US 2015.0050713A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0050713 A1 Malakhov et al. (43) Pub. Date: Feb. 19, 2015 (54) TECHNOLOGY FOR THE PREPARATION OF Publication Classification MCROPARTICLES (51) Int. Cl. (71) Applicant: Ansun Biopharma, Inc., San Diego, CA CI2N 9/96 (2006.01) (US) CI2N 7/00 (2006.01) CI2N IS/II3 (2006.01) (72) Inventors: Michael P. Malakhov, San Francisco, C07D 305/.4 (2006.01) CA (US); Fang Fang, Rancho Santa Fe, C07K 4/765 (2006.01) CA (US) C07K9/00 (2006.01) (52) U.S. Cl. CPC ................ CI2N 9/96 (2013.01); C07K 14/765 (21) Appl. No.: 14/341,502 (2013.01); C07K9/008 (2013.01): CI2N 15/113 (2013.01); C07D 305/14 (2013.01); (22) Filed: Jul. 25, 2014 CI2N 7/00 (2013.01); C12N 2770/00051 (2013.01) O O USPC ........... 435/188: 530/363; 530/350, 530/367; Related U.S. Application Data 530/385:536/16.8; 536/13.7: 540/336; 530/317; (63) Continuation of application No. 13/874,424, filed on 536/24.5; 530/328; 552/203; 549/510; 435/238 Apr. 30, 2013, now abandoned, which is a continuation (57) ABSTRACT of application No. 13/250,653, filed on Sep. 30, 2011, Mi h duced b tacti lution of a now abandoned, which is a continuation of application 1crospneres are produced by contacting a sol No. 12/179.520, filed on Jul 24, 2008, now aban- macromolecule or small molecule in a solvent with an ant1 doned sa- 1 ws s s Solvent and a counterion, and chilling the solution. The microspheres are useful for preparing pharmaceuticals, (60) Provisional application No. 60/961,872, filed on Jul. nutraceuticals, cosmetic products and the like of defined 24, 2007. dimensions. US 2015/0050713 A1 Feb. 19, 2015 TECHNOLOGY FOR THE PREPARATION OF solid microparticle formulation. Other methods, such as MICROPARTICLES directly precipitating a compound out of Solution by adding an antisolvent, can generate microparticles in an uncontrolled RELATED APPLICATIONS manner that results in uneven-sized and/or aggregated micro particles. 0001. This application is a continuation and claims prior 0006. Accordingly, there is a need for a method for pro ity to U.S. application Ser. No. 13/874,424, filed Apr. 30, ducing protein and other macromolecular microparticles, and 2013, which claims priority to U.S. application Ser. No. Small-molecule microparticles, which does not require com 13/250,653, filed Sep. 30, 2011, which claims priority to U.S. plex or specialized equipment and that produces uniform application Ser. No. 12/179,520, filed Jul. 24, 2008, which sized microparticles for delivery. There further is a need for a claims priority to U.S. provisional application Ser. No. method of producing microparticles of a compound that con 60/961,872, entitled “TECHNOLOGY FOR THE PREPA tain high concentrations of the compound relative to other RATION OF MICROPARTICLES” to Fang et al. filed Jul. components of the microparticles, that are stable and main 24, 2007. This application also is related to International PCT tain their activity for long periods of time when stored at application No. (Attorney Dkt. No. 21865-005 WO1/ ambient temperature, and that do not contain a significant 6505PC) filed on the same day herewith. Each of these appli amount of inactive compound. There also is a need for a cations is incorporated by reference herein in its entirety. method of producing microparticles of compounds where 0002 This application is related to International PCT Substantially all of the compound present in the starting mate application Serial No. (Attorney Docket No. 21865 rial (e.g., a solution of the compound) is recovered in the 004WO1/6504PC, filed Jan. 24, 2007), and to U.S. applica microparticle formulation, with minimal loss. There also is a tion Ser. No. 1 1/657,812, filed Jan. 24, 2007 (Attorney need for microparticles containing these properties for Docket No. 21865-004001/6504). This application also is 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 21865.005001SeqList.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 US 2015/0050713 A1 Feb. 19, 2015 chemical parameters. As would be apparent to one of skill in 1000 or 1000 to about or at 300,000 or 300,000 Daltons; about the art, the compositions and methods provided herein or at 1000 or 1000 to about or at 200,000 or 200,000 Daltons; include any and all permutations and combinations of the about or at 1000 or 1000 to about or at 100,000 or 100,000 ingredients, steps and/or parameters described below. Daltons; about or at 1000 or 1000 to about or at 50,000 or 0010. The methods provided herein can include the steps 50,000 Daltons; about or at 1000 or 1000 to about or at 25,000 of: or 25,000 Daltons; about or at 1000 or 1000 to about or at 0.011 a) adding a counterion to a solution containing the 15,000 or 15,000 Daltons; about or at 1000 or 1000 to about compound in a solvent; or at 10,000 or 10,000 Daltons; about or at 1000 or 1000 to 0012 b) adding an antisolvent to the solution; and about or at 5,000 or 5,000 Daltons; about or at 1000 or 1000 0013 c) gradually cooling the solution to a temperature to about or at 3,000 or 3000 Daltons; or about or at 1000 or below about 25° C., whereby a composition containing 1000 to about or at 2,000 or 2000 Daltons. microparticles of the compound is formed. In the method, 0018. The macromolecule can be a polynucleotide, a steps a), b) and c) can be performed simultaneously, sequen nucleic acid, a polypeptide, a glycopeptide, a protein, a car tially, intermittently, or in any order.