivered when sodium sulfate is turbulence produced during the actuation through dry powder 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 respiratory Sodium Potassium Magnesium Calcium cut-off, 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 s10 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) 6.5-8.6 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 19.57%. 20.21% 17.01% 12.65% bronchi 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) O.25-0.54 alveoli 1.82% 2.31% 3.44% 2.16% US 2009/00982O7 A1 Apr. 16, 2009 52

(0695) 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 (0696. 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. various metal Sulfates as counterions were comparable to that The production of dry DAS181 microspheres was undertaken 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. (0697 A. Stability of DAS181 Microspheres without Sug aS TABLE 9 (0698. 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 Example 1 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% S2.76% 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 2 mM 116.67% 93.20% 87.12% 40.48% Sialidase Activity with 100% 102.0% 99.9% Sulfate reference to time O

TABLE 11

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

Throat >10 Oral cavity 19.57 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 Stage 2 3.3-44 trachea 10.67 + 0.23 10.70- 0.35 9.30 - 0.82 bronchi 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 O23 1.73 O.O6 2.37 - O.O6 Stage 6 0.25-0.54 alveoli O.33 OO6 O40. O.10 O.73 OO6

Table 11: Aerodynamic particle distribution was assessed by Andersen Cascade Impaction and expressed as % of total DAS181 protein recovered. Capsules were filled with 10 mg of DAS181 dry powder and actuated using Cyclohaler dry powder inhaler as delivery device. Air flow rate was 60 Liters per minute. Assays were performed in triplicate, mean and stan dard deviation are shown. US 2009/00982O7 A1 Apr. 16, 2009

(0699 B. Stability of DAS181 Microspheres Formulated (degradation products) was quantitated by size exclusion with Sugars HPLC. The results in Table 12B (below) again demonstrate (0700. The sialidase activity of DAS181 in the dry powder that Trehalose and Sucrose Sugars provided significant pro microsphereformulations containing Sugars and in the unlyo tection. Results are expressed as % of oligomers at 0 months, philized microsphere formulations stored at -80° C., were 1 month, 2 months or 8 months. measured using the fluorescent substrate 4-MU-NANA as described in Example 1 and as incorporated by reference TABLE 12B herein. The dry powder formulations containing no Sugar or various sugars as indicated below in Table 12 were stored at % of oligomers +42°C. for 4 weeks (forced degradation). The results are shown in Table 12. Relative to unlyophilized formulations HPMC Capsules Unencapsulated stored at -80° C., the formulation containing no Sugar Sugar t = 0 t = 1 t = 2 t = 8 t = 1 t = 2 t = 8 retained almost 80% of its activity. The addition of various Sucrose O.O 4.2 8.9 1S.O O.O 1.1 O.9 sugars increase the stability so that about 88-98% of the Trehalose O.O 4.8 8.6 14.3 O.O 1.O 1.O activity is retained, depending on the Sugar. None O.O S.O 9.4 18.6 O.9 2.4 3.5 Formulations contained 15% w/w Sugar, 5% w/w Glycine, 2 mM Acetate TABLE 12 pH 6.0, and 2 mM MgSO Percent Sialidase Activity Sugar Remaining after 4 weeks at 42°C. EXAMPLE 13 No Sugar 79.82 Sorbitol 91.23 Mannitol 89.47 Preparation of Microspheres Using a Variety of Trehalose 97.37 Classes of Compounds Sucrose 88.60 0704. The methods provided herein can be used to prepare 0701. The stabilizing effect of higher percentages of Sug microspheres using a variety of classes of compounds. In ars on the DAS181 microspheres was investigated. The pres addition to proteins such as DAS181, BSA, trypsin, hemo ence of glycine in the reaction cocktails served to prevent globin, DNase I, lysozyme, ovalbumin, RNAse A, hexahisti crystallization of the Sugars during the manufacture of the dine-tagged human proteinase inhibitor 8 (PI8, having the DAS181 microspheres. The study showed that up to 15% of sequence of amino acids set forth in SEQ ID NO:15), red Trehalose, Sucrose, Sorbitol, or Mannitol, when combined fluorescent protein (RFP) and green fluorescent protein with 5% glycine, can be incorporated into the DAS181 micro (GFP), as described in the above Examples, this Example sphere-forming reactions without forming crystals during demonstrates that the method can be used to prepare micro lyophilization. The microspheres were indistinguishable spheres of: from the ones produced without Sugars, based on their 0705 A. The antibiotics Vancomycin, Tobramycin, appearance under light microscopy and scanning electron Kanamycin and Ampicillin microscopy (SEM). The aerodynamic particle size distribu tion of the microspheres also remained unaffected. 0706 B. A nucleic acid siRNA 0702 5 mg of the resulting DAS181 dry powder was 0707 C. A virus Tobacco Mosaic Virus placed in clear size 3 HPMC capsules and stored at 37°C. The 0708. D. The peptides—leuprolide and somatostatin percentage amount of high molecular weight DAS181 oligo 0709. The microspheres prepared from A-D above were mers (degradation products) was quantitatively analyzed by compared to those of the protein, DAS181. size exclusion HPLC. The results in Table 12A demonstrate the protective effect of the sugars, with Trehalose and Sucrose 0710 Preparation of microspheres: For each of the Com providing the best protection. Results are expressed as % of pounds listed in A-Dabove, and for DAS181, cocktail solu oligomers at 0 months, 1 month, 2 months or 3 months. tions containing 2 mg/ml of compound dissolved in an aque ous solvent and various counterions, antisolvents and pH, as TABLE 12A listed below, were prepared in a 96-well microtiter plate (0.1 ml cocktail/well) at room temperature. Control solutions con % of oligomers tained either solvent orantisolvent alone, with or without the counterion. Cocktails were cooled by placing in a freezer. The O months 1 month 2 months 3 months chilled plates were transferred onto pre-chilled (-45° C.) Mannitol O.O 6.O1 9.79 12.59 shelves of a Millrock Lab Series Lyophilizer, and the vacuum Trehalose O.O 3.75 S.82 6.79 Sucrose O.O 3.92 5.67 8.16 was applied. The frozen cocktail solutions were allowed to Sorbitol O.O 4.88 7.35 9.59 lyophilize for 16 hours. 0711. The lyophilized powders from the bottoms of the 0703. The stabilizing effect of Sucrose and Trehalose on wells were transferred onto glass slides and analyzed by light DAS181 microsphereformulations was further confirmed by microscopy for appearance. The quality of the product micro the following experiments. Formulations containing the Sug spheres was scored based on the uniformity of the micro ars and a Sugar-free control were produced and either left as spheres, the absence of undesirable non-microsphere par bulk powders (unencapsulated) or placed into clear size 3 ticles (glass-like crystalline forms), and the absence of HPMC capsules. The samples were stored at 37° C. The aggregates. Table 13 below provides an exemplary scoring percentage of high molecular weight DAS181 oligomers system, based on appearance. US 2009/00982O7 A1 Apr. 16, 2009 54

TABLE 13 TABLE 14-continued Scoring System for Assessing Quality of Microspheres Combinations Used to Produce Microspheres of Different Compounds Presence of A. Antibiotics Glass like Presence? Quality of crystals and/or Microsphere Score Microspheres Aggregates Counterion Antisolvent pH Quality O Absent Exclusive 2 mMNa- 15% 7.0 4 1 Almost absent Almost exclusive Glutamate isopropanol 2 SC3Ce Highly dominant 2 mM Arginine 15% 7.0 4 3 observable Dominant HCJNaOH isopropanol 4 Present in large quantities Present in smaller 2 mM Arginine 15% 9.0 O relative to glass-like crystals or quantities than HCJNaOH isopropanol aggregates the microspheres 2 mM Itaconic Acid- 15% 4.0 9 5 Dominant Scarce Na isopropanol 6 Almost uniform Observable, but 2 mM Itaconic Acid- 15% 7.0 7 minimal Na isopropanol 7 Uniform Observable, but 2 mMNa-Citrate 5% in- S.O 7 very minimal propanol 8 Uniform Very few 2 mMNa- 5% in- 4.0 5 9 Very uniform Almost absent Glutamate propanol 10 Perfect and uniform Absent 2 mM Itaconic Acid- 5% in- 7.0 9 Na propanol 2 mM Itaconic Acid- 15% in- 4.0 8 0712 Table 14 below shows the various combinations of Na Compound:propanol Ampicillin compound, solvent, antisolvent and counterion that were o used to generate microspheres, and the quality of the resulting 2 mMNa-Citrate 5% S.O 5 microspheres. 2 mMNa- propanol 4.0 4 Glutamate isopropanol TABLE 1.4 2 mM Itaconic Acid- 59 4.0 3 Na isopropanol Combinations Used to Produce Microspheres of Different Compounds 2 mMNa-Citrate 15% S.O 2 1sopropano A. Antibiotics 2 mMNa- 15% 4.0 3 Glutamate isopropanol Microsphere 2 mM Itaconic Acid- 15% 4.0 7 Counterion Antisolven pH Quality Na isopropanol 2 mMNa-Citrate 5% in- S.O 3f4 Compound: Tobramycin propanol 2 mMNa- 5% in- 4.0 3-5 2 mMNa-Citrate 59 S.O 4 Glutamate propanol isopropano 2 mMNa- 15% in- 4.0 4 2 mMNa- 59 4.0 2 Glutamate propanol Glutamate isopropano 2 mM Itaconic Acid- 15% in- 4.0 3 2 mM Arginine 59 7.0 5 Na propanol HCJNaOH isopropano 2 mM Itaconic Acid- 15% in- 7.0 7 2 mM Itaconic Acid- 59 4.0 9 Na propanol Na isopropano Compound: Vancomycin 2 mMNa-Citrate 15% S.O 3 isopropano 2 mMNa-Citrate 59 S.O 4 2 mMNa- 15% 4.0 2 isopropano Glutamate isopropano 2 mMNa- 59 4.0 4 2 mM Arginine 15% 7.0 3 Glutamate isopropano HCJNaOH isopropano 2 mMNa- 59 7.0 7 2 mM Arginine 15% 9.0 5 Glutamate isopropano HCJNaOH isopropano 2 mM Arginine 59 7.0 7 2 mM Itaconic Acid- 15% 4.0 8 HCJNaOH isopropano Na isopropano 2 mM Arginine 59 9.0 6 2 mM Itaconic Acid- 15% in- 4.0 8 HCJNaOH isopropano Na propanol 2 mM Itaconic Acid- 59 4.0 8 Compound: Kanamycin Na isopropano 2 mM Itaconic Acid- 59 7.0 8 2 mMNa-Citrate 5% S.O 8 Na isopropano 1sopropano l? 0. 2 M Na- 59 4.0 3 2 mMNa-Citrate Rio 8O S.O 4 Glutamate isopropano 2 mM Itaconic Acid- 5% 4.0 8 2 mMNa- 15% 4.0 4 Na isopropanol Glutamate SOO.80 2 mM Itaconic Acid- 59 7.0 8 2 mMNa- 15% 7.0 3 Na isopropanol Glutamate SOO.80 2 mMNa-Citrate 15% S.O 9 2 mM Arginine 15% 7.0 4 isopropanol HCJNaOH isopropano 2 mMNa- 15% 4.0 4 2 mM Arginine 15% 9.0 4 Glutamate isopropanol HCJNaOH isopropano US 2009/00982O7 A1 Apr. 16, 2009 55

TABLE 14-continued -continued Combinations Used to Produce Microspheres of Different Compounds B. Nucleic Acid - siRNA A. Antibiotics Microsphere Counterion Antisolven pH Quality Microsphere Counterion Antisolvent pH Quality 2 mMNa-Citrate 59 S.O 2 isopropano 2 mM Itaconic Acid- 15% 4.0 7 2 mMNa-Citrate 59 7.0 2 Na isopropanol isopropano 2 mM Itaconic Acid- 15% 7.0 3 2 mMNa- 59 4.0 Na isopropanol Glutamate isopropano 2 mMNa-Citrate 5% in- S.O 9 2 mMNa- 59 7.0 O8O Glutamate isopropano 2 mMNa- 5% in- 4.0 6 2 mM Arginine 59 7.0 Glutamate O8O HCJNaOH isopropano 2 mMNa- 5% in- 7.0 8 2 mM Arginine 59 9.0 Glutamate O8O HCJNaOH isopropano 2 mM Arginine 5% in- 7.0 3 2 mM Itaconic Acid- 59 4.0 HCJNaOH O8O Na isopropano 2 mM Arginine 5% in- 9.0 4 2 mM Itaconic Acid- 59 7.0 HCJNaOH O8O Na isopropano 2 mM Itaconic Acid- 5% in- 4.0 5 2 mM Pivalic Acid 59 4.0 Na O8O isopropano 2 mM Itaconic Acid- 5% in- 7.0 2 2 mM Pivalic Acid 59 S.O Na O8O isopropano 2 mMNa-Citrate 15% in- S.O 9 2 MPEI7SOOOO 59 O8O isopropano 2 mMNa- 15% in- 4.0 7 2 MPEI 2SOOO 59 Glutamate O8O isopropano 2 mMNa- 15% in- 7.0 4 2 MPEI 2000 59 Glutamate O8O isopropano 2 mM Arginine 15% in- 9.0 4 2 mMNa- 59 4.0 HCJNaOH O8O Sulfate, Na-Acetate isopropano 2 mM Itaconic Acid- 5% in- 7.0 5 2 mMNa- 59 6.O Na O8O Sulfate, Na-Acetate isopropano 2 mMNa-Citrate 59% 4.0 isopropano 2 mMNa-Citrate 59% S.O 0713 The experiments with Vancomycin were also per isopropano formed on a larger scale (20 mg Vancomycin), namely, at 2 2 mMNa-Citrate 59% 7.0 mg/ml Vancomycin in a 5 ml total Volume; and at 10 mg/ml isopropano 2 mMNa- 59% 7.0 Vancomycin in a 2 ml total Volume. All cocktail mixes tested Glutamate isopropano produced very high quality microspheres of Vancomycin as 2 mM Arginine 59% 7.0 described below: HCJNaOH isopropano 2 mM Arginine 59% 9.0 HCJNaOH isopropano 2 mM Itaconic Acid- 59% 4.0 Na isopropano 20 mg Vancomycin 2 mM Pivalic Acid 59% 4.0 isopropano Microsphere 2 mM Pivalic Acid 59% S.O Counterion Antisolvent pH Quality isopropano 2 MPEI7SOOOO 59% 5 mMNa-Citrate 15% in- S.O 9 isopropano propanol 2 MPEI 2SOOO 59% 5 mM Na-glutamate 5% in- 7.0 9 isopropano propanol 2 mMNa- 59% 4.0 5 mMNa-Citrate 15% in- S.O 10 Sulfate, Na-Acetate isopropano propanol 2 mMNa- 59% 6.O 5 mM Na-glutamate 5% in- 7.0 9 Sulfate, Na-Acetate isopropano propanol Compound:siRNA (2 mg/ml)

None 59% 10 isopropano 2 mM Arginine 59% 7.0 7/8 HCJNaOH isopropano B. Nucleic Acid - siRNA 2 mM Arginine 59% 9.0 HCJNaOH isopropano Microsphere 2 mM Itaconic Acid- 59% 4.0 Counterion Antisolvent pH Quality Na isopropano 2 MPEI 2SOOO 59% Compound: siRNA isopropano 1 mMPEI 2SOOO 59% 2 mMNa-Citrate 59 4.0 2 isopropano isopropanol OS MPEI 2SOOO 59% isopropano US 2009/00982O7 A1 Apr. 16, 2009 56

-continued -continued

B. Nucleic Acid - siRNA C. Virus - Tobacco Mosaic Virus Compound: Tobacco Mosaic Virus Microsphere 0.5 mg/ml Counterion Antisolven pH Quality Microsphere O.1 MPEI 2SOOO 15% 7 Counterion Antisolvent pH Quality SOO.80 2 mM Arginine 30% 7.0 5 2 mMNa- None 7.0 3 HCJNaOH isopropano Glutamate 2 mM Arginine 30% 9.0 5.6 2 mM Arginine None 7.0 6 HCJNaOH isopropano HCJNaOH 2 mM Itaconic Acid- 30% 4.0 8 2 mM Arginine None 9.0 2 Na isopropano HCJNaOH 2 MPEI 2SOOO 30% 7 2 mMNa- None 4.0 4 isopropano sulfate Na-acetate 1 mMPEI 2SOOO 30% 8 2 mMNa- None 6.O 6 isopropano sulfate Na-acetate OS MPEI 2SOOO 30% 9 isopropano * As noted above, microspheres of good quality could be formed with O.1 MPEI 2SOOO 30% 6 tobacco mosaic virus, even in the absence of antisolvent. Some crystallinity isopropano was observed, but depending on the choice of counterion (e.g., pivalic acid), Compound: siRNA (0.25 mg/ml) uniform microspheres could be obtained without the addition of antisolvent. 2 mM Arginine 15% 7.0 O HCJNaOH isopropano 2 mM Arginine 15% 9.0 O HCJNaOH isopropano D. Peptides - Sonatostatin and Leuprolide 2 mM Arginine 30% 7.0 6 HCJNaOH isopropano Microsphere 2 mM Arginine 30% 9.0 3 Counterion Antisolvent pH Quality HCJNaOH isopropano Compound:siRNA (5 mg/ml) Compound: Somatostatin

2 mM Arginine 15% 7.0 3 2 mMNa-citrate 59 4.0 5 HCJNaOH isopropano isopropano 2 mM Itaconic acid- 15% 4.0 5 2 mMNa-citrate 5% S.O 5 Na isopropano so O8O 2 mM Arginine 30% 7.0 4 2 mMNa-citrate 5% 7.0 5 HCJNaOH isopropano 2 mM Arginine propano 7.0 5 2 mM Arginine 30 0. 4.0 4 i.S.S is bro 8O HCJNaOH SOO.80 2 mM Arginine 59 9.0 5 HCJNaOH isopropano 2 mM Itaconic Acid- 59 4.0 5 Na isopropano 2 mM Itaconic Acid- 59 7.0 3 Na isopropano C. Virus - Tobacco Mosaic Virus 2 mM Pivalic Acid- 59 4.0 5 Compound: Tobacco Mosaic Virus Na isopropano (0.5 mg/ml) 2 mM Pivalic Acid- 59 7.0 6 Na isopropano Microsphere 2 mMNa- 59 4.0 6 Counterion Antisolven pH Quality Glutamate isopropano 2 mMNa- 59 7.0 4 None 59 8 Glutamate isopropano isopropano 2 MPEI7SOOOO 4 2 mMNa-Citrate 59 4.0 9 2 MPEI 2SOOO 4 isopropano 2 MPEI 2000 3 2 mMNa-Citrate 59 S.O 4 2 mMNa- 59 4.0 8 isopropano sulfate Na-acetate isopropano 2 mM Pivalic Acid- 59 S.O 6 2 mMNa- 59 6.O 5 Na isopropano sulfate Na-acetate isopropano 2 mMNa- 59 7.0 7 2 mMNa-citrate 15% 4.0 5 Glutamate isopropano isopropano 2 mM Arginine 59 7.0 8 2 mMNa-citrate 15% S.O 5 HCJNaOH isopropano isopropano 2 mM Arginine 59 9.0 8 2 mMNa-citrate 15% 7.0 4 HCJNaOH isopropano isopropano 2 mMNa- 59 4.0 10 2 mM Arginine 15% 7.0 5 sulfate, Na-acetate isopropano HCJNaOH isopropano 2 mMNa- 59 6.O 7 2 mM Arginine 15% 9.0 7 sulfate, Na-acetate isopropano HCJNaOH isopropano 2 mMNa-Citrate None:* 4.0 5 2 mM Itaconic Acid- 15% 4.0 5 2 mMNa-Citrate None S.O 2 Na isopropano 2 mM Pivalic Acid- None S.O 8 2 mM Itaconic Acid- 15% 7.0 6 Na Na isopropano US 2009/00982O7 A1 Apr. 16, 2009 57

-continued -continued D. Peptides - Sonatostatin and Leuprolide D. Peptides - Somatostatin and Leuprolide Microsphere Microsphere Counterion Antisolvent pH Quality Counterion Antisolvent pH Quality 2 mM Pivalic Acid 15% 4.0 6 Na isopropanol 2 mMNa 15% 4.0 7 2 mM Pivalic Acid 15% 7.0 6 sulfate Na-acetate isopropanol Na isopropanol 2 mMNa 15% 6.O 5 2 mMNa 15% 4.0 Glutamate isopropanol sulfate Na-acetate isopropanol 2 mMNa 15% 7.0 3 Glutamate isopropanol 2 MPEI7SOOOO 2 MPEI 2SOOO 2 MPEI 2000 2 mMNa 15% 4.0 E. DAS181 Protein sulfate, Na-acetate isopro Compound: DAS181 2 mMNa 15% 6.O sulfate, Na-acetate isopro Microsphere Compound: Counterion Antisolven pH Quality 2 mMNa-citrate 59 4.0 2 mMNa-citrate 59 4.0 7 isopro isopropano 2 mMNa-citrate 59 S.O 2 mMNa-citrate 59 S.O 7 isopro isopropano 2 mMNa-citrate 59 7.0 2 mMNa-citrate 59 7.0 5 isopro isopropano 2 mM Arginine 59 7.0 2 mM Arginine 59 7.0 4 HCJNaOH isopro HCJNaOH isopropano 2 mM Arginine 59 9.0 2 mM Arginine 59 9.0 5 HCJNaOH isopro HCJNaOH isopropano 2 mM Itaconic Acid 59 4.0 2 mM Itaconic Acid 59 4.0 5 Na isopro Na isopropano 2 mM Itaconic Acid 59 7.0 2 mM Itaconic Acid 59 7.0 5 Na isopro Na isopropano 2 mM Pivalic Acid 59 4.0 2 mM Pivalic Acid 59 4.0 5 Na isopro Na isopropano 2 mM Pivalic Acid 59 7.0 2 mM Pivalic Acid 59 7.0 7 Na isopro Na isopropano 2 mMNa 59 4.0 2 mMNa 59 4.0 6 Glutamate isopro Glutamate isopropano 2 mMNa 59 7.0 2 mMNa 59 7.0 6 Glutamate isopro Glutamate isopropano 2 MPEI7SOOOO 2 MPEI7SOOOO 8 2 MPEI 2SOOO 2 MPEI 2SOOO 10 2 MPEI 2000 2 MPEI 2000 7 2 mMNa 59 4.0 2 mMNa 59 4.0 8 sulfate, Na-acetate isopro sulfate Na-acetate isopropano 2 mMNa 59 6.O 2 mMNa 59 6.O 7 sulfate, Na-acetate isopro sulfate Na-acetate isopropano 2 mMNa-citrate 59 4.0 5.6 2 mMNa-citrate 59% 4.0 5.6 isopro isopropano 2 mMNa-citrate 59 S.O 2 mMNa-citrate 59% S.O 7 isopro isopropano 2 mMNa-citrate 59 7.0 2 mMNa-citrate 59% 7.0 6 isopro isopropano 2 mM Arginine 59 7.0 2 mM Arginine 59% 7.0 4 HCJNaOH isopro HCJNaOH isopropano 2 mM Arginine 59 9.0 2 mM Arginine 59% 9.0 7 HCJNaOH isopro HCJNaOH isopropano 2 mM Itaconic Acid 59 4.0 2 mM Itaconic Acid 59% 4.0 7 Na isopro Na isopropano 2 mM Itaconic Acid 59 7.0 2 mM Itaconic Acid 59% 7.0 7 Na isopro Na isopropano 2 mM Pivalic Acid 59 4.0 2 mM Pivalic Acid 59% 4.0 6 Na isopro Na isopropano 2 mM Pivalic Acid 59 7.0 2 mM Pivalic Acid 59% 7.0 7 Na isopro Na isopropano 2 mMNa 59 4.0 2 mMNa 59% 4.0 6 Glutamate isopro Glutamate isopropano 2 mMNa 59 7.0 2 mMNa 59% 7.0 5 Glutamate isopro Glutamate isopropano 2 MPEI7SOOOO 2 MPEI7SOOOO 8 2 MPEI 2SOOO 2 MPEI 2SOOO 9 2 MPEI 2000 2 MPEI 2000 6 US 2009/00982O7 A1 Apr. 16, 2009 58

of macromolecules (see Examples 2-4), can readily be opti -continued mized by varying parameters, such as the concentrations of compound, counterion, and/or antisolvent, in a variety of E. DAS181 Protein permutations in high-throughput format. By conducting these Compound: DAS181 reactions in high-throughput format, conditions that are opti Microsphere mal for microsphere formation of any compound can rapidly Counterion Antisolvent pH Quality be identified. 2 mMNa- 15% 4.0 7 0718 96-well plates containing cocktail solutions of Tet sulfate, Na-acetate isopropanol racycline, Kanamycin or Ampicillin under various concen 2 mMNa- 15% 6.O 8 sulfate, Na-acetate isopropanol tration conditions were set up as described in Example 1. 2 mMNa-citrate 5% in- S.O 10 Arginine was used as counterion and isopropanol was used as O8O antisolvent. Concentrations of each of the cocktail compo 2 mM Arginine 5% in- 7.0 3 nents—the compound, the counterion and the antisolvent— HCJNaOH O8O 2 mM Arginine 5% in- 9.0 7 were varied as shown below in Table 15, and the effect on HCJNaOH O8O microsphere quality assessed. 2 mM Itaconic Acid- 5% in- 4.0 5 Na O8O TABLE 1.5 2 mM Itaconic Acid- 5% in- 7.0 2 Na O8O Arginine 2 mMNa- 5% in- 4.0 6 Concentration Isopropanol Microsphere Glutamate O8O (mg/ml) (%) Quality 2 mMNa- 5% in- 7.0 4 Glutamate O8O Tetracycline 2 mMNa-citrate 15% in- S.O 8.9 Concentration O8O (mg/ml) 2 mM Arginine 15% in- 9.0 6 HCJNaOH O8O 25 60 O 1 2 mM Itaconic Acid- 15% in- 4.0 4 25 60 10 3 Na O8O 25 60 2O 4 2 mM Itaconic Acid- 15% in- 7.0 2 25 60 30 7 Na O8O 25 60 40 5 2 mMNa- 15% in- 4.0 6.7 25 60 50 6 Glutamate O8O 2O 48 60 6 2 mMNa- 15% in- 7.0 4 15 36 70 1 Glutamate O8O 25 60 50 5 25 50 50 9 25 40 50 9 0714 Results: These experiments demonstrate that by 25 30 50 9 selecting the appropriate combination of: (a) type and (b) 25 2O 50 7 concentration of compound, counterion and antisolvent, 25 10 50 7 microspheres of good quality (at least 6, as high as 10) can be 25 5 50 7 25 O 50 9 obtained using a wide variety of macromolecules and Small O 15 25 7 molecules. Depending on the particular combination of com 5 15 25 9 pound, counterion and antisolvent, the quality of the micro 10 15 25 8.9 spheres often was as good or better than the quality of micro 15 15 25 8.9 spheres obtained using the sialidase fusion protein, DAS181, 2O 15 25 7 under comparable conditions. 25 15 25 8 0715. In control cocktail reactions containing no com 30 15 25 9 pounds, it was noted that some counterions, such as polyeth 31.25 15 25 7 yleneimine (PEI) and Na-acetate/sulfate, could form micro Kanamycin spheres under certain conditions. Without being bound by any Concentration theory, depending on the compound of interest, such counte (mg/ml) rions potentially could act as “primers' or “carriers’ that help 25 60 O O to nucleate and/or facilitate the formation of higher quality 25 60 10 O microspheres, relative to those obtained with “non-nucleat 25 60 2O O ing counterions. For example, formation of a compound 25 60 30 3 (see, for example, in Table 14 above, microspheres formed 25 60 40 O 25 60 50 O from siRNA and DAS181, using PEI as counterion). 2O 48 60 6 0716. The results further demonstrate that under certain 25 60 50 O conditions, microspheres can be formed in the absence of 25 50 50 O counterion and/or antisolvent. For example, in the case of 25 40 50 2 siRNA, very high quality (scale of 10) microspheres were 25 30 50 4 obtained when no counterion was added. Similarly, tobacco 25 2O 50 7 mosaic virus formed microspheres in the absence of counte 25 10 50 10 rion and, in Some instances, in the absence of antisolvent. 25 5 50 7 25 O 50 8 EXAMPLE 1.4 O 15 25 O 5 15 25 4 Size and Quality of Microspheres as a Function of 10 15 25 8 Concentration of the Cocktail Components (Com 15 15 25 7 pound, Counterion, Antisolvent) 2O 15 25 7 0717. This Example demonstrates that the size and quality 25 15 25 8 of the microspheres of small molecule compounds, like those US 2009/00982O7 A1 Apr. 16, 2009 59

0723. When the kanamycin microspheres were studied as TABLE 15-continued a function of varying arginine concentration, it was found that Arginine decreasing the arginine concentration resulted in higher qual Concentration Isopropanol Microsphere ity, Smaller microspheres. At less than 10 mg/ml arginine, (mg/ml) (%) Quality however, the microspheres became larger and tended to 30 15 25 7 agglomerate more. In the absence of arginine, high quality 31.25 15 25 9 microspheres were again obtained, although some aggrega Ampicillin Concentration tion was present. (mg/ml) 0724. When varying the concentration of kanamycin, it 25 60 O O was found that as the concentration of kanamycin was 25 60 10 3 increased, the quality of the microspheres increased and the 25 60 2O O size decreased. 25 60 30 O 25 60 40 O 0725 Ampicillin: Although microspheres were obtained 25 60 50 6 using amplicillin, their hygroscopic nature made it difficult to 15 36 70 O 25 60 50 2 unambiguously assign their quality. In general, several con 25 2O 50 O ditions produced distinct microspheres, with the best quality 25 10 50 1 microspheres being observed at high (50%) antisolvent con 25 5 50 1 25 O 50 2 centration. 0726. This example demonstrates that a variety of small molecule antibiotics can produce microspheres by the meth Results: ods provided herein. The example also demonstrates that 0719 Tetracycline: With tetracycline, the absence of anti under certain conditions, the addition of a counterion may not Solvent resulted in few, ifany, microspheres being formed. As be necessary for microparticle formation. This was observed the concentration of antisolvent was increased, the quality of for all three antibiotics tested. Without being bound by theory, the microspheres increased, reaching a maximum at about it is possible that the compounds themselves function as 30% isopropanol. Increasing the isopropanol concentration counterions, or that the preparations used in the experiments beyond 30% resulted in a decrease in overall microsphere contained excipients/impurities/bulking agents that served as quality and the formation of larger, chunky agglomerations of counterions. microspheres and crystalline solids. At isopropanol concen trations of 60% and 70%, the cocktail mixture was found to precipitate before freezing, resulting in large amounts of EXAMPLE 1.5 aggregates and crystals. A few microspheres were formed at the highest isopropanol concentrations, but they were not of Preparation of Microspheres from Water-Insoluble uniform size. Molecules 0720 Microsphere quality was then assessed as a function of counterion (arginine) concentration. It was found that at Paclitaxel constant antisolvent and tertracycline concentration, the microsphere size distribution decreases and its overall quality 0727. The chemotherapeutic agent, Paclitaxel, has a log P increases as the concentration of arginine is decreased, with value that is higher than 3 (log of the octanol/water partition 30 mg/ml arginine giving the Smallest size distribution as coefficient) (Bombuwala et al., Beilstein J. Org. Chem. 2006, visualized by light microscopy. It was interesting to note that 2:13), which is a value that is representative of a large portion in the absence of counterion, Small microspheres with very of the Small molecule drugs that are currently on the market. little size variation were observed. Their overall quality was Hence, the identification of conditions for Paclitaxel micro high, although some amount of aggregation was present. sphere formation should be applicable to a large number of 0721 Microsphere quality was also assessed by increas therapeutically relevant compounds. ing tetracycline concentration at constant antisolvent and arginine concentrations. In the absence of tetracycline, hygro 0728 Paclitaxel, being water-insoluble, was dissolved in scopic arginine microspheres were formed. As the tetracy one of the following organic solvents: Isopropanol, t-Butyl cline concentration was increased, the microsphere size was Alcohol or DMSO. A 20 mg/ml stock solution of paclitaxel in found to increase to a maximum at a concentration of 25-30 each of the organic solvents was used to generate cocktail mg/ml tetracycline, then decreased again as the concentration solutions in 96-well plates, where the net concentration of of tetracycline was further increased. At 25-30 mg/ml tetra paclitaxel in each well (i.e., reaction) was 2 mg/ml paclitaxel. cycline, aggregation also was minimal; the aggregation With ispropanol, a 20 mg/ml slurry was obtained and used as increased as the tetracycline concentration was further the stock solution, because the solubility of paclitaxel in increased. isopropanol is lower than int-Butyl Alcohol and DMSO. 2 0722 Kanamycin: The formation of microspheres from mMNa-citrate buffer, pH 5.0, was used as the antisolvent and kanamycin required at least 25-30% antisolvent (isopro counterion. The various experimental conditions are listed panol); below this concentration, hygroscopic crystals were below in Table 16. The plates containing the cocktail solu formed. At 60% isopropanol, better results were obtained, tions with varying concentrations of antisolvent/buffer rela although there was precipitation prior to freezing, which tive to the concentration of organic Solvent, were then placed compromised microsphere quality. in a -80° C. freezer for lyophilization. US 2009/00982O7 A1 Apr. 16, 2009 60

Optical microscopy data did however reveal the presence of TABLE 16 microspheres, with the best microspheres being observed when 50% DMSO was used. Compound: Paclitaxel (2 mg/ml Citrate Buffer EXAMPLE 16 (mM) Solvent (%) Effect of Drug, Antisolvent and Counterion Ratios 90% isopropanol on the Quality of Microspheres 75% isopropanol 50% isopropanol 0732 Experiments were performed to evaluate the effect 25% isopropanol of antisolvent and counterion concentration variation on the 10% isopropanol formation of microspheres. The peptides leuprolide and 100% isopropanol 50% isopropanol Somatostatin, and the antibiotics Vancomycin and tobramy 90% t-butanol cin, were tested under a variety of conditions for forming 75% t-butanol microspheres. Table 17 describes the conditions under which 50% t-butanol the reactions were performed. Samples were analyzed in 25% t-butanol 10% t-butanol 96-well plates as described in the previous Examples. 100% t-butanol 50% t-butanol TABLE 17 90% DMSO 75% DMSO Isopropanol Microsphere 50% DMSO (%) Quality 25% DMSO 10% DMSO Compound: Leuprolide (2 mg/ml) 100% DMSO 50% DMSO Na-glutamate, pH 7.0 (mM) 2 50 9 Results: 2 40 7 2 30 8 2 2O 9 0729. With all three organic solvents, it was found that 2 10 5 paclitaxel precipitated if the solvent concentration was 25% 2 5 7 or less. Optical microscopy of the lyophilized samples 2 2.5 8 2 O 3 showed that with the organic solvent isopropanol, the micro 17 5 5 particles increased in quality as the concentration of isopro 15 5 5 panol was lowered from 90%, with the best microspheres 12.5 5 7 10 5 7 being formed in 50% isopropanol. When the concentration of 7.5 5 7 isopropanol was lowered below 50%, crystals were observed, 5 5 6 potentially due to precipitation of paclitaxel prior to freezing. 2.5 5 7 When 50% isopropanol was used with no citrate counterion, O 5 6 the microspheres showed a higher tendency to aggregate than Compound: Somatostatin (2 mg/ml) in the presence of 2 mM citrate. With 100% isopropanol and Na-Sulfate, Na no citrate counterion, the samples appeared to have high Acetate, pH 4.0 crystallinity and were aggregated, although many Small (mM) microparticles were also observed. 2 50 9 2 40 8 0730. With t-butanol, the optimum solvent concentration 2 30 9 was higher than isopropanol, with best results being observed 2 2O 7 at 90% t-butanol and aggregation and rod-like formations 2 10 67 2 5 6 increasing as the concentration of Solvent was decreased. At 2 2.5 34 25% and 10% t-butanol, significant crystallinity was 2 O 3 observed, likely due to precipitation of paclitaxel before 17 5 6 15 5 8 freezing. In 100% t-butanol with no citrate counterion, the 12.5 5 9 microspheres were almost as high in quality as the best qual 10 5 7 ity microspheres observed at 90% t-butanol and 2 mM citrate, 7.5 5 7 as discussed above. When the concentration of t-butanol was 5 5 67 2.5 5 6 lowered to 50% in the absence of citrate counterion, high O 5 7 quality microspheres were still present, although the aggre Compound: Vancomycin (2 mg/ml) gation increased. Na-citrate, pH 5.0 0731. The results obtained with the organic solvent (mM) DMSO showed higher amounts of crystallinity in general, along with aggregated microspheres of lower quality, relative 2 50 7 2 40 9 to isopropanol and t-butanol. This could be due to the high 2 30 8 boiling point of DMSO, as the water in the solutions likely 2 2O 10 evaporated/Sublimed first upon lyophilization, leaving nearly 2 10 7 pure DMSO solution from which the paclitaxel crystallized. US 2009/00982O7 A1 Apr. 16, 2009 61

tin was also found to form microspheres in the absence of TABLE 17-continued counterion, but they were aggregated and of varying (not uniform) size. Isopropanol Microsphere 0736. In the case of Vancomycin, with changing antisol (%) Quality 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 2 O 7 tration was further dropped down to 0% isopropanol, a high 17 5 6 degree of crystallinity was present, with a few aggregated 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 7.5 5 9.8 mM citrate, and the amount of aggregation decreased as the 5 5 9 counterion concentration was decreased. The best micro 2.5 5 9.8 spheres formed below 7.5 mM citrate, but as the counterion O 5 7 concentration was further dropped down to Zero, the amount Compound: Tobramycin (2 mg/ml) of aggregation again increased. Itaconic acid-Na, 0737. In the case oftobramycin, as the antisolvent concen pH 4.0 (mM) tration was varied, with 50% isopropanol there was a signifi cant amount of crystallinity and aggregation, although micro 2 50 7 2 40 6.7 spheres were also detected. As the antisolvent concentration 2 30 8.9 was reduced from 40% to 10%, the microspheres formed 2 2O 8 were found to be well-separated and of high quality. When the 2 10 9 antisolvent concentration was further decreased from 5% to 2 5 7/8 2 2.5 7/8 0%, the amount of aggregation again increased and at 0% 2 O 4f S there was a high degree of crystallinity along with significant 17 5 Crystals numbers of aggregated microspheres. 15 5 Crystals 12.5 5 Crystals 10 5 Crystals EXAMPLE 17 7.5 5 Crystals 5 5 Crystals Aerodynamic Particle Size Distribution of Vancomy 2.5 5 7 cin Microspheres for Inhalation O 5 8 0738. As described herein, the methods provided herein can be used to produce microspheres in any desired size 0733 Results: range, including a range of about 0.5 micron to about 6-8 0734. In the leuprolide group, decreasing antisolvent con microns for delivery via inhalation. centration reduced the aggregation of microspheres with an (0739 A. Preparation of Microspheres optimum at 10% isopropanol, and the aggregation increased 0740 Vancomycin was dissolved in aqueous buffer at a again as the isopropanol concentration was further reduced final concentration of 10 mg/ml. The cocktail contained 5 downto 0%. When the counterion concentration was varied at mM sodium citrate pH 5.0 as counterion and 15% V/v n-pro constant antisolvent concentration (5%), 17 mM counterion panol as anti-solvent. A 2 ml aliquot of cocktail was frozen in showed a high degree of crystal formation. The crystal for a 10-ml lyophilization vial placed in a -80° C. freezer for 1 mation decreased as the counterion (buffer) concentration hour. The frozen vial was transferred onto a -45° C. lyo was decreased, until at 10 mM, the microspheres are evenly philizer shelf and freeze dried for 36 hours. sized and well separated. As the buffer concentration was 0741. B. Aerodynamic Particle Size Distribution of further decreased beyond 10 mmM, the aggregation began to Microspheres increase again, with a moderate degree of crystallinity being 0742 The microspheres prepared as described in Example observed at 0 mM glutamate. 5 were tested by Cascade Impaction using a New Generation 0735. In the case of somatostatin, even-sized well-sepa Impactor. The deposition of pharmaceuticals in the respira rated microspheres were observed at 50% isopropanol. The tory tract can be predicted by the aerodynamic behavior of level of aggregation increased as the concentration of antisol particles (microspheres) on the stages/collection plates of the vent was decreased, to an optimum of 10% isopropanol. cascade impactor. Below 10% isopropanol, crystals began to appear and con 0743. The microspheres (10 mg) were loaded into HPMC tinued to increase as the antisolvent concentration was (hydroxypropyl methylcellulose) capsule. The capsule was decreased to 0% isopropanol, where a majority of the sample placed into a CycloHaler (PharmaChemie) dry powder was crystalline with only a few aggregated microspheres. inhaler and Subjected to cascade impaction. The collection When the counterion concentration was varied at constant plates of the impactor representing various areas/stages of antisolvent concentration (5%), at 17 mM Sulfate/Acetate deposition post-inhalation (trachea, primary and secondary and 5% isopropanol, microspheres were present, but a high bronchi, terminal bronchi, alveoli, etc.) were coated with degree of crystallinity also was observed. As the concentra silicon spray to prevent bouncing of the microspheres. The tion of counterion was decreased, the amount of crystals microspheres from the stages and collection plates were present decreased until well separated microspheres were recovered into a phosphate buffered saline containing 0.1% detected at 12.5 mM counterion concentration. As the sulfatef Tween, and the amount of deposited Vancomycin recovered acetate concentration was further decreased, aggregation from each stage and collection plate was quantified by mea increased again and microsphere size decreased. Somatosta Suring absorbance at 280 nm. US 2009/00982O7 A1 Apr. 16, 2009 62

0744 Results: The geometric size of microspheres was under the bag to equilibrate for approximately 30 seconds, assessed by light microscopy and found to be in the range of before opening and spreading onto the glass slide. 1.0-3.0 microns. As shown in Table 18 below, the aerody 0748 Prostaglandin is unstable at pH values lower than 8: namic particle size was consistent with the observed geomet therefore, the following basic buffers were used in this experi ric size. The results demonstrate that methods provided ment: Polyethyleneimine (PEI), Triethylamine (TEA) and herein can produce microspheres for delivery into deep lungs, Arginine. Ciprostene is not highly soluble in aqueous solu and that the microspheres produced by methods provided tions, therefore n-propanol was added to the buffer, in herein have good disagglomeration and flowability properties amounts that rendered the compound soluble. The solvent/ (provide a higher delivered dose). antisolvent system for the prostaglandins was water/n-pro panol/t-butanol (water/aqueous buffer being more of the TABLE 18 “antisolvent component for Ciprostene, which has poor Results of Cascade Impaction Analyses of Vancomycin solubility in water, and n-propanol/Tert-Butyl alcohol (t-bu Microspheres tanol, tBA) being more of the “antisolvent component for PGI2, which has higher solubility in water). The results are Component of Corresponding Expected Deposition Percent summarized in Table 19 below: the Cascade Size Cut-Off in Respiratory Deposition of Impactor (microns) Airways Vancomycin TABLE 19 Capsule + NA NA 37.57 device Buffer? Microsphere Throat >10 oral cavity 10.48 Counterion pH (% n-propanol) (% t-butanol) Quality 1 >8.06 Oral cavity pharynx 3.33 2 4.46-8.06 pharynx 7.71 Compound: Prostaglandin I2 3 2.82-4.46 tracheabronchi 15.47 4 1.66-2.82 secondary bronchi 16.69 2 mM Arginine 9 2O O 7 5 0.94-1.66 terminal bronchi 6.38 2 mM Arginine 9 30 O 5 alveoli 2 mM Arginine 9 2O 5 7/8 6 0.55-0.94 alveoli 1.59 2 mM Arginine 9 2O 30 5 7 0.34-0.55 alveoli O.S1 2 mM Arginine 9 2O 55 5.6 8 &O.34 alveoli 0.27 2 mM Arginine 9 2O 70 4 2 mM TEA 11 2O O O 2 mM TEA 11 30 O 2.3 2 mM TEA 11 2O 5 7/8 2 mM TEA 11 2O 30 8 EXAMPLE 1.8 2 mM TEA 11 2O 55 7 2 mM TEA 11 2O 70 8 Preparation of Microspheres Using Prostaglandin 2 MPEI 10.75 2O O 7 2 MPEI 10.75 30 O 8 2 MPEI 10.75 2O 5 7 0745 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 requiring the drug to be administered through continuous 2 mM Arginine 9 2O 5 8 infusion in order to have an appreciable effect. Therefore, it is 2 mM Arginine 9 2O 30 6.7 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 the pharmacokinetic effects associated with clearance rates 2 mM TEA 11 30 O 3f4 and stability in the bloodstream. This example demonstrates 2 mM TEA 11 2O 5 8.7 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 0746 The experiments were performed using PGI, and an 2 MPEI 10.75 2O O 6 analog of PGI, Ciprostene, at a concentration of 2 mg/ml. 2 MPEI 10.75 30 O 6.7 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. 0747. Because the resulting prostaglandin microspheres Results: are hygroscopic, upon microsphere initiation, the humidity was maintained at low levels during the experiments, using a 0749. With PGI, several conditions were identified for nitrogen gas tank attached to the backfill system on the lyo good quality microsphere formation, with several ratings philizer. Each of the reaction tubes was flushed with N. In above 6 and a maximum rating of 8. When the buffer/coun addition, the backfill valve on the lyophilizer was left open to terion was Arginine, lower concentrations of n-propanol and continually flush a low humidity atmosphere over the t-butanol favored better microsphere formation alcohol con samples. The microscope used to visualize the resulting centrations, crystal formation was observed. When the buffer/ microspheres was contained within a plastic bag that was counterion was TEA, on the other hand, higher concentra continually purged with dry N. Sample tubes were placed tions of t-butanol favored higher quality microsphere US 2009/00982O7 A1 Apr. 16, 2009

formation. When the buffer? counterion was PEI, the best like crystals were observed, although there were some quality microspheres were obtained at a higher concentration microparticles present. With Tobacco Mosaic Virus, high of n-propanol (30%), and in the absence oft-butanol. quality microspheres, with a rating of 9, were formed in both (0750 Ciprostene is a more stable analog of PGI, and it flash freeze cases. It therefore appears that the formation of also appeared to be less hygroscopic. With Arginine buffer, no Tobacco Mosaic Virus microspheres, under the conditions particular concentration-dependent trend was observed, but tested, was not highly affected by the rate of freezing. several Solvent conditions produced high quality micro 0755 With Vancomycin, on the other hand, the quality of spheres with ratings of 8 (see, e.g. 20% n-Propanol/5% t-bu the microspheres decreased as the freezing rate was tanol and 20% n-Propanol/55% t-butanol). With TEA, the increased. While the Vancomycin cocktail produced a micro quality of microspheres obtained with n-propanol in the sphere rating of 9/10 under normal freezing conditions, as absence of t-butanol was low. The quality of microspheres described in Example 13, the 200 ul flash freeze sample increased as t-butanol was added to the cocktail solution, with provided lower quality microspheres with a rating of 7 and a maximum at about 5% t-butanol. As the concentration of observed aggregation. The PCR tube flash freeze produced t-butanol was increased even further, increasing amounts of ever lower quality microspheres, with a rating of 5, higher aggregation was observed. PEI proved to be the best counte amounts of aggregation and a significant amount of rod-like rion for ciprostene, with a maximum microsphere quality crystals. Thus, in the case of Vancomycin, faster freeze rates rating of 9 at 20% n-propanol/5% t-butanol. As the concen resulted in lower quality microspheres. Similarly, with Tetra tration oft-butanol was further increased, increasing amounts cycline, while microsphere ratings of 8/9 were obtained under of aggregation were observed. normal freezing conditions (see Example 14), both flash 0751. The results demonstrate that high quality micro freeze conditions produced lower quality microspheres of spheres of prostaglandin can be formed under a variety of rating 5/6, with significant aggregation. conditions, which should facilitate a stable formulation for 0756. The results demonstrate that the freezing rate can pulmonary delivery. have an impact on the quality of microspheres generated according to the methods provided herein. The impact, how EXAMPLE19 ever, is dependent on the compound forming the micro Effect of Cooling Rate on the Quality of Micro spheres. As shown in this example, for Some compounds, spheres such as Paclitaxel, DAS181, Vancomycin and Tetracycline, if the freezing rate is too rapid, the microspheres can get trapped 0752. This example demonstrates that a controlled cooling in crystalline phases or aggregate before having the opportu rate, during which the cocktail solutions from which the nity to grow to a reasonable size. microspheres are produced are maintained at specific tem peratures for defined periods of time, as opposed to flash freezing, produces higher quality microspheres with desired EXAMPLE 20 characteristics. Flash freeze experiments were conducted Efficiency of Nucleic Acid Incorporation into Micro with five different cocktails that previously produced excel spheres lent microspheres under standard freezing conditions per formed according to the methods provided herein. The com 0757. To assess the process yield for nucleic acid incorpo pound/counterion/antisolvent conditions were as follows: ration into microspheres, the following experiment was con 1) Paclitaxel/citrate pH 5.0/90% t-butanol (see Example 15) ducted. One mg of yeast tRNA (Sigma, Type X-SA) in 0.5 ml 2) DAS181/citrate pH 5/5% n-propanol (see Example 13) Volume (2 mg/ml final concentration in the cocktail) was 3) Tobacco Mosaic Virus/Na sulfate-Na acetate pH 4/5% combined with isopropanol (IPA: 40% final concentration) isopropanol (see Example 13) and sodium citrate (100 mM final concentration) at pH 8.0. 4) Vancomycin/citrate pH 5/5% n-propanol (see Example 13) Formation of microparticles from the resulting cocktail was 5) Tetracycline/Arginine/25-30% isopropanol (see Example induced by placing the cocktail on ice. The microspheres 14) were fixed by the addition of 10 ml (20 volumes) of IPA, and (0753 Experiments were performed with 200ul of each of pelleted by centrifugation at 5000 rpm for 3 min. The pellet the above cocktail solutions in a 2 ml lyophilization bottle was dried in a vacuum. Microscopic analysis confirmed the (first flash freeze condition), and 25 ul of each of the above formation of high quality microspheres, 1-2 micron in size, cocktail solutions in a PCR tube (second flash freeze condi and the absence of aggregated material or crystals. tion). The samples in the lyophilization bottles took approxi (0758. The amount of tRNA recovered in the pellet and the mately 15 seconds to freeze. The samples in the PCR tubes supernatant was quantitated by UV absorption at 260 nm. It took approximately 3 seconds or less to freeze. was found that 78% of the tRNA was packaged into the microparticles and 22% tRNA remained in the supernatant. Results: This result demonstrated that tRNA, and likely other nucleic acids such as DNA and siRNA, can be efficiently condensed 0754 Microscopic analysis of the samples showed that in and packaged into a microsphere formulation. most cases, the freezing rate has a significant effect on the formation of microspheres. The Paclitaxel samples were mostly crystalline in both cases after the flash freeze, EXAMPLE 21 although there was evidence that microspheres were begin siRNA that is Incorporated into Microspheres ning to form. The DAS181 cocktail showed high quality Retains its Activity microspheres, with a rating of 9, when flash frozen in the lyophilization bottle. The quality of the DAS181 micro 0759 Experiments were performed to assess if the method spheres, however, was reduced to a rating of 5 in the faster of producing microspheres as provided herein inhibits the freezing PCR tube experiment; a significant amount of rod activity of the molecules incorporated in the microspheres. US 2009/00982O7 A1 Apr. 16, 2009 64

0760 Preparation of siRNA Microspheres 0761 The exemplary molecule used in this experiment is TABLE 20-continued double stranded GAPDH siRNA (sense sequence 5'-UGGU UUACAUGUUCCAAUAUU-3' (SEQ ID. NO: 27); anti siRNA Gene Silencing Activity when used alone or when incorporated sense sequence 5'-UAUUGGAACAUGUAAACCAUU-3' into microspheres. (SEQID NO: 28); with two “UU” overhangs at each 3'-end). Sample Microspheres containing GAPDH siRNA in various cocktail No. Formulation Cocktail % Activity formulations, as described below, were produced: 6 10 mM (Lysine, Citric acid, 1:1 molar ratio), 13020 0762. 1: 2 mM Arginine, pH 7.0, 15% IPA, 2 mg/ml 15% 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 0763. 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 0764 3:2 mM Itaconic 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 0765 4: 10 mM (Glutamic acid, Lysine, Alanine, 3:2:5 described herein, were reconstituted in water to 10uM siRNA. The negative molar ratio), 5% IPA, 1 mg/ml. controls are composed of each formulation without the siRNA. For the posi tive control, lyophilized GAPDH siRNA was reconstituted to 10uM siRNA. 0766 5: 10 mM (Lysine, Citric acid, 1:4 molar ratio), 15% Each siRNA sample was transfected into Hep-2 cells using lipid-based IPA, 1 mg/ml siRNA, siPORT TM NeoFXTM transfection reagent (Applied Biosystems #AM4510). 0767 6: 10 mM (Lysine, Citric acid, 1:1 molar ratio), 15% At 48 hr post transfection, GAPDH enzyme activity was measured using the IPA, 1 mg/ml siRNA KDalert TM GAPDH Assay Kit (Applied Biosystems #AM1639). Fluores cence readings in the negative controls (no siRNA used in transfections) 0768 7: 10 mM Alanine, 15% IPA, 1 mg/ml siRNA were used to set the baseline. The changes of fluorescent reading in the posi Control formulations contained all cocktail ingredients with tive controls (siRNA not subjected to lyophilization) were set as 100% activ the exception of siRNA. A lyophilized siRNA control con ity for siRNA. tained no excipients and 15% IPA. 0769 The resulting cocktails were chilled to form micro spheres and frozen in a single step by placing the vial onto the EXAMPLE 22 shelf of a -80° C. freezer. Lyophilization was performed overnight at shelf temperature of +10°C. and a vacuum of 150 Microspheres Containing Nucleic Acids as Active mTorr. Agents and Gelatin as a Carrier (0770 Activity of siRNA in Microsphere Formulations 0772. This Example demonstrates that the methods pro (0771. The siRNA microspheres isolated from the lyo vided herein can be used to prepare microspheres containing philization were then reconstituted and transfected to Hep-2 cells. As a positive control, the same amount of GAPDH gelatin, and the gelatin can act as a carrier for other active siRNA in the original buffer was lyophilized, reconstituted, agents in the microspheres. The gelatin-containing micro and transfected, without formation of microspheres. At 48 hr spheres are stable, and they retain their stability when nucleic post transfection, the level of GAPDH in the Hep-2 cells was acids are incorporated along with the gelatin. Microspheres measured using a fluorescent enzymatic assay. The results were prepared containing gelatin from a variety of sources as (Table 20) demonstrated that siRNAs processed into micro follows: spheres had gene-silencing activity that was equivalent to or, 0773 A. Gelatin from bovine skin, Type B (Sigma, in Some instances, even greater than that of the corresponding G9382) positive control (i.e., 100% or more gene-silencing activity). 0774 B. Gelatin from porcine skin, Type A (Sigma, Microscopic analyses confirmed the formation of high qual G2500) ity microspheres. (0775 C. Gelatin from coldwater fish skin (Sigma, G.7041) 0776 Preparation of Microspheres Containing Gelatin: TABLE 20 for Each of the Gelatin compounds listed in A-C above, siRNA Gene Silencing Activity when used alone or when incorporated cocktail solutions containing from 2.5 mg/ml to 25 mg/ml of into microspheres. gelatin dissolved in aqueous solvent, counterions at different pH, and IPA as antisolvent at different concentrations, as Sample listed below, were prepared in a 96-well microtiter plate (0.1 No. Formulation Cocktail % Activity ml cocktail/well) at room temperature. The cocktails in the Lyophilized siRNA positive control 10O2O 96-well plates were cooled by placing in a freezer. The chilled (no microsphere) plates were transferred onto pre-chilled (-45° C.) shelves of 1 2 mM Arginine, pH 7.0, 15% IPA, 2 mg/ml siRNA 7S 25 2 2 mM PEI (25,000 mol wit, branched, Sigma), 11S 15 a Millrock Lab Series Lyophilizer, and a vacuum was applied. pH 10, 15% IPA, 2 mg/ml siRNA The frozen cocktail solutions were allowed to lyophilize for 3 2 mM Itaconic Acid, pH 8.0, 15% IPA, 1102 16 hours. 2 mg/ml siRNA 1 neg Same as 1, but no siRNA (negative control) O (0777. The lyophilized powders from the bottoms of the 2 neg Same as 2, but no siRNA (negative control) O wells were transferred onto glass slides and analyzed by light 3 neg Same as 3, but no siRNA (negative control) O microscopy for appearance. The quality of the product micro 4 10 mM (Glutamic acid, Lysine, Alanine, 13S 25 spheres was scored based on the uniformity of the micro 3:2:5 molar ratio), 5% IPA, 1 mg/ml siRNA spheres, the absence of undesirable non-microsphere par 5 10 mM (Lysine, Citric acid, 1:4 molar ratio), 12S25 ticles (glass-like crystalline forms), and the absence of 15% IPA, 1 mg/ml siRNA aggregates. The scoring system as described in Table 13 was used. US 2009/00982O7 A1 Apr. 16, 2009

0778 Table 21 below shows the various combinations of compound, solvent, antisolvent and counterion that were TABLE 21-continued used to generate microspheres, and the quality of the resulting microspheres. Gelatin Microspheres Concentration TABLE 21 of Microsphere Compound Counterion Antisolven pH Quality Gelatin Microspheres 25 mg/m 20 mM Citric Acid 30% 3.5 6 Concentration isopropano of Microsphere 2.5 mg/m 20 mM Tris 59 8 1 Compound Counterion Antisolvent pH Quality isopropano 2.5 mg/m 20 mM Tris 10% 8 1 Compound: Gelatin from isopropano bovine skin, Type B 2.5 mg/m 20 mM Tris 20% 8 8 isopropano 2.5 mg/ml 20 mM Citric Acid 10% 3.5 2 2.5 mg/m 20 mM Tris 30% 8 7 isopropano isopropano 2.5 mg/ml 20 mM Citric Acid 20% 3.5 3 5 mg/m 20 mM Tris 10% 8 5 isopropano isopropano 2.5 mg/ml 20 mM Citric Acid 30% 3.5 8 5 mg/m 20 mM Tris 30% 8 5 isopropano isopropano 10 mg/ml 20 mM Citric Acid 59 3.5 6 10 mg/m 20 mM Tris 10% 8 1 isopropano isopropano 10 mg/ml 20 mM Citric Acid 10% 3.5 5 10 mg/m 20 mM Tris 20% 8 5 isopropano isopropano 10 mg/ml 20 mM Citric Acid 20% 3.5 2 10 mg/m 20 mM Tris 30% 8 1 isopropano isopropano 10 mg/ml 20 mM Citric Acid 30% 3.5 2 25 mg/m 20 mM Tris 20% 8 9 isopropano isopropano 25 mg/ml 20 mM Citric Acid 30% 3.5 1 25 mg/m 20 mM Tris 30% 8 9 isopropano isopropano Compound: Gelatin from porcine skin, Type A (0779 Preparation of Microspheres Containing Gelatin 2.5 mg/m 20 mM Citric Aci 59 3.5 1 isopropano and Nucleic Acids: for each of the three gelatin compounds 2.5 mg/m 20 mM Citric Aci 10% 3.5 2 listed in A-C above, cocktail solutions containing 15 mg/ml isopropano of gelatin and various concentrations of tRNA dissolved in 2.5 mg/m 20 mM Citric Aci 20% 3.5 2 isopropano aqueous solvent, with counterions at different pH, and IPA as 5 mg/m 20 mM Citric Aci 10% 3.5 6 antisolvent at different concentrations, as listed below, were isopropano prepared in a 96-well microtiterplate (0.1 ml cocktail?well) at 5 mg/m 20 mM Citric Aci 20% 3.5 5 room temperature. tRNA used in this experiment was type isopropano 5 mg/m 20 mM Citric Aci 30% 3.5 2 X-SA, from Bakers Yeast (Sigma, R8759). The cocktail solu isopropano tions were cooled by placing in a freezer. The chilled plates 10 mg/m 20 mM Citric Aci 20% 3.5 1 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 1 isopropano frozen cocktail solutions were allowed to lyophilize for 16 Compound: Gelatin from hours. cold water fish skin 0780. 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 3 isopropano microscopy for appearance. The quality of the product micro 2.5 mg/m 20 mM Citric Aci 10% 3.5 2 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 2 isopropano ticles (glass-like crystalline forms), and the absence of 2.5 mg/m 20 mM Citric Aci 30% 3.5 5 aggregates. isopropano 5 mg/m 20 mM Citric Aci 59 3.5 6 isopropano 5 mg/m 20 mM Citric Aci 10% 3.5 6 Concentration of Microsphere isopropano tRNA Counterion Antisolvent pH Quality 5 mg/m 20 mM Citric Aci 20% 3.5 8 isopropano Compound: Gelatin from bovine 5 mg/m 20 mM Citric Aci 30% in- 3.5 9 skin, Type B with tRNA propanol 10 mg/m 20 mM Citric Aci 59 3.5 1 2 mg/ml 10 mM Citric Acid 10% 3.5 7 isopropano isopropanol 10 mg/m 20 mM Citric Aci 20% 3.5 7 2 mg/ml 10 mM Citric Acid 20% 3.5 5 isopropano isopropanol 10 mg/m 20 mM Citric Aci 30% 3.5 5 2 mg/ml 10 mM Citric Acid 30% 3.5 4 isopropano isopropanol 25 mg/m 20 mM Citric Aci 20% 3.5 8 2 mg/ml 10 mM Citric Acid 40% 3.5 5 isopropano isopropanol US 2009/00982O7 A1 Apr. 16, 2009 66

-continued -continued

Concentration of Microsphere Concentration of Microsphere tRNA Counterion Antisolven pH Quality tRNA Counterion Antisolvent pH Quality 1 mg/m OmM Citric Aci 10% 3.5 1 Compound: Gelatin from cold isopropano water fish skin with tRNA 1 mg/m OmM Citric Aci 20% 3.5 3 isopropano 2 mg/m OmM Citric Aci 10% 3.5 5 1 mg/m OmM Citric Aci 30% 3.5 isopropano isopropano 2 mg/m OmM Citric Aci 20% 3.5 5 1 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 2 mg/m OmM Citric Aci 30% 3.5 4 0.5 mg/m OmM Citric Aci 10% 3.5 isopropano isopropano 2 mg/m OmM Citric Aci 40% 3.5 5 isopropano 0.5 mg/m OmM Citric Aci 20% 3.5 isopropano 1 mg/m OmM Citric Aci 10% 3.5 4 isopropano 0.5 mg/m OmM Citric Aci 30% 3.5 1 mg/m OmM Citric Aci 20% 3.5 4 isopropano isopropano 0.5 mg/m OmM Citric Aci 40% 3.5 1 mg/m OmM Citric Aci 30% 3.5 6 isopropano isopropano 0.1 mg/m OmM Citric Aci 30% 3.5 1 mg/m OmM Citric Aci 40% 3.5 4 isopropano isopropano 0.1 mg/m OmM Citric Aci 40% 3.5 0.5 mg/m OmM Citric Aci 10% 3.5 10 isopropano isopropano 2 mg/m 0 mM Tris 30% 0.5 mg/m OmM Citric Aci 20% 3.5 8 isopropano isopropano 2 mg/m 0 mM Tris 40% 0.5 mg/m OmM Citric Aci 30% 3.5 8 isopropano isopropano 1 mg/m 0 mM Tris 40% 0.5 mg/m OmM Citric Aci 40% 3.5 7 isopropano isopropano 0.5 mg/m 0 mM Tris 40% 0.1 mg/m OmM Citric Aci 20% 3.5 7 isopropano isopropano Compound: Gelatin from porcine 0.1 mg/m 0 mM Citric Aci 30% 3.5 7 skin, Type A with tRNA isopropano 0.1 mg/m OmM Citric Aci 40% 3.5 7 2 mg/m OmM Citric Aci 10% 3.5 isopropano isopropano 2 mg/m 0 mM Tris 20% 8 6 2 mg/m OmM Citric Aci 20% 3.5 isopropano isopropano 2 mg/m 0 mM Tris 30% 8 5 2 mg/m OmM Citric Aci 30% 3.5 isopropano isopropano 2 mg/m 0 mM Tris 40% 8 6 2 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 10% 8 4 1 mg/m OmM Citric Aci 10% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 20% 8 5 1 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 30% 8 7 0.5 mg/m OmM Citric Aci 10% 3.5 isopropano isopropano 1 mg/m 0 mM Tris 40% 8 4 0.5 mg/m OmM Citric Aci 20% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 10% 8 2 0.5 mg/m OmM Citric Aci 30% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 20% 8 8 0.5 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 30% 8 8 0.1 mg/m OmM Citric Aci 30% 3.5 isopropano isopropano 0.5 mg/m 0 mM Tris 40% 8 4 0.1 mg/m OmM Citric Aci 40% 3.5 isopropano isopropano 0.1 mg/m 0 mM Tris 10% 8 2 2 mg/m 0 mM Tris 30% isopropano isopropano 0.1 mg/m 0 mM Tris 20% 8 4 2 mg/m 0 mM Tris 40% isopropano isopropano 0.1 mg/m 0 mM Tris 30% 8 3 1 mg/m 0 mM Tris 30% isopropano isopropano 0.1 mg/m 0 mM Tris 40% 8 5 1 mg/m 0 mM Tris 40% isopropano isopropano 0.5 mg/m 0 mM Tris 30% isopropano Results: These experiments demonstrate that by selecting the 0.5 mg/m 0 mM Tris 40% isopropano appropriate parameters, stable gelatin microspheres can be 0.1 mg/m 0 mM Tris 40% obtained. Further, active agents such as nucleic acids can be SOO8O incorporated into the gelatin matrix to produce a drug product with defined potency. US 2009/00982O7 A1 Apr. 16, 2009 67

EXAMPLE 23 Preparation of Microspheres Using a Polysaccharide -continued as a Carrier Concentration of Microsphere 0781. This Example demonstrates that the methods pro Compound Counterion Antisolven pH Quality vided herein can be used to prepare microspheres containing 5 mg/m 30 mM Citric Aci 30% 3.5 2 polysaccharides. The polysaccharides in turn can be carriers isopropano for therapeutic agents or active agents incorporated into the 1 mg/m 30 mM Citric Aci 59 3.5 2 microspheres. The following compounds were tested: isopropano 1 mg/m 30 mM Citric Aci 20% 3.5 0782 A) Dextran Sulfate Sodium Salt (Sigma, D 6924) isopropano 0783 B) Hydroxypropyl-B-cyclodextrin (Tokyo Chemi 10 mg/m 10 mM Citric Aci 59 5.2 cal Industry Co., Ltd, HO979) isopropano 10 mg/m 10 mM Citric Aci 10% 5.2 0784 Preparation of microspheres: for the compounds S) isopropano and B) above, cocktail solutions containing from 0.5 mg/ml to 10 mg/m 10 mM Citric Aci 20% 5.2 10 mg/ml of compound, with counterions at different pH, and isopropano 10 mg/m 10 mM Citric Aci 30% 5.2 IPA as antisolvent at different concentrations, as listed below, isopropano were prepared in a 96-well microtiter plate (0.1 ml cocktail/ 5 mg/m 10 mM Citric Aci 59 5.2 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 10% 5.2 chilled (-45° C.) shelves of a Millrock Lab Series Lyo isopropano 5 mg/m 10 mM Citric Aci 20% 5.2 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 20% 5.2 0785. The lyophilized powders from the bottoms of the isopropano 10 mg/m 30 mM Citric Aci 30% 5.2 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 59 5.2 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 10% 5.2 ticles (glass-like crystalline forms), and the absence of isopropano 5 mg/m 30 mM Citric Aci 20% 8 aggregates. isopropano 5 mg/m 30 mM Citric Aci 30% 5.2 isopropano 10 mg/m 30 mM Tris 59 Concentration isopropano of Microsphere 10 mg/m 30 mM Tris 10% Compound Counterion Antisolvent pH Quality isopropano 10 mg/m 30 mM Tris 20% Compound: Dextran isopropano Sulfate 10 mg/m 30 mM Tris 30% isopropano 5 mg/m OmM Citric Acid 59 3.5 4 5 mg/m 30 mM Tris 59 isopropano isopropano 5 mg/m 0 mM Citric Acid 10% 3.5 5 5 mg/m 30 mM Tris 20% isopropano isopropano 5 mg/m 0 mM Citric Acid 20% 3.5 2 5 mg/m 30 mM Tris 30% isopropano isopropano 5 mg/m 0 mM Citric Acid 30% 3.5 6 10 mg/m 0 mM Tris 59 isopropano isopropano 1 mg/m OmM Citric Acid 59 3.5 2 10 mg/m 0 mM Tris 30% isopropano isopropano 1 mg/m 0 mM Citric Acid 10% 3.5 4 5 mg/m 0 mM Tris 59 isopropano isopropano 1 mg/m 0 mM Citric Acid 20% 3.5 3 5 mg/m 0 mM Tris 10% isopropano isopropano 5 mg/m 0 mM Tris 59 8 2 5 mg/m 0 mM Tris 20% isopropano isopropano 5 mg/m 0 mM Tris 10% 8 2 5 mg/m 0 mM Tris 30% isopropano isopropano 5 mg/m 0 mM Tris 20% 8 2 1 mg/m 0 mM Tris 59 isopropano isopropano 10 mg/m 30 mM Citric Acid 59 3.5 2 1 mg/m 0 mM Tris 10% isopropano isopropano 10 mg/m 30 mM Citric Acid 10% 3.5 3 1 mg/m 0 mM Tris 20% isopropano isopropano 10 mg/m 30 mM Citric Acid 20% 3.5 2 1 mg/m 0 mM Tris 30% isopropano isopropano 5 mg/m 30 mM Citric Acid 59 3.5 2 10 mg/m 30 mM Tris 10% isopropano isopropano 5 mg/m 30 mM Citric Acid 10% 3.5 3 10 mg/m 30 mM Tris 30% isopropano isopropano 5 mg/m 30 mM Citric Acid 20% 3.5 2 5 mg/m 30 mM Tris 59 isopropano isopropano US 2009/00982O7 A1 Apr. 16, 2009

0797 Table 24 below shows the various combinations of -continued compound, Solvent, and antisolvent that were used to gener ate microspheres, and the quality of the resulting micro Concentration of Microsphere spheres. Compound Counterion Antisolvent pH Quality TABLE 24 5 mg/ml 30 mM Tris 30% 11 2 isopropanol Compound: Amino Acid Microspheres Hydroxypropyl-B- Amino Acids: cyclodextrin Antisolvent pH Microsphere Quality 5 mg/ml 10 mM Citric Acid 59 3.5 1 Compound: Alanine isopropanol 5 mg/ml 10 mM Citric Acid 20% 3.5 2 5% isopropano 7 9 isopropanol 10% isopropano 7 2 5 mg/ml 10 mM Citric Acid 30% 3.5 1 20% isopropano 7 8 isopropanol 30% isopropano 7 5 1 mg/ml 10 mM Citric Acid 10% 3.5 1 5% isopropano 6 7 isopropanol Compound: Glutamic Acid 1 mg/ml 10 mM Citric Acid 20% 3.5 4 isopropanol 5% isopropano 3.2 6 1 mg/ml 10 mM Citric Acid 30% 3.5 5 10% isopropano 3.2 3 isopropanol 20% isopropano 3.2 6 0.5 mg/ml 10 mM Citric Acid 59 3.5 2 Compound: Tryptophan isopropanol 5 mg/ml 10 mM Tris 20% 8 1 5% isopropano 7 5 isopropanol 10% isopropano 7 4 20% isopropano 7 7 30% isopropano 7 4 0786 Results: These experiments demonstrate that by 20% isopropano 6 2 selecting the appropriate combination of: (a) type and (b) 30% isopropano 6 7 concentration of compound, counterion and antisolvent, Compound: Methionine polysaccharide microspheres can be obtained. 5% isopropano 7 3 10% isopropano 7 4 EXAMPLE 24 20% isopropano 7 1 30% isopropano 7 2 Amino Acid Microspheres 5% isopropano 5.7 2 10% isopropano 5.7 7 0787. This Example demonstrates that the methods pro 30% isopropano 5.7 1 vided herein can be used to prepare microspheres containing Compound: Phenylalanine various amino acids, which could be active agents or thera peutic agents themselves, or serve as carriers for other active 10% isopropano 7 6 20% isopropano 7 8 agents and therapeutic agents. Microspheres of the following 30% isopropano 7 5 amino acids were prepared: 5% isopropano 5.5 6 0788 A. Alanine 10% isopropano 5.5 5 0789 B. Glutamic Acid 20% isopropano 5.5 3 0790 C. Tryptophan 30% isopropano 5.5 7 0791. D. Methionine Compound: Glycine 0792 E. Phenylalanine 5% isopropano 7 7 0793 F. Glycine 10% isopropano 7 6 20% isopropano 7 4 0794. G. Lycine 30% isopropano 7 4 0795 Preparation of Amino Acid Microspheres: for Each 5% isopropano 6 5 of the Compounds listed in A-G above, cocktail solutions 10% isopropano 6 5 containing 20 mM amino acid dissolved in aqueous solvent, 20% isopropano 6 3 at different pH, and ispropanol (IPA) as antisolvent at differ 30% isopropano 6 6 ent concentrations, as listed below, were prepared in a 96-well Compound: Lysine microtiter plate (0.1 ml cocktail/well) at room temperature. 5% isopropano 7 3 Cocktails were cooled by placing in a freezer. The chilled 20% isopropano 7 7 30% isopropano 7 3 plates were transferred onto pre-chilled (-45° C.) shelves of 5% isopropano 5.5 4 a Millrock Lab Series Lyophilizer, and the vacuum was 10% isopropano 5.5 5 applied. The frozen cocktail solutions were allowed to lyo 30% isopropano 5.5 7 philize for 16 hours. 0796. The lyophilized powders from the bottoms of the wells were transferred onto glass slides and analyzed by light 0798 Results: These experiments demonstrate that by microscopy for appearance. The quality of the product micro selecting the appropriate combination of: (a) type and (b) spheres was scored based on the uniformity of the micro concentration of amino acid, counterion and antisolvent, spheres, the absence of undesirable non-microsphere par microspheres made of amino acids can be obtained. ticles (glass-like crystalline forms), and the absence of 0799. Since modifications will be apparent to those of skill aggregates. The scoring system as described in Table 13 was in this art, it is intended that this invention be limited only by used. the scope of the appended claims. US 2009/00982O7 A1 Apr. 16, 2009 69

SEQUENCE LISTING

NUMBER OF SEO ID NOS: 28

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

<4 OO SEQUENCE: 1.

Met Thir Ser His Ser Pro Phe Ser Arg Arg Arg Lell Pro Ala Luell Luell 1. 5 10 15

Gly Ser Luell Pro Lell Ala Ala Thir Gly Lieu. Ile Ala Ala Ala Pro Pro 2O 25 3 O

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

Glin Wall Asn Ala Pro Ala Asp Gly Leu 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 7s

Ala Pro Ala Ser Thir Asn Lell Ser Gly Asn Wall Ser Arg Trp 85 9 O 95

Arg Asn Wall Pro Ala Gly Thir Thir Lys Thr Asp Thir Gly Luell Ala OO OS 1O

His Thir Wall Thir Ala Glu Asp Lieu Lys Ala Gly Gly Phe Thir Pro 15 2O 25

Glin Ile Ala Glu Wall Ala Wall Glu yr Ala Gly Ala Luell 3O 35 4 O

Thir Pro Glu Thir le Gly Ala Thr Ser Pro Wall Ala Asn SO 55 160

Luell Arg Wall Glu Ile Thir Pro Ser Ser Ser Glin Glu Asn Tyr 70 7s

Luell Gly Asp Thir Wall Ser Thir Wall Wall Arg Ser Wall Ser 85 90

Thir Ile Asn Wall Ala Ala Thr Glu Ser Ser Phe Asp Asp Luell 2 OO 2O5

Gly Arg Glin His Trp Gly Gly Lieu Lys Pro Gly Gly Ala Wall 210 215 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 Lieu. Thir Ala Thir Gly Thir Asp 245 250 255

Gly Ala Thir Luell Glin Thir Lell Thir Ala Thr 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 28O 285

Thir Glu Luell Pro Ala Ser Met Ser Glin Ala Glin His Lell Ala Ala Asn 290 295 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 2009/00982O7 A1 Apr. 16, 2009 70

- Continued

34 O 345 350 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 37O 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 410 415 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 4 4 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 5 OO 5 OS 510 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 sfs Llys Val Lieu. Lieu Lleu Ser His Ser Pro Asn Pro Arg Pro Trp Ser Arg 58O 585 590 Asp Arg Gly. Thir Ile Ser Met Ser Cys Asp Asp Gly Ala Ser Trp Thr 595 6 OO 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 660 665 670 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 690 695 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. 75O US 2009/00982O7 A1 Apr. 16, 2009 71

- Continued

Glu Thir Ser Ser Ala Pro Ala Ala Glu Pro Thir Glin Ala Pro Thr Wall 75s 760 765

Ala Pro Ser Wall Glu Pro Thir Glin Ala Pro Gly Ala Glin Pro Ser Ser 770 775 78O

Ala Pro Pro Gly Ala Thir Gly Arg Ala Pro Ser Wall Wall Asn Pro 78s 79 O 79.

Lys Ala Thir Gly Ala Ala Thir Glu Pro Gly Thir Pro Ser Ser Ser Ala 805 810 815

Ser Pro Ala Pro Ser Arg Asn Ala Ala Pro Thir Pro Pro Gly Met 82O 825 830

Glu Pro Asp Glu Ile Asp Arg Pro Ser Asp Gly Thir Met Ala Glin Pro 835 84 O 845

Thir Gly Gly Ala Ser Ala Pro Ser Ala Ala Pro Thir Glin Ala Ala 850 855 860

Ala Gly Ser Arg Lell Ser Arg Thir Gly Thr Ala Lell Lieu. Ile Luell 865 87O 88O

Gly Luell Ala Gly Wall Ala Wall Wall Gly Gly Tyr Lell Lell Lieu. Arg Ala 885 890 895

Arg Arg Ser Asn 9 OO

<210 SEQ ID NO 2 <211 LENGTH: 394 &212> TYPE : PRT <213> ORGANISM: Actinomyces viscosus

<4 OO SEQUENCE: 2

Gly Asp His Pro Ala Thir Pro Ala Pro Ala Pro Asp Ala Ser Thir 1. 10 15

Glu Luell Pro Ala Met Ser Glin Ala Glin His Lell Ala Ala Asn Thir 2O 25 3 O

Ala Thir Asp Asn Arg Ile Pro Ala Ile Thir Thir Ala Pro Asn Gly 35 4 O 45

Asp Luell Luell 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 Wall Glin Arg Arg Ser Thir 65 70 7s

Asp Gly Gly Thir rp Ser Ala Pro Thir Ile His Gln Gly Thir 85

Thir Gly Wall Gly Ser Asp Pro Ser Wall Wall Asp OS 1O

Glin Thir Gly Thir le Phe Asn Phe His Wall Ser Glin 2O 25

Trp Gly Gly Ser Gly Gly Thir Asp Pro Glu Asn Arg Gly Ile 4 O

Glin Ala Glu Wall Thir Ser Thir Asp ASn Gly Trp Thir Trp Thir 55 160

Arg Thir Ile Thir Asp Ile Thr Lys Pro Trp Thr Ala 7s

Phe Ala Ala Ser Glin Gly Ile Glin le Glin His Gly Pro His 90

Gly Arg Luell Wall Glin Thir Ile Arg Thir Ala Gly Gly Ala US 2009/00982O7 A1 Apr. 16, 2009 72

- Continued

195 2 OO 2O5 Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Llys Thir Trp Glin 210 215 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 290 295 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 345 350 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. 37O 375 38O Gly Glu Gln Cys Gly Glin Llys Pro Ala Glu 385 390

<210 SEQ ID NO 3 <211 LENGTH: 24 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 3 Asn Gly Arg Arg Ile Cys Lieu. Asp Lieu. Glin Ala Pro Lieu. Tyr Lys Llys 1. 5 10 15 Ile Ile Llys Llys Lieu. Lieu. Glu Ser 2O

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

<4 OO SEQUENCE: 4 Gly Arg Glu Lieu. Cys Lieu. Asp Pro Lys Glu Asn Trp Val Glin Arg Val 1. 5 10 15 Val Glu Lys Phe Lieu Lys Arg Ala Glu Asn. Ser 2O 25

<210 SEQ ID NO 5 <211 LENGTH: 34 &212> TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO SEQUENCE: 5 Glin Ile His Phe Phe Phe Ala Lys Lieu. Asn. Cys Arg Lieu. Tyr Arg Llys 1. 5 10 15 US 2009/00982O7 A1 Apr. 16, 2009 73

- Continued

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

Llys Ser

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

<4 OO SEQUENCE: 6 Glu Lieu. Arg Val Arg Lieu Ala Ser His Lieu. Arg Llys Lieu. Arg Lys Arg 1. 5 10 15 Lieu. Lieu. Arg Asp Ala Asp Asp Lieu. Glin Lys Arg Lieu Ala Val Tyr Glin 2O 25 3 O Ala Gly

<210 SEQ ID NO 7 <211 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 10

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

<4 OO SEQUENCE: 8 Lys Arg Llys Llys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg 1. 5 10 15 Llys Llys Lys Asn. Pro 2O

<210 SEQ ID NO 9 <211 LENGTH: 4 OO &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OO SEQUENCE: 9 Met Gly Glu Lieu Pro Ala Ser Met Ser Glin Ala Glin His Lieu Ala Ala 1. 5 10 15 Asn Thr Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile Thr Thr Ala Pro 2O 25 3 O 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 9 O 95 Val Asp His Glin Thr Gly. Thir Ile Phe Asin Phe His Val Lys Ser Tyr US 2009/00982O7 A1 Apr. 16, 2009 74

- Continued

OS

Glin Gly Trp Gly Ser Arg Gly Gly Asp Pro Glu Asn Arg

Ile Ile Glin Ala Glu Wall Ser Thir Ser Asp Asn Gly Trp Thir 35

Thir His Arg Thir le Thir Ala Asp Ile Asp Pro Trp SO 160

Ala Arg Phe Ala Ala Ser Gly Glin Gly Glin Ile Glin His Gly 65 70

Pro His Ala Gly Arg Lell Wall Glin Glin Tyr Ile Arg Thir Ala Gly 8O 85

Ala Wall Glin Ala Wall Ser Wall Ser Asp Asp His Gly Thir 95 2 OO 2O5

rp Glin Ala Gly Thir Pro Ile Gly Thir Gly Met Asp Glu Asn Wall 210 215 22O

Wall Glu Luell Ser Asp Gly Ser Luell Met Luell ASn Ser Arg Ala Ser Asp 225 23 O 235 24 O

Gly Ser Gly Phe Lys Wall Ala His Ser Thir Asp Gly Gly Glin Thir 245 250 255

Trp Ser Glu Pro Wall Ser Asp Asn Luell Pro Asp Ser Wall 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 Wall Luell Lell Lell Ser His Ser Pro ASn Pro Arg Pro Trp Ser 290 295 3 OO

Arg Asp Arg Gly Thir Ile Ser Met Ser Asp Asp Gly Ala Ser Trp 3. OS 310 315

Thir Thir Ser Wall Phe His Glu Pro Phe Wall Gly Tyr Thir Thir Ile 3.25 330 335

Ala Wall Glin Ser Asp Gly Ser Ile Gly Luell Luell Ser Glu Asp Ala His 34 O 345 350

Asn Gly Ala Asp Tyr Gly Gly Ile Trp Arg Asn Phe Thir Met Asn 355 360 365

Luell Gly Glu Glin Cys Gly Glin Pro Ala Arg 375 38O

Gly Gly Asn Gly Lys Asn Arg Arg Asn Arg Asn Pro 385 390 395 4 OO

SEQ ID NO 10 LENGTH: 422 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic construct

<4 OO SEQUENCE: 10

Met Val Lys Arg Llys Lys Gly Gly Lys ASn Gly Asn Arg Arg 1. 5 10 15

Asn Arg Llys Llys Llys Asn Pro Gly Gly Gly Gly Ser Gly Asp His Pro 25 3 O

Glin Ala Thr Pro Ala Pro Ala Pro Asp Ala Ser Thir Glu Luell Pro Ala 35 4 O 45

Ser Met Ser Glin Ala Glin His Lieu Ala Ala ASn Thir Ala Thir Asp Asn US 2009/00982O7 A1 Apr. 16, 2009 75

- 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 9 O 95 Pro Asn Pro Asn His Ile Val Glin Arg Arg Ser Thr Asp Gly Gly Lys OO OS 1O Thir Trp Ser Ala Pro Thr Tyr Ile His Glin Gly Thr Glu Thr Gly Lys 15 2O 25 Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp His Glin Thr Gly 3O 35 4 O Thir Ile Phe Asin Phe His Val Lys Ser Tyr Asp Gln Gly Trp Gly Gly 45 SO 55 160 Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg Gly Ile Ile Glin Ala Glu 65 70 7s Val Ser Thr Ser Thr Asp Asn Gly Trp Thir Trp Thr His Arg Thr Ile 8O 85 90 Thir Ala Asp Ile Thr Lys Asp Llys Pro Trp Thr Ala Arg Phe Ala Ala 95 2 OO 2O5 Ser Gly Glin Gly Ile Glin Ile Glin His Gly Pro His Ala Gly Arg Lieu. 210 215 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 290 295 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 345 350 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 37O 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 410 415 Gly Glin Llys Pro Ala Glu 42O

<210 SEQ ID NO 11 <211 LENGTH: 415 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence US 2009/00982O7 A1 Apr. 16, 2009 76

- Continued

&220s FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OO SEQUENCE: 11 Met Gly Glu Lieu Pro Ala Ser Met Ser Glin Ala Glin His Lieu Ala Ala 1. 5 10 15 Asn Thr Ala Thr Asp Asn Tyr Arg Ile Pro Ala Ile Thr Thr Ala Pro 2O 25 3 O 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 9 O 95 Val Asp His Glin Thr Gly. Thir Ile Phe Asin Phe His Val Lys Ser Tyr OO OS 1O Asp Glin Gly Trp Gly Gly Ser Arg Gly Gly Thr Asp Pro Glu Asn Arg 15 2O 25 Gly Ile Ile Glin Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thr 3O 35 4 O rp Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Llys Pro Trp 45 SO 55 160 Thir Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin Ile Gln His Gly 65 70 7s Pro His Ala Gly Arg Lieu Val Glin Glin Tyr Thr Ile Arg Thr Ala Gly 8O 85 90 Gly Ala Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Llys Thr 95 2 OO 2O5 Trp Glin Ala Gly Thr Pro Ile Gly Thr Gly Met Asp Glu Asn Llys Val 210 215 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 290 295 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 345 350 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 37O 375 38O US 2009/00982O7 A1 Apr. 16, 2009 77

- 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 410 415

<210 SEQ ID NO 12 <211 LENGTH: 404 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OO SEQUENCE: 12 Met Gly His His His His His His Leu Glu Gly Asp His Pro Glin Ala 1. 5 10 15 Thr Pro Ala Pro Ala Pro Asp Ala Ser Thr Glu Lieu Pro Ala Ser Met 2O 25 3 O 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 9 O 95 Ser Ala Pro Thr Tyr Ile His Glin Gly Thr Glu Thr Gly Lys Llys Val OO OS 1O Gly Tyr Ser Asp Pro Ser Tyr Val Val Asp His Glin Thr Gly Thr Ile 15 2O 25 Phe Asin Phe His Val Lys Ser Tyr Asp Glin Gly Trp Gly Gly Ser Arg 3O 35 4 O Gly Gly. Thir Asp Pro Glu Asn Arg Gly Ile Ile Glin Ala Glu Val Ser 45 SO 55 160 Thir Ser Thr Asp Asn Gly Trp Thir Trp Thr His Arg Thr Ile Thr Ala 65 70 7s Asp Ile Thir Lys Asp Llys Pro Trp Thir Ala Arg Phe Ala Ala Ser Gly 8O 85 90 Glin Gly Ile Glin Ile Gln His Gly Pro His Ala Gly Arg Lieu Val Glin 95 2 OO 2O5 Gln Tyr Thr Ile Arg Thr Ala Gly Gly Ala Val Glin Ala Val Ser Val 210 215 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 290 295 3 OO Asn Ala Ala Pro Asp Asp Pro Arg Ala Lys Val Lieu Lleu Lieu. Ser His 3. OS 310 315 32O US 2009/00982O7 A1 Apr. 16, 2009 78

- 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 345 350 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 37O 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

<210 SEQ ID NO 13 <211 LENGTH: 416 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OO SEQUENCE: 13 Met Lys Arg Llys Llys Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn 1. 5 10 15 Arg Llys Llys Lys Asn Pro Gly Asp His Pro Glin Ala Thr Pro Ala Pro 2O 25 3 O 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 9 O 95 Val Glin Arg Arg Ser Thr Asp Gly Gly Llys Thir Trp Ser Ala Pro Thr OO OS 1O Tyr Ile His Glin Gly Thr Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp 15 2O 25 Pro Ser Tyr Val Val Asp His Glin Thr Gly Thr Ile Phe Asin Phe His 3O 35 4 O Val Lys Ser Tyr Asp Glin Gly Trp Gly Gly Ser Arg Gly Gly. Thir Asp 45 SO 55 160 Pro Glu Asn Arg Gly Ile Ile Glin Ala Glu Val Ser Thr Ser Thr Asp 65 70 7s Asn Gly Trp Thir Trp Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys 8O 85 90 Asp Llys Pro Trp Thr Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin 95 2 OO 2O5 le Gln His Gly Pro His Ala Gly Arg Lieu Val Glin Glin Tyr Thr Ile 210 215 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 2009/00982O7 A1 Apr. 16, 2009 79

- 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 290 295 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 345 350 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 37O 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 410 415

<210 SEQ ID NO 14 <211 LENGTH: 10 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Random primer sequence for N-terminal optimization (aa) &220s FEATURE: <221 NAME/KEY: VARIANT <222> LOCATION: 2, 3 <223> OTHER INFORMATION: Xaa = Any Amino Acid <4 OO SEQUENCE: 14 Met Xaa Xala Lys Arg Llys Llys Lys Gly Gly 1. 5 10

<210 SEQ ID NO 15 <211 LENGTH: 374 &212> TYPE: PRT <213> ORGANISM: Homo sapiens &220s FEATURE: <223> OTHER INFORMATION: protease inhibitor 8 (PI8) (Serpin B8) <4 OO SEQUENCE: 15 Met Asp Asp Lieu. Cys Glu Ala Asn Gly. Thir Phe Ala Ile Ser Lieu. Phe 1. 5 10 15 Lys Ile Lieu. Gly Glu Glu Asp Asn. Ser Arg Asn Val Phe Phe Ser Pro 2O 25 3 O 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 9 O 95 US 2009/00982O7 A1 Apr. 16, 2009 80

- Continued Thr Cys Asp Phe Lieu Pro Asp Phe Lys Glu Tyr Cys Gln Llys Phe Tyr OO OS 1O Glin Ala Glu Lieu. Glu Glu Lieu. Ser Phe Ala Glu Asp Thr Glu Glu. Cys 15 2O 25 Arg Llys His Ile Asn Asp Trp Val Ala Glu Lys Thr Glu Gly Lys Ile 3O 35 4 O Ser Glu Val Lieu. Asp Ala Gly Thr Val Asp Pro Lieu. Thir Lys Lieu Val 45 SO 55 160 Lieu Val Asn Ala Ile Tyr Phe Lys Gly Lys Trp Asn. Glu Glin Phe Asp 65 70 7s Arg Llys Tyr Thr Arg Gly Met Lieu. Phe Llys Thr Asn. Glu Glu Lys Llys 8O 85 90 Thr Val Glin Met Met Phe Lys Glu Ala Lys Phe Lys Met Gly Tyr Ala 95 2 OO 2O5 Asp Glu Val His Thr Glin Val Lieu. Glu Lieu Pro Tyr Val Glu Glu Glu 210 215 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 290 295 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 345 350 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 37O

<210 SEQ ID NO 16 <211 LENGTH: 4176 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: &223> OTHER INFORMATION: Plasmic vector PTrce 9A

<4 OO 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 2009/00982O7 A1 Apr. 16, 2009 83

- Continued

Thr Asp Gly Gly Lys Thir Trp Ser Ala Pro Thr Tyr Ile His Glin Gly

Thr Glu Thr Gly Lys Llys Val Gly Tyr Ser Asp Pro Ser Tyr Val Val OO OS 1O Asp His Glin Thr Gly Thr Ile Phe Asin Phe His Val Lys Ser Tyr Asp 15 2O 25 Glin Gly Trp Gly Gly Ser Arg Gly Gly. Thir Asp Pro Glu Asn Arg Gly 3O 35 4 O le Ile Glin Ala Glu Val Ser Thr Ser Thr Asp Asn Gly Trp Thir Trp 45 SO 55 160 Thr His Arg Thr Ile Thr Ala Asp Ile Thr Lys Asp Llys Pro Trp Thr 65 70 7s Ala Arg Phe Ala Ala Ser Gly Glin Gly Ile Glin Ile Gln His Gly Pro 8O 85 90 His Ala Gly Arg Lieu Val Glin Glin Tyr Thir Ile Arg Thr Ala Gly Gly 95 2 OO 2O5 Ala Val Glin Ala Val Ser Val Tyr Ser Asp Asp His Gly Lys Thir Trip 210 215 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 290 295 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 345 350 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 37O 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 410 415

<210 SEQ ID NO 18 <211 LENGTH: 116 &212> TYPE: PRT <213> ORGANISM: Homo sapiens &220s FEATURE: <223> OTHER INFORMATION: Preprosomatostatin

<4 OO SEQUENCE: 18 Met Lieu. Ser Cys Arg Lieu. Glin Cys Ala Lieu Ala Ala Lieu. Ser Ile Val 1. 5 10 15 US 2009/00982O7 A1 Apr. 16, 2009 84

- Continued

Lieu Ala Lieu. Gly Cys Val Thr Gly Ala Pro Ser Asp Pro Arg Lieu. Arg 2O 25 3 O 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 9 O 95 Ala Pro Arg Glu Arg Lys Ala Gly Cys Lys Asn. Phe Phe Trp Llys Thr 1 OO 105 11O Phe Thir Ser Cys 115

<210 SEQ ID NO 19 <211 LENGTH: 92 &212> TYPE: PRT <213> ORGANISM: Homo sapiens &220s FEATURE: <223> OTHER INFORMATION: Prosomatostatin

<4 OO SEQUENCE: 19 Ala Pro Ser Asp Pro Arg Lieu. Arg Glin Phe Lieu Gln Lys Ser Lieu. Ala 1. 5 10 15 Ala Ala Ala Gly Lys Glin Glu Lieu Ala Lys Tyr Phe Lieu Ala Glu Lieu 2O 25 3 O 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 9 O

<210 SEQ ID NO 2 O <211 LENGTH: 28 &212> TYPE: PRT <213> ORGANISM: Homo sapiens &220s FEATURE: <223> OTHER INFORMATION: somatostatin 28 (SS-28)

<4 OO SEQUENCE: 2O Ser Ala Asn. Ser Asn Pro Ala Met Ala Pro Arg Glu Arg Lys Ala Gly 1. 5 10 15 Cys Lys Asn Phe Phe Trp Llys Thr Phe Thr Ser Cys 2O 25

<210 SEQ ID NO 21 <211 LENGTH: 14 &212> TYPE: PRT <213> ORGANISM: Homo sapiens &220s FEATURE: <223> OTHER INFORMATION: somatostatin 14 (SS-14)

<4 OO SEQUENCE: 21 US 2009/00982O7 A1 Apr. 16, 2009 85

- Continued Ala Gly Cys Lys Asn Phe Phe Trp Llys Thr Phe Thr Ser Cys 1. 5 10

<210 SEQ ID NO 22 <211 LENGTH: 9 &212> TYPE: PRT <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Leuprolide &220s FEATURE: <221 NAME/KEY: MOD RES <222> LOCATION: 1 <223> OTHER INFORMATION: pyroglutamic acid &220s FEATURE: <221 NAME/KEY: MOD RES <222> LOCATION: 6 <223> OTHER INFORMATION: d-leucine &220s FEATURE: <221 NAME/KEY: MOD RES <222> LOCATION: 10 <223> OTHER INFORMATION: Proline residue bound to ethylamide (NHEt) <4 OO SEQUENCE: 22 Glu. His Trp Ser Tyr Lieu Lleu. Arg Pro 1. 5

<210 SEQ ID NO 23 <211 LENGTH: 21 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic siRNA NP1496 sense strand &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 2O, 21 &223> OTHER INFORMATION: n = cT

<4 OO SEQUENCE: 23 ggaucullalulu ulculu.cggagn in 21

<210 SEQ ID NO 24 <211 LENGTH: 21 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic siRNA NP-1496 antisense strand &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 2O, 21 &223> OTHER INFORMATION: n = ct

<4 OO SEQUENCE: 24

Cuccgaagaa aluaagaluccin in 21

<210 SEQ ID NO 25 <211 LENGTH: 21 &212> TYPE: DNA <213> ORGANISM: Artificial Sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic siRNA PA-2087 sense strand &220s FEATURE: <221 NAMEAKEY: misc feature <222> LOCATION: 2O, 21 &223> OTHER INFORMATION: n = cT

<4 OO SEQUENCE: 25 gcaauugagg agugcclugan in 21 US 2009/00982O7 A1 Apr. 16, 2009 86

- 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: dT

<4 OO 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 OO> 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 OO SEQUENCE: 28 ulauluggalaca lugulaalaccalu u. 21

1. A method of making microparticles of a compound, 9. The method of claim 1, wherein steps a) and b) are comprising: performed simultaneously, sequentially, intermittently, or in a) adding a counterion to a solution containing the com any order, followed by step c). pound in a solvent; 10. The method of claim 1, wherein steps b) and c) are b) adding an antisolvent to the solution; and performed simultaneously, sequentially, intermittently, or in c) gradually cooling the solution to a temperature below any order, preceded by step a). about 25°C., whereby a composition containing micro 11-65. (canceled) particles comprising the compound is formed, wherein 66. The method of claim 1, wherein the antisolvent is steps a), b) and c) are performed simultaneously, selected from among water, buffered solutions, aliphatic sequentially, intermittently, or in any order. alcohols, aromatic alcohols, chloroform, polyhydric Sugar 2. The method of claim 1, wherein the counterion is not a alcohols, aromatic hydrocarbons, aldehydes, ketones, esters, polymer. ethers, dioxanes, alkanes, alkenes, conjugated dienes, dichlo romethane, carbon tetrachloride, dimethylformamide 3. The method of claim 1, wherein the antisolvent is not a (DMF), dimethyl sulfoxide (DMSO), acetonitrile, ethyl polymer. acetate, polyols, polyimides, polyimines, polyesters, polyal 4. The method of claim 1, wherein prior to step a), the dehydes and mixtures thereof. compound is dissolved in the solvent at a temperature of 67-79. (canceled) about or at 30° C. or below. 80. The method of claim 1, wherein the counterion is a 5. The method of claim 4, wherein prior to step a), the polymer. compound is dissolved in the solvent at a temperature of 81. The method of claim 80, wherein the polymer is the about or at 25° C. or below. counterion and the antisolvent. 6. The method of claim 1, wherein steps a) and b) are 82. The method of claim 81, wherein the polymer is poly performed at ambient temperature. ethylene glycol (PEG) or polyethyleneimine (PEI). 7. The method of claim 1, wherein none of the solutions of 83. The method of claim 1, wherein the microparticles are steps a)-c) are heated and/or maintained at a temperature obtained by precipitation, by phase separation or by colloid above about or at 30° C. formation. 8. The method of claim 1, wherein the compound is not a 84. The method of claim 1, wherein the pH of the solution protein or a polypeptide. is from about 4.0 or 4.0 to about 9.0 or 9.0. US 2009/00982O7 A1 Apr. 16, 2009

85. The method of claim 1, wherein the resulting micro 103. The method of claim 102, wherein the temperature is particle composition further comprises micro-carriers, acid between about 0°C. or 0°C. to about -2°C. or -2°C. to from resistant coating agents, protease-resistant coating agents, about -150° C. or -150° C. to about -165° C. or -165° C. enteric coating agents, bulking agents, excipients, inactive 104-114. (canceled) ingredients, stability enhancers, taste and/or odor modifiers 115. The method of claim 1, wherein the gradual cooling is or masking agents, Vitamins, Sugars, therapeutic agents, anti at a rate of from about or at 0.01° C./min or 0.01° C./min to oxidants, immuno-modulators, trans-membrane transport about or at 20° C./min or 20° C./min. modifiers, anti-caking agents, chitosans or flowability 116-118. (canceled) enhancers. 119. The method of claim 1, wherein the size of the micro 86-100. (canceled) particles is from about or at 0.001 um or 0.001 um to about or 101. The method of claim 1, wherein the temperature is at 50 um or 50 Lum. between about or at 4°C. to about or at -200° C. 120-208. (canceled) 102. The method of claim 101, wherein the temperature is between about or at 2°C. to about or at -170° C. c c c c c