(11) Numéro de publication:
`
`(11) Publication number:
`
`(11) Veréffentlichungsnummer:
`
`EP 2 928 500 AO
`
`Internationale Anmeldung veréffentlicht durch die
`
`Weltorganisation fur geistiges Eigentum unter der Nummer:
`
`WO 2014/089160 (Art. 153(3) EPU).
`
`International application published by the World
`
`Intellectual Property Organization under number:
`
`WO 2014/089160 (Art. 153(3) EPC).
`
`Demandeinternationale publiée par |’Organisation
`
`Mondiale de la Propriété Intellectuelle sous le numéro:
`
`WO 2014/089160 (art. 153(3) CBE).
`
`

`

`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`12 June 2014 (12.06.2014)
`
`WIPO!IPCT
`
`\=
`
`(10) International Publication Number
`WO 2014/089160 Al
`
`GD)
`
`International Patent Classification:
`A61K 47/30 (2006.01)
`B82Y 5/00 (2011.01)
`B82Y¥ 40/00 (2011.01)
`
`QD
`
`International Application Number:
`
`PCT/US2013/073019
`
`(22)
`
`International Filing Date:
`
`4 December 2013 (04.12.2013)
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`(81)
`
`Filing Language:
`
`Publication Language:
`
`English
`
`English
`
`Priority Data:
`61/733,216
`
`4 December 2012 (04.12.2012)
`
`US
`
`Applicant: PHOSPHOREX, INC.
`Street, Hopkinton, MA 01748 (US).
`
`[US/US]; 94 South
`
`Inventor: WU, Bin; 12206 Main Campus Drive, Lexing-
`ton, MA 02421 (US).
`
`Agents: LU, Yu et al.; McCarter & English, LLP, 265
`Franklin Street, Boston, MA 02110 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available). AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`
`HN, HR, HU,ID,IL,IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM,PA,PL, PG, PII, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`Zw.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FL FR, GB, GR, HR, HU,IE,IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`Declarations under Rule 4.17:
`
`as to applicant's entitlement to apply for and be granted a
`patent (Rule 4.17(ii))
`
`as to the applicant's entitlement to claim the priority of the
`earlier application (Rule 4.17(iii))
`Published:
`
`with international search report (Art. 21(3))
`
`(54) Title: MICROPARTICLES AND NANOPARTICLES HAVING NEGATIVE SURFACE CHARGES
`
`(57) Abstract: This invention provides methods for producing a polymer particle which contains unusually high negative charges on
`the surface of the particle. Preferably, the polymer is pharmaceutically acceptable. The negative charges can be conferred by chemic-
`al groups such as carboxyl, sulfonate, nitrate, fluorate, chloride, iodide, persulfate, and many others, with carboxyl group being pre-
`ferred. The invention also provides polymerparticle produced by the methodsof the invention.
`
`wo2014/089160A1IIITINNIITANMIMTANTVNRAATTATA
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`

`

`WO 2014/089160
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`PCT/US2013/073019
`
`MICROPARTICLES AND NANOPARTICLES HAVING
`
`NEGATIVE SURFACE CHARGES
`
`REFERENCE TO RELATED APPLICATION
`
`This application claims the benefit of priority to U.S. Provisional Application No.
`
`61/733,216, filed on December4, 2012.
`
`BACKGROUNDOF THE INVENTION
`
`Certain carboxylated particles, such as carboxylated polystyrene, PLGA, or
`
`diamondparticles, when administered to subjects, may ameliorate certain conditions,
`
`such as pathological inflammatory immune responses (see WO 2012/065153).
`
`10
`
`Inflammatory diseases and disorders are conditions in which an abnormal or
`
`otherwise deregulated inflammatory response contributes to the etiology or severity of
`
`disease. Examples include autoimmunediseases such as rheumatoid arthritis, multiple
`
`sclerosis, and diabetes, infectious diseases such as tuberculosis and various forms of
`
`meningitis and encephalitis including West Nile Virus encephalitis and other disorders
`
`15
`
`include atherosclerosis and ischemic reperfusion.
`
`Manyof these diseases are characterized by a mononuclearcell infiltration at a
`
`site of tissue injury or other insult. Examples of mononuclear cells that have been
`
`observedin these infiltrations include lymphocytes, especially T lymphocytes, and cells
`
`of the mononuclear phagocyte system (MPScells) such as monocytes, macrophages,
`
`20
`
`dendritic cells, microglial cells and others.
`
`However, itis Applicant’s belief that carboxylated PLGAparticles produced
`
`using conventional means are frequently not biocompatible and thus PLGAparticles
`
`resulting from such manufacturing processes may not be safe for use on humans and
`
`animals. In addition, it is Applicant’s belief that PLGA particles produced using
`
`25
`
`conventional means may not contain sufficient number of COOH groups for attaching
`
`API’s or other chemical entities to microparticles and nanoparticles.
`
`There is a need to prepare negatively charged (e.g., carboxylated PLGA)
`
`microparticles and nanoparticles with enhanced therapeutic properties.
`
`

`

`WO 2014/089160
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`PCT/US2013/073019
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`SUMMARYOF THE INVENTION
`
`Oneaspect of the invention provides a method for the preparation of a
`
`composition comprising microparticles or nanoparticles having negative surface charges,
`
`said method comprising producing said microparticles or nanoparticles with a
`
`pharmaceutically acceptable polymer using either an emulsion process or a precipitation
`
`process: (a) wherein said emulsion process or said precipitation process is carried out in
`
`an aqueous solution having a pH that promotesionization of said pharmaceutically
`
`acceptable polymer; (b) wherein said pharmaceutically acceptable polymerhas an
`
`average molecular weight of from about 500 to about 1,000,000 Da, about 500 to about
`
`10
`
`50,000 Da, or preferably from about 1,000 to about 50,000 Da, or about 1,000 to about
`
`30,000 Da; and/or, (c) wherein said pharmaceutically acceptable polymer contains
`
`multiple negatively charged terminal groups.
`
`In certain embodiments, the emulsion process comprises: (1) dissolving the
`
`pharmaceutically acceptable polymerin a first solvent to form a polymer solution; (2)
`
`15
`
`emulsifying the polymersolution in a solution of a second solvent to form an emulsion,
`
`wherein the first solvent is not miscible or partially miscible with the second solvent, and
`
`wherein the solution of the second solvent optionally comprises a pharmaceutically
`
`acceptable negatively charged agent; and, (3) removingthe first solvent to form said
`
`microparticles or nanoparticles having negative surface charges; wherein said solution of
`
`20
`
`the second solvent is optionally said aqueoussolution.
`
`In certain embodiments, the precipitation process comprises: (1) dissolving the
`
`pharmaceutically acceptable polymerin a first solvent to form a polymersolution; (2)
`
`preparing a solution of a second solvent, wherein the first solvent is miscible with the
`
`second solvent, and wherein the solution of the second solvent optionally comprises a
`
`25
`
`pharmaceutically acceptable negatively charged agent and optionally comprises a
`
`surfactant; and, (3) combining (e.g., adding) the polymersolution to the solution of the
`
`second solvent while mixing, thus forming said microparticles or nanoparticles having
`
`negative surface charges; wherein said solution of the second solvent is optionally said
`
`aqueoussolution.
`
`comprising: (1) dissolving the pharmaceutically acceptable polymerinafirst solvent to
`
`In certain embodiments, the emulsion process is a double emulsion process
`
`form a polymer solution; (2) adding a small amountof a solution of a second solvent to
`
`the polymer solution to form a mixture, wherein the first solvent is not miscible or
`
`

`

`WO 2014/089160
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`PCT/US2013/073019
`
`partially miscible with the second solvent, and wherein the solution of the second solvent
`
`optionally comprises an active pharmaceutical ingredient (API); (3) emulsifying the
`
`mixture to form a first emulsion; (4) emulsifying the first emulsion in the solution of the
`
`second solvent to form a second emulsion, wherein the solution of the second solvent
`
`optionally comprises a pharmaceutically acceptable negatively charged agent, and
`
`optionally further comprises a surfactant; and, (5) removing thefirst solvent to form said
`
`microparticles or nanoparticles having negative surface charges; wherein said solution of
`
`the second solvent is optionally said aqueoussolution.
`
`In certain embodiments, the pharmaceutically acceptable negatively charged agent
`
`10
`
`is incorporated into said microparticles or nanoparticles to increase negative surface
`
`charges on said microparticles or nanoparticles.
`
`In certain embodiments, the pharmaceutically acceptable negatively charged agent
`
`is incorporated into said microparticles or nanoparticles to increase the numbers of
`
`carboxyl groups on said microparticles or nanoparticles.
`
`15
`
`In certain embodiments, the pharmaceutically acceptable negatively charged agent
`
`comprises polyacrylic acid, or poly(ethylene-alt-maleic acid) (PEMA).
`
`In certain embodiments, the pharmaceutically acceptable polymeris a naturally
`
`occurring polymer.
`
`In certain embodiments, the naturally occurring polymeris cellulose, dextrin,
`
`20
`
`hyluronic acid, gelatin, polysaccharide, amino acid, or polyhydroxyalkanoates.
`
`In certain embodiments, the pharmaceutically acceptable polymeris a synthetic
`
`polymer.
`
`In certain embodiments, the synthetic polymer is polyacrylic acid,
`
`polymethacrylic acid, polylactic acid, polyglycolic acid, polyhydroxybutytic acid,
`
`25
`
`polylactide, polyglycolide, poly(actide-co-glycolide) polycaprolactone, polyanhydride,
`
`or poly(lactide-co-glycolide) or PLGA,orasalt, derivative, copolymer, or mixture
`
`thereof. Preferably, the synthetic polymeris a biodegradable polymer.
`
`In certain embodiments, the synthetic polymer is a PLGA polymer having an L/G
`
`ratio of from about 95/5 to 5/95, preferably from 85/15 to 15/85, and most preferably
`
`30
`
`about 50/50.
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`

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`WO 2014/089160
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`PCT/US2013/073019
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`In certain embodiments, the microparticles or nanoparticles have a zeta potential
`
`of from about -5 mV to about -200 mV,preferably from about -15 mV to about -100 mV,
`
`most preferably from -35 mV to -85 mV.
`
`In certain embodiments, the first solvent is a volatile solvent.
`
`In certain embodiments, the polymer is a PLGA polymer, and the volatile solvent
`
`is methylene chloride, ethyl acetate, or chloroform.
`
`In certain embodiments, the solution of the second solvent comprises a surfactant.
`
`In certain embodiments, said solution of the second solvent is a mixture of the
`
`first and second solvent. In certain embodiments, the volumeratio of the first solvent to
`
`10
`
`the second solventin said solution of the second solvent is about 0.1:99.9, about 1:99,
`
`about 5:95, about 7.5:92.5; about 7.8:92.2; about 8:92, or about 10:90.
`
`In certain embodiments, the surfactant comprises organic or inorganic
`
`pharmaceutical excipients; various polymers; oligomers; natural products; nonionic,
`
`cationic, zwitterionic, or ionic surfactants; and mixtures thereof.
`
`In certain embodiments, the polymer is a PLGA polymer, and the surfactantis /
`
`comprises polyvinyl alcohol, polyvinylpyrrolidone, a Tween series surfactant, Pluronic
`
`F-68, Poloxamerseries, or Triton X-100 andits derivatives.
`
`In certain embodiments, the emulsifying step comprises homogenization,
`
`mechanicalstirring, and/or microfluidization.
`
`20
`
`In certain embodiments, the first solvent is removed through solvent exchange
`
`and/or evaporation.
`
`In certain embodiments, the pharmaceutically acceptable negatively charged agent
`
`is a carboxyl-containing agent.
`
`In certain embodiments, the carboxyl-containing agent comprises hyaluronic acid;
`
`25
`
`gelatin; polysaccharide; polyacrylic acid; polymethacrylic acid; hydroxyethylmethacrylic
`
`acid; amino acid; or a salt, derivative, copolymer, or mixture thereof.
`
`In certain embodiments, the pH that promotes ionization of the pharmaceutically
`
`acceptable polymeris between about 4-14, 6-14, 6-10, or about 8-12.
`
`In certain embodiments, a base (e.g., sodium hydroxide, potassium hydroxide,
`
`30
`
`sodium bicarbonate, sodium carbonate, potassium bicarbonate, or potassium carbonate)is
`
`used to adjust pH of the aqueoussolution.
`
`

`

`WO 2014/089160
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`PCT/US2013/073019
`
`In certain embodiments, the second solvent is water, and the first solvent miscible
`
`with water is or comprises acetone, tetrahydrofuron (THF), acetonitrile, dimethyl
`
`sulfoxide (DMSO), or dimethylformamide (DMF).
`
`In certain embodiments, the multiple negatively charged terminal groups are
`
`carboxyl terminal groups.
`
`In certain embodiments, the polymer containing multiple negatively charged
`
`carboxyl terminal groups is produced by using a carboxyl-functional initiator in the
`
`preparation of the polymer.
`
`In certain embodiments, the carboxyl-functional initiator is an o-hydroxyl acid,
`
`10
`
`preferably lactic acid or glycolic acid.
`
`In certain embodiments, the polymer containing multiple negatively charged
`
`carboxyl terminal groups is produced by grafting a carboxyl-containing entity to the
`
`polymer.
`
`In certain embodiments, the carboxyl-containing entity is or comprises polyacrylic
`
`15
`
`acid, polymethacrylic acid, poly(hydroxyethyl methacrylic acid), poly(maleic acid),
`
`polyanhydrides, or a salt, derivative, copolymer, or mixture thereof.
`
`In certain embodiments, the polymer containing multiple negatively charged
`
`carboxyl terminal groups is produced by converting a functional group on the polymerto
`
`carboxyl group.
`
`20
`
`In certain embodiments, the functional group is a hydroxyl group, and wherein the
`
`hydroxyl group is converted to the carboxyl group by reacting with an anhydride(e.g¢.,
`
`dihydrofuran-2,5-dione).
`
`In certain embodiments, the polymer containing multiple negatively charged
`
`carboxyl terminal groups is produced by using an initiator that contains multiple carboxyl
`
`25
`
`groups for generating hyperbranched polymercontaining multiple negatively charged
`
`carboxyl terminal groups.
`
`In certain embodiments, the microparticles or nanoparticles have average particle
`
`sizes of from about | nm to about 1000 um, preferably from about 10 nm to about 100
`
`um, more preferably from about 20 nm to about 5 um, and most preferably from about
`
`50 nm to about 2 um.
`
`

`

`WO 2014/089160
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`PCT/US2013/073019
`
`In certain embodiments, the pharmaceutically acceptable polymer is PLGA, and
`
`wherein the microparticles or nanoparticles are PEGylated.
`
`In certain embodiments, the microparticles or nanoparticles are PEGylated by
`
`mixing polyethylene glycol (PEG) or PEG-containing entity during the preparation of the
`
`microparticles and nanoparticles.
`
`In certain embodiments, the microparticles or nanoparticles are PEGylated by
`
`using copolymers of PEG and PLGA.
`
`In certain embodiments, the microparticles or nanoparticles are PEGylated by
`
`physically absorbing PEG polymers or polymers containing PEG units onto the PLGA
`
`10
`
`microparticles and nanoparticles.
`
`In certain embodiments, the microparticles or nanoparticles are PEGylated by
`
`conjugating PEG units to the surface of the PLGA microparticles or nanoparticles via
`
`covalent bonds.
`
`In certain embodiments, an API (active pharmaceutical ingredient) is covalently
`
`15
`
`attached to the surface of the microparticles or nanoparticles via covalent bonds.
`
`In certain embodiments, the method further comprises chemically conjugating a
`
`biomolecule (e.g., a peptide or a protein) to the surface of the microparticles or
`
`nanoparticles.
`
`Anotheraspect of the invention provides a composition comprising microparticles
`
`20
`
`or nanoparticles having negative surface charges, wherein the composition is prepared
`
`according to any one of the methods described herein.
`
`In certain embodiments, the composition is free from other API (e.g., attached
`
`peptide or antigenic moieties).
`
`Another aspect of the invention provides a pharmaceutical composition
`
`25
`
`comprising any of the subject composition, and a pharmaceutically accepted carrier or
`
`excipient.
`
`Another aspect of the invention provides a methodof treating a disease or
`
`condition in a subject, wherein the disease or condition is treatable with microparticles or
`
`nanoparticles with negative surface charge, comprising administering a composition or a
`
`30
`
`pharmaceutical composition comprising the microparticles or nanoparticles to the subject,
`
`thereby treating the disease or condition.
`
`

`

`WO 2014/089160
`
`PCT/US2013/073019
`
`In certain embodiments, the disease or condition is characterized by an
`
`inflammatory immuneresponse.
`
`In certain embodiments, the disease or condition is multiple sclerosis (MS),
`
`psoriasis, rheumatoid arthritis, type 1 diabetes.
`
`In certain embodiments, the method further comprises administering a second
`
`therapeutic agent knownto be effective for treating the disease or condition.
`
`It should be understood that any embodiments described herein can be combined
`
`with any other embodiments, including embodiments described only under one aspect of
`
`the invention, and embodiments described only in the examples.
`
`10
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`This invention described herein provides polymerparticles (microparticles and
`
`nanoparticles) which contain unusually high or at least increased negative charges on the
`
`surface of said particles compared to those produced using conventional methods.
`
`Preferably, polymers used for the preparation of the particles are pharmaceutically
`
`15
`
`acceptable materials.
`
`Asused herein, “pharmaceutically acceptable” includes those compounds,
`
`materials, compositions, and/or dosage forms which are, within the scope of sound
`
`medical judgment, suitable for medical or veterinary use when in contact with the tissues
`
`of human beings and animals, without causing excessive toxicity, irritation, allergic
`
`20
`
`response, or other problems or complications, commensurate with a reasonable
`
`benefit/risk ratio. Preferably, a pharmaceutically acceptable material (e.g., polymer or
`
`microparticles / nanoparticles produced therefrom) is suitable or approved for human
`
`medical use.
`
`As used herein, “microparticles” are roughly round, sphere, or sphere-like in
`
`25
`
`shape, and are generally within the size range of, e.g., between about 1-1,000 um,or
`
`between about 10-100 um. The subject microparticles may also include particles that are
`
`less likely to clump in vivo.
`
`Asused herein, “nanoparticles” are roughly round, sphere, or sphere-like in shape,
`
`and are generally within the size range of, e.g., between about 1-1,000 nm, between about
`
`30
`
`10-1,000 nm, or between about 50-1,000 nm, or between about 100-500 nm. The subject
`
`nanoparticles may also include particles that are less likely to clump in vivo.
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`

`

`WO 2014/089160
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`PCT/US2013/073019
`
`It is not necessary that each microparticle or nanoparticle be uniform in size,
`
`although they are generally of a size sufficient to trigger phagocytosis in an antigen
`
`presenting cell (APC) or other MPScell. Thus in one embodiment, the subject
`
`microparticles and nanoparticles have a diameter sufficient to trigger phagocytosis in an
`
`antigen presenting cell (APC) or other MPScell.
`
`Asusedherein, “about” generally means up to +10% of the particular term being
`
`modified.
`
`The negative charge can be, for example, in the form of a carboxylate, sulfonate,
`
`nitrate, fluorate, chloride, iodide, persulfate, and many other negatively charged chemical
`
`10
`
`groups. In certain embodiments, the negative charge is mainly conferred by carboxyl
`
`groups. The subject microparticles or nanoparticles having net negative surface charges,
`
`and may or may not contain somepositive surface charges.
`
`A preferred pharmaceutically acceptable polymer useful for the preparation of the
`
`subject microparticles and nanoparticles is PLGA. PLGAistypically prepared by ring-
`
`15
`
`opening polymerization of lactide and glycolide. In this reaction, Stannous octoate is
`
`usually used as the catalyst, although other catalysts may also be used. An initiator, such
`
`as an alcohol, is often used to initiate the polymerization reaction. If no initiator is
`
`intentionally added, trace amount of polar compound containing an active proton, such as
`
`alcohol and water, may serveas the initiator. Polymerization usually results ina PLGA
`
`20
`
`polymer with a carboxyl group at the chain terminal, as illustrated below:
`
`R-OH + L (lactide monomer) + G (glycolide monomer) = PLGA-COOH
`
`Therefore, each PLGA polymer molecule is typically linear, and typically contains
`
`a single COOHgroupat the chain terminal. Consequently, conventional PLGAparticles
`
`prepared from such PLGA polymers only have small amount of COOH groups on the
`
`25
`
`surface, and the negative charge thereon may not be sufficient for certain uses, such as
`
`treating inflammatory diseases. In addition, there may not be sufficient numbers of
`
`COOH groups for covalently attaching API’s or other chemical moiety such as protein
`
`ligands or other targeting agents to the surface of said microparticles and nanoparticles.
`
`Such protein ligands or other targeting agents may bind to a receptor or a binding partner
`
`on the surface ofa target cell, tissue, organ, or location.
`
`The instant invention provides various methods or combinations thereof for
`
`producing PLGAparticles with additional negatively charged groups (e.g., carboxyl
`
`groups) on the PLGAparticle surfaces. Such PLGAparticles with increased net negative
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`

`

`WO 2014/089160
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`PCT/US2013/073019
`
`surface charges are particularly useful, for example, to treat certain diseases (such as
`
`inflammatory diseases) andto facilitate the conjugation of API’s or other chemical entity
`
`to the microparticles and nanoparticles.
`
`The invention described herein provides several basic methods for the preparation
`
`of particles with highly negative surface charges. These methods are not mutually
`
`exclusive, and may be combined with one another to produce additive or even synergistic
`
`effects to produce microparticles and nanoparticles with highly negatively charged
`
`surfaces.
`
`Thusin one aspect, the invention provides a method for the preparation of a
`
`10
`
`composition comprising microparticles or nanoparticles having negative surface charges,
`
`the method comprising producing the microparticles or nanoparticles with a
`
`pharmaceutically acceptable polymer using either an emulsion process or a precipitation
`
`process, wherein the method comprises any one or more features described below, or
`
`combination thereof.
`
`15
`
`Specifically, one feature of the methods of the invention comprises carrying out
`
`the emulsion process or the precipitation process in an aqueoussolution having a pH that
`
`promotes ionization of the pharmaceutically acceptable polymer. For example, the
`
`pharmaceutically acceptable polymer may comprise a carboxyl group that becomes
`
`ionized (e.g., carries a negative charge) at a basic pH. In another example, the
`
`20
`
`pharmaceutically acceptable polymer may comprise a chemical moiety having a low pKa
`
`such that the moiety becomesionized at a relatively acidic pH (e.g., pH 5 or6).
`
`While not wishing to be bound byany particular theory, the ionized groups or
`
`moieties, compared to their non-ionized forms, tend more to be exposed on the surface of
`
`the eventually formed microparticles or nanoparticles, and tend less to be buried inside
`
`25
`
`the eventually formed microparticles or nanoparticles.
`
`Another feature of the methods of the invention comprises using pharmaceutically
`
`acceptable polymers having a low average molecular weight. As described herein, PLGA
`
`is typically prepared by ring-opening polymerization of lactide and glycolide using
`
`Stannous octoate as the catalyst and an alcohol as an initiator. Polymerization usually
`
`results in a linear PLGA polymerwith a single carboxyl group at the chain terminal.
`
`Thus, by using a PLGA polymer having a lower molecular weights, or shorter polymer
`
`chains, relatively higher carboxyl group density can be reached in the nanoparticles and
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`

`

`WO 2014/089160
`
`PCT/US2013/073019
`
`microparticles. Here, carboxyl group density can be defined as numberof carboxyl
`
`groups per gram of polymer.
`
`In certain embodiments, the average molecular weight of the pharmaceutically
`
`acceptable polymeris within a desired range.
`
`The low end ofthe range is preferably no less than about 100, 200, 300, 400, 500,
`
`600, 700, 800, 900, 1000, 1200, 1500, 2000, 2500, or 3000 Da. The desired range can
`
`has a low endof any of the above values.
`
`The high end of the range is preferably no more than 50,000, 40,000, 35,000,
`
`30,000, 25,000, 20,000, 15,000, 10,000, 7,500, or 5,000 Da. The desired range can has a
`
`10
`
`high end of any of the above values.
`
`For instance, the desired range may be from about 500 to about 50,000 Da, or
`
`from about 1,000 to about 30,000 Da.
`
`In certain pharmaceutically acceptable polymers, such as PLGA,average
`
`molecular weight is expressed in other physical properties such as inherent viscosity.
`
`15
`
`Inherent Viscosity (IV) is a viscometric method for measuring molecular size. IV is
`
`based on the flow time of a polymersolution through a narrow capillary relative to the
`
`flow time of the pure solvent through the capillary. For certainly measuresin the instant
`
`application, the solvent used is typically chloroform, and the polymer concentration is
`
`about 0.5 % (w/v). The temperature at which the viscosity is measured is about 30°C.
`
`20
`
`The units of IV are typically reported in deciliters per gram (dL/g). Thus, for example,
`
`the desired pharmaceutically acceptable polymer (such as PLGA) that maybe used in the
`
`instant invention may have an inherent viscosity of from about 0.01 to about 20 dL/g, or
`
`from about 0.05 to about 2.0 dL/g.
`
`Yet another feature of the methodsof the invention comprises using
`
`25
`
`pharmaceutically acceptable polymers containing multiple (7.e., two or more, > 2, > 3, > 4,
`
`>5,> 10, = 20, > 50, => 75, => 100, or a range between any of the two recited values, efc.)
`
`potentially negatively charged terminal groups. In certain embodiments, the multiple
`
`negatively charged terminal groups are carboxyl terminal groups.
`
`Polymers containing multiple carboxyl groups can be obtained by a variety of
`
`30
`
`means, including: 1) using a carboxyl-functional initiator in the preparation of the
`
`polymer. Common carboxyl-functional initiators include but are not limited to a-
`
`hydroxyl acid. For example, lactic acid, glycolic acid; 2) grafting carboxyl-containing
`
`entities to the polymer chain; 3) converting other functional groups on the PLGA polymer
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`WO 2014/089160
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`PCT/US2013/073019
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`to carboxyl groups via a chemical reactions. For example, hydroxyl groups on PLGA
`
`polymers may be converted to carboxyl groups by reacting the hydroxyl groups with an
`
`anhydride (e.g., dihydrofuran-2,5-dione). An exemplary reaction is depicted in the
`
`scheme below:
`
`HO
`
`ie
`
`8
`om
`
`G
`B
`aL
`oy
`yen 8
`PF
`i
`i
`Ss a Bt
`a
`G
`
`1 SEEMED
`¥
`
`o
`a
`*
`4 oe
`y
`wy
`sy
`oh
`yk
`ng ae A
`olf
`fm
`A
`a
`es,
`AS eS Pes a
`a
`ies “Sey hs “
`jer
`sok “SO
`2.
`tt
`
`BG
`Dug <8
`
`
`
`and, 4) using hyperbranched PLGA polymersthat contain multiple carboxyl-
`
`containing arms obtained by, for example, using an initiator that contains multiple
`
`carboxyl groups on its molecule.
`
`The number of carboxyl group on each (PLGA) polymeris preferably from 1 to
`
`10
`
`100, more preferably from 2 to 10 (e.g., 2, 3, 4,5, 6, 7, 8, 9, or 10).
`
`The methods of the invention can comprise any one or more features described
`
`herein.
`
`Anyart-recognized emulsion process may be used in the methods of the invention.
`
`In certain embodiments, the subject microparticles and nanoparticles (e.g., PLGA
`
`15
`
`microparticles and nanoparticles) can be prepared by an emulsification process
`
`comprising the following steps (not necessarily in this order): 1) dissolving the
`
`pharmaceutically acceptable polymer (e.g., PLGA)inafirst solvent (e.g., methylene
`
`chloride) to form a polymersolution; 2) emulsifying the polymersolution (e.g., PLGA
`
`solution) in a solution of a second solvent(e.g., an aqueous solution, or an organic solvent)
`
`20
`
`to form an emulsion, wherein the first solvent is not miscible or partially miscible with
`
`the second solvent, and wherein the solution of the second solvent optionally comprises a
`
`pharmaceutically acceptable negatively charged agent; and, 3) removingthe first solvent
`
`to form the microparticles or nanoparticles having negative surface charges, wherein the
`
`solution of the second solvent is optionally the aqueoussolution.
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`In certain embodiments, in the emulsification process, the weight ratio of the
`
`PLGAsolution to the aqueous solution is typically from 1:1,000 to 10:1, preferable from
`
`1:100 to 1:1.
`
`Asused herein, miscibility is defined to be the property of liquids to mix inall
`
`proportions, forming a homogeneous solution. Substances / liquids are said to be
`
`immiscible or not miscible, if in some proportion, they do not form a solution.
`
`Exemplary solvents miscible with water include acetone, tetrahydrofuron (THF),
`
`acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF).
`
`Anotherart-recognized emulsion process is commonly knownas double emulsion
`
`10
`
`process, which maybe particularly useful when an active pharmaceutical ingredient (API),
`
`such as a protein-based therapeutics prepared in an aqueous solution,is first emulsified
`
`with a pharmaceutically acceptable polymer solution to form a first emulsion such that
`
`the protein-based therapeutics is encapsulated within the polymer solution. Then the
`
`polymer, and the therapeutics encapsulated therein, is again emulsified in a larger volume
`
`15
`
`of solvent to form a second emulsion (e.g., the water-in-oil-in- water or w/o/w type double
`
`emulsion), before the microparticle or nanoparticle is formed.
`
`For example, in the above described w/o/w technique, a relatively small amount of
`
`aqueous protein solution may be introduced into a relatively larger amount of organic
`
`solvent, such as methylene chloride or ethyl acetate, that dissolves the hydrophobic
`
`20
`
`polymer PLGA. Thefirst emulsion is then formed using a suitable method, e.g., probe
`
`sonication or homogenization. After formation of the first emulsion, a second emulsion is
`
`formed by introducing the first emulsion into an aqueous solution containing an
`
`emulsifier, e.g., polyvinyl alcohol. Again, a homogenization methodis used to form the
`
`second emulsion. This is next followed by a period of solvent evaporation leading to the
`
`25
`
`hardening of the polymer, typically by stirring for some hours. As a result, the protein
`
`solution is trapped into the relative hydrophobic matrix of the PLGA polymer forming
`
`small inclusions. Finally, the microparticles ornanoparticles formed are collected,
`
`washed(e.g., with distilled water) via repeated centrifugationorfiltration, followed by
`
`dehydration, typically by lyophilization.
`
`Thusin certain embodiments, the subject microparticles and nanoparticles(e.g.,
`
`PLGAmicroparticles and nanoparticles) can be prepared by a double emulsification
`
`process comprising the following steps (not necessarily in this order): 1) dissolving the
`
`pharmaceutically acceptable polymer (e.g., PLGA)in a first solvent (e.g., methylene
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`WO 2014/089160
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`chloride) to form a polymersolution; 2) adding a relatively small amountof a solution of
`
`a second solvent into the polymer solution to form a mixture, whereinthe first solventis
`
`not miscible orpartially miscible with the second solvent, and wherein the solution of the
`
`second solvent optionally comprises an active pharmaceutical ingredient (APD); 3)
`
`emulsifying the mixture to form a first emulsion; 4) emulsifying the first emulsion in the
`
`solution of the second solvent to form a second emulsion, wherein the solution of the
`
`second solvent optionally comprises a pharmaceutically acceptable negatively charged
`
`agent, and optionally further comprises a surfactant; and, 5) removingthe first solvent to
`
`form said microparticles or nanoparticles having negative surface charges; wherein said
`
`10
`
`solution of the second solventis optionally said aqueous solution.
`
`In certain embodiments, the volume of the small amount of the solution of the
`
`second solvent addedto the polymersolution for the generation of the first emulsion is
`
`typically from 0.01% to 50%, preferable from 0.1% to 10%, based on the volumeof the
`
`PLGAsolution.
`
`15
`
`In certain embodiments, the volumeratio of