Extended release drug-loaded microparticles

Abstract

Described are extended release drug-loaded microparticles, pharmaceutical compositions comprising them, methods of making them, and therapeutic methods using them.

Description

EXTENDED RELEASE DRUG-LOADED MICROPARTICLESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 528,015, filed on July 20, 2023, the entire contents of which is incorporated by reference herein.FIELD

[0002] Described are extended release drug-loaded microparticles, pharmaceutical compositions comprising them, methods of making them, and therapeutic methods using them.BACKGROUND

[0003] The formulation of drugs in injectable compositions that provide an extended release profile can be challenging. Formulations based on polymeric microparticles, such as poly(D,L- lactide-co-glycolide) (PLGA) microparticle-based parenteral formulations, have been described for various drugs, but only 19 PLGA microsphere-based pharmaceutical products are available in the market. See, e.g., Li et al., Bridging the gap between fundamental research and product development of long acting injectable PLGA microspheres, Exp. Op. Drug. Deliv. (2022).

[0004] Triptorelin is a synthetic decapeptide agonist analog of gonadotropin releasing hormone (GnRH) and reversibly represses luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Triptorelin may be used in the treatment of hormone-responsive cancers, such as breast cancer or prostate cancer; in the management of endometriosis, female infertility and uterine fibroids; and in treatment of precocious puberty.

[0005] Injectable formulations of triptorelin are approved for use in treating prostate cancer (TRELSTAR®, Verity Pharmaceuticals) and central precocious puberty (TRIPTODUR®, Arbor Pharmaceuticals, LLC). U.S. Patent 10,166,181 describes injectable pharmaceutical compositions comprising a mixture of triptorelin PLGA microparticles, where each type of triptorelin microparticle comprises triptorelin pamoate in a different copolymer of lactic and glycolic acid(PLGA), e.g., PLGA containing approximately 85% lactide and 15% glycolide or PLGA containing approximately 75% lactide and 25% glycolide, where the mixture of microparticles is said to provide prolonged release of triptorelin after injection. U.S. Patent No. 7,252,842 discloses microparticles containing hydrophilic active agents, including triptorelin-loaded microparticles comprising PLGA containing approximately 85% lactide and 15% glycolide. However, microparticles prepared in accordance with Example 28 of U.S. Patent No. 7,252,842 had low core loading (on the order of 2%) and poor encapsulation efficiency (on the order of 17%) and a mean particle size < 20 pm. Thus, those microparticles likely would not be suitable for development of a one month, three month or six month depot formulation because a large quantity of microparticles would need to be administered to achieve the target dose.

[0006] There remains a need for extended-release injectable pharmaceutical compositions, such as injectable pharmaceutical compositions that provide drug release over an extended period of time after injection, such as for 1 week, 1 month, 6 weeks, 3 months or 6 months.SUMMARY

[0007] Provided in one aspect is a drug-loaded microparticle comprising:(a) a drug;(b) a polymer component comprising one or more polymers selected from ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymers having a lactide: glycolide ratio of about 50:50, ester-terminated PLGA polymers having a lactide: glycolide ratio of about 75:25, and ester- terminated polylactide (PLA) polymers;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 1 week after administration by subcutaneous or intramuscular injection.

[0008] In some embodiments, the microparticle releases the drug in vivo for a period of at least one month after administration by subcutaneous injection. In some embodiments, the microparticlereleases the drug in vivo for a period of at least three months after administration by subcutaneous injection. In some embodiments, the microparticle releases the drug in vivo for a period of at least six months after administration by subcutaneous or intramuscular injection.

[0009] Provided in one aspect is a drug-loaded microparticle comprising:(a) a drug;(b) one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 1 week after administration by subcutaneous or intramuscular injection.

[0010] Provided in one aspect is a drug-loaded microparticle comprising:(a) a drug;(b) one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0011] Provided in one aspect is a drug-loaded microparticle comprising:(a) a drug;(b) one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 6 weeks after administration by subcutaneous or intramuscular injection.

[0012] The one or more ester-terminated PLGA polymers may be selected from one or more of RG502, RG503 and RG504 (either as single polymer or as mixtures thereof). The one or more ester-terminated PLGA polymers may comprise one ester-terminated PLGA polymer. The ester- terminated PLGA polymer may be RG503 or the ester-terminated PLGA polymer may be RG504. The one or more ester-terminated PLGA polymers may comprise two different ester-terminated PLGA polymers. The two different ester-terminated PLGA polymers may be present in a weight ratio of lower molecular weight PLGA to higher molecular weight PLGA of from about 99: 1 to about 51:49, such as a ratio of about 99: 1, about 90: 10, about 80:20, about 70:30, about 60:40, about 55:45, or about 51:49. The two different ester-terminated PLGA polymers may be RG504 and RG503. The two different ester-terminated PLGA polymers may be RG503 and RG502. The weight ratio of triptorelin to one or more ester-terminated PLGA-polymers may be about 4 / 100 to about 10 / 100. The weight ratio of FSH to ester-terminated PLGA polymer(s) may be from about 5 / 1000 to about 5 / 100, or higher.

[0013] Provided in another aspect is a drug-loaded microparticle comprising:(a) a drug;(b) an ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 3 months after administration by subcutaneous or intramuscular injection.

[0014] The ester-terminated PLGA polymer may be RG753S. The weight ratio of triptorelin to ester-terminated PGLA-polymer may be about 5 / 100 to about 10 / 100. The weight ratio of FSH to ester-terminated PLGA polymer(s) may be from about 5 / 1000 to about 5 / 100, or higher.

[0015] Provided in another aspect is a drug-loaded microparticle comprising:(a) a drug;(b) two different ester-terminated PLA polymers;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 6 months after administration by subcutaneous or intramuscular injection.

[0016] The two different ester-terminated PLA polymers may be present in a weight ratio of lower molecular weight PLA to higher molecular weight PLA of from about 90: 10 to about 60:40, such as a ratio of about 90: 10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35 or about 60:40. The two different ester-terminated poly lactide PLA polymers may be R203S and R205S. The weight ratio of triptorelin to PLA polymers may be about 5 / 100 to about 15 / 100. The weight ratio of FSH to PLA polymers may be from about 5 / 1000 to about 5 / 100, or higher.

[0017] In some embodiments, the drug is one or more selected from a small molecule drug, a peptide drug, and a protein drug. In some embodiments, the drug is one or more selected from selected from triptorelin, follicle stimulating hormone (FSH), leuteinizing hormone (LH), human chorionic gonadotrophin (hCG), atosiban, insulin, abiraterone, barusiban, degarelix, desmopressin, ganirelix (e.g., ganirelix acetate), gemcitabine, gonadorelin (e.g., gonadorelin acetate), menotropin or human menopausal gonadotropin (hMG), merotocin, progesterone, quinagolide, somatostatin, somatropin, and tolterodine. In some embodiments, the drug is triptorelin, optionally wherein the triptorelin is triptorelin acetate. In some embodiments, the drug is FSH, optionally wherein the FSH is recombinant FSH, optionally wherein the FSH is selected from follitropin alfa, follitropin beta, and follitropin delta. In further specific embodiments, the FSH is follitropin delta. In some embodiments, the drug is two or more drugs. In some embodiments, the drug comprises FSH (e.g., follitropin delta) and LH and / or hCG.

[0018] In any drug-loaded microparticles described herein, the surfactant may comprise or be a poloxamer (e.g., Poloxamer 188) or a polyvinyl alcohol. In any drug-loaded microparticles described herein, the alkaline salt may comprise or be sodium chloride. In any drug-loaded microparticles described herein, the polyol may comprise or be sucrose or polyethylene glycol.

[0019] The mean particle size of the drug-loaded microparticles as described herein may be from about 20 pm to about 180 pm, including from about 30 pm to about 150 pm, such as from about 30 pm to about 120 pm, including from about 30 pm to about 80 pm.

[0020] The drug-loaded microparticles as described herein may further comprise a residual content of components used to make the microparticle. Thus, the drug-loaded microparticles may further comprise a residual content of acid, such as acetic acid or citric acid, of from about 0.01% w / w to about 1.0% w / w. The drug-loaded microparticles may have a polyol content of from about 0.1% w / w to about 5% w / w, such as a sucrose content of from about 0.20% w / w to about 5% w / w or such as a polyethylene glycol content of 0.20% w / w to about 5% w / w. The drug-loaded microparticles may further comprise a residual content of a buffering salt, such as citric acid, of from about 0.01% w / w to about 1.0% w / w. The drug-loaded microparticles may further comprise a residual organic solvent content of about 50 ppm to about 20000 ppm, such as an ethyl acetate content of from about 50 ppm to about 20000 ppm. The drug-loaded microparticles may have a surfactant content of from about 0% w / w to about 4% w / w.

[0021] Also provided herein are pharmaceutical compositions comprising any drug-loaded microparticles as disclosed herein. The pharmaceutical composition may be formulated for subcutaneous injection. The pharmaceutical composition may be formulated for intramuscular injection. The pharmaceutical composition may comprise drug-loaded microparticles and one or more pharmaceutically acceptable carriers, diluents, and / or excipients. The pharmaceutical composition may comprise drug-loaded microparticles and a pharmaceutically acceptable diluent selected from carboxymethylcellulose, dextran, and hyaluronic acid and salts thereof. The pharmaceutical composition may comprise drug-loaded microparticles and a pharmaceutically acceptable diluent comprising hyaluronic acid and / or a salt thereof, optionally wherein the hyaluronic acid and / or salt thereof has an average molecular weight of from about 1 MDa to about 5 MDa, further optionally wherein the hyaluronic acid and / or salt thereof has an average molecular weight of about 3.2 MDa, further optionally wherein the composition comprises from about 0.01% (w / v) to about 5% (w / v) of the hyaluronic acid and / or salt thereof, based on the volume of thepharmaceutical composition, including from about 1% (w / v) to about 3 % (w / v), or about 1.5 mg hyaluronic acid and / or a salt thereof per mb pharmaceutical composition.

[0022] A pharmaceutical composition as described herein may be formulated to contain an amount of the drug-loaded microparticles that provides a therapeutically effective amount of the drug in a single dose. The pharmaceutical composition may be formulated to provide a therapeutic mean plasma concentration of the drug for a period of at least 1 week after administration of a single dose by subcutaneous or intramuscular injection, for a period of at least 1 month after administration of a single dose by subcutaneous or intramuscular injection, for a period of at least 6 weeks after administration of a single dose by subcutaneous or intramuscular injection, for a period of at least 3 months after administration of a single dose by subcutaneous or intramuscular injection, or for a period of at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

[0023] A pharmaceutical composition comprising triptorelin-loaded microparticles formulated to release triptorelin in vivo for a period of about 1 week after subcutaneous or intramuscular injection may be formulated to contain an amount of the triptorelin-loaded microparticles that provides about 1 mg of triptorelin in a single dose. A pharmaceutical composition comprising triptorelin- loaded microparticles formulated to release triptorelin in vivo for a period of about 1 month after subcutaneous or intramuscular injection may be formulated to contain an amount of the triptorelin- loaded microparticles that provides 3.75 mg of triptorelin in a single dose. A pharmaceutical composition comprising triptorelin-loaded microparticles formulated to release triptorelin in vivo for a period of about 3 months after subcutaneous or intramuscular injection may be formulated to contain an amount of the triptorelin-loaded microparticles that provides 11.25 mg of triptorelin in a single dose. A pharmaceutical composition comprising triptorelin-loaded microparticles formulated to release triptorelin in vivo for a period of about 6 months after subcutaneous or intramuscular injection may be formulated to contain an amount of the triptorelin-loaded microparticles that provides 22.5 mg of triptorelin in a single dose. Such pharmaceutical compositions respectively may provide a mean plasma concentration of triptorelin of at least 0.1ng / mL for a period of at least 1 week, at least 1 month, at least 6 weeks, at least 3 months, or at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

[0024] A pharmaceutical composition comprising FSH-loaded microparticles formulated to release FSH in vivo for a period of about 1 week after subcutaneous or intramuscular injection may be formulated to contain an amount of the FSH-loaded microparticles that provides about 85 pg of FSH (e.g., follitropin delta) or more in a single dose. A pharmaceutical composition comprising FSH-loaded microparticles formulated to release FSH in vivo for a period of about 1 month after subcutaneous or intramuscular injection may be formulated to contain an amount of the FSH- loaded microparticles that provides about 0.35 mg of FSH (e.g., follitropin delta) or more in a single dose. A pharmaceutical composition comprising FSH-loaded microparticles formulated to release FSH in vivo for a period of about 6 weeks after subcutaneous or intramuscular injection may be formulated to contain an amount of the FSH-loaded microparticles that provides about 0.5 mg of FSH (e.g., follitropin delta) or more in a single dose. A pharmaceutical composition comprising FSH-loaded microparticles formulated to release FSH in vivo for a period of about 3 months after subcutaneous or intramuscular injection may be formulated to contain an amount of the FSH-loaded microparticles that provides about 1 mg of FSH (e.g., follitropin delta) or more in a single dose. A pharmaceutical composition comprising FSH-loaded microparticles formulated to release FSH in vivo for a period of about 6 months after subcutaneous or intramuscular injection may be formulated to contain an amount of the FSH-loaded microparticles that provides about 2 mg of FSH (e.g., follitropin delta) or more in a single dose. Such pharmaceutical compositions may respectively provide a mean plasma concentration of FSH of at least 14 mIU / ml for a period of at least 1 week, at least 1 month, at least 6 weeks, at least 3 months, or at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

[0025] Also provided herein are methods of treatment, comprising administering a pharmaceutical composition as disclosed herein to a subject in need thereof by subcutaneous or intramuscular injection. The method may comprise injection of a single dose that provides a therapeutically effective amount of the drug. The method may provide a therapeutic mean plasma concentration of the drug for a period of at least 1 week after administration of a single dose by subcutaneous orintramuscular injection, for a period of at least 1 month after administration of a single dose by subcutaneous or intramuscular injection, for a period of at least 6 weeks after administration of a single dose by subcutaneous or intramuscular injection, for a period of at least 3 months after administration of a single dose by subcutaneous or intramuscular injection, or for a period of at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

[0026] For triptorelin embodiments, a weekly method may comprise injection of a single dose that provides about 1 mg triptorelin, a monthly method may comprise injection of a single dose that provides 3.75 mg of triptorelin, a 3 -month method may comprise injection of a single dose that provides 11.25 mg of triptorelin, and a 6-month method may comprise injection of a single dose that provides 22.5 mg of triptorelin. Such methods may respectively provide a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 1 week, at least 1 month, at least 6 weeks, at least 3 months, or at least 6 months, after subcutaneous or intramuscular injection of a single dose. The subject may be in need of treatment of one or more of hormoneresponsive cancer, endometriosis, female infertility, uterine fibroids, and central precocious puberty. The subject may be in need of treatment of hormone-responsive breast cancer or hormoneresponsive prostate cancer.

[0027] For FSH embodiments, a weekly method may comprise injection of a single dose that provides about 85 pg of FSH (e.g., follitropin delta) or more in a single dose, a monthly method may comprise injection of a single dose that provides about 0.35 mg of FSH (e.g., follitropin delta) or more in a single dose, a 6- week method may comprise injection of a single dose that provides about 1 mg of FSH (e.g., follitropin delta) or more in a single dose, a 3-month method may comprise injection of a single dose that provides about 1 mg of FSH (e.g., follitropin delta) or more in a single dose, and a 6-month method may comprise injection of a single dose that provides about 2 mg of FSH (e.g., follitropin delta) or more in a single dose. Such methods may respectively provide a mean plasma concentration of at least 14 mIU / ml FSH for a period of at least 1 week, at least 1 month, at least 6 weeks, at least 3 months, or at least 6 months after administration of a single dose by subcutaneous or intramuscular injection. The subject may be in need of treatment of female infertility or male infertility.

[0028] Also provided are triptorelin-loaded microparticles as described herein and compositions comprising them for use in treating one or more of hormone-responsive cancer, endometriosis, female infertility, uterine fibroids, and central precocious puberty. Also provided are uses triptorelin-loaded microparticles as described herein and compositions comprising them in the preparation of a medicament for treating one or more of hormone-responsive cancer, endometriosis, female infertility, uterine fibroids, and central precocious puberty.

[0029] Also provided are FSH-loaded microparticles as described herein and compositions comprising them for use in treating female infertility or male infertility. Also provided are uses of FSH-loaded microparticles as described herein and compositions comprising them for preparation of a medicament for treating female infertility or male infertility.

[0030] Also provided herein are kits comprising (a) a container containing drug-loaded microparticles as disclosed herein, and (b) instructions for preparation of a pharmaceutical composition comprising the microparticles and a pharmaceutically acceptable carrier, such as may be suitable for intramuscular or subcutaneous injection.

[0031] Also provided are methods of making drug-loaded microparticles comprising:(a) adding (i) a drug solution comprising a drug in an aqueous solvent to (ii) a polymer solution comprising one or polymers selected from ester-terminated poly(D,L-lactide-co- glycolide) (PLGA) polymers having a lactide: glycolide ratio of about 50:50, ester-terminated PLGA polymer having a lactide:glycolide ratio of about 75:25, and ester-terminated polylactide (PLA) polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the drug solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of drug- loaded microparticles comprising the drug and the one or more polymers selected from ester- terminated PLGA polymers having a lactide:glycolide ratio of about 50:50, ester-terminated PLGA polymers having a lactide:glycolide ratio of about 75:25, and ester-terminated PLA polymers.

[0032] Also provided herein are methods of making triptorelin-loaded microparticles as disclosed herein comprising:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising a mixture of one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50 in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of an alkaline salt and a polyol to the dispersion, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the one or more PLGA polymers.

[0033] Also provided herein are methods of making triptorelin-loaded microparticles as disclosed herein comprising:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising an ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25 in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLGA polymer.

[0034] Also provided herein are methods of making triptorelin-loaded microparticles as disclosed herein comprising:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising a mixture of two different ester-terminated PLA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of an alkaline salt and a polyol to the dispersion, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLA polymers.

[0035] In accordance with any of the methods, the triptorelin may be triptorelin acetate. The triptorelin solution may have a pH of about 4.

[0036] Also provided herein are methods of making FSH-loaded microparticles as disclosed herein comprising:(a) adding (i) a FSH solution comprising FSH in an aqueous solvent (such as an aqueous buffer solution)to (ii) a polymer solution comprising a mixture of one or more ester-terminated PLGA polymers having a lactide: glycolide ratio of about 50:50 in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of an alkaline salt and a polyol to the dispersion, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the one or more PLGA polymers.

[0037] Also provided herein are methods of making FSH-loaded microparticles as disclosed herein comprising:(a) adding (i) a FSH solution comprising FSH in an aqueous solvent (such as an aqueous buffer solution) to (ii) a polymer solution comprising an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 75:25 in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLGA polymer.

[0038] Also provided herein are methods of making FSH-loaded microparticles as disclosed herein comprising:(a) adding (i) a FSH solution comprising FSH in aqueous solvent (such as an aqueous buffer solution) to (ii) a polymer solution comprising a mixture of two different ester-terminated PLA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of an alkaline salt and a polyol to the dispersion, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLA polymers.

[0039] In accordance with any of the methods, the aqueous solvent may be an aqueous acidic buffer, such as an aqueous acetic acid buffer or an aqueous citric acid buffer. The phase inversion solution may comprise a buffer substance, such as a buffer salt. The phase inversion solution may comprise a poloxamer (e.g., Poloxamer 188) and / or polyvinyl alcohol as a surfactant. The phase inversion solution may comprise sodium chloride as an alkaline salt. The phase inversion solution may comprise sucrose and / or polyethylene glycol as a polyol.

[0040] In accordance with any of the methods, the method may further comprise, after step (b), adding a wash phase solution, optionally comprising an aqueous buffer and a surfactant. The wash phase solution may comprise a citrate buffer and a surfactant such as a poloxamer (e.g., Poloxamer 188) or polyvinyl alcohol.

[0041] In accordance with any of the methods, the method may further comprise, after step (b), one or more of removing the solvent, such as by vacuum, separating the microparticles from the suspension, drying the microparticles, optionally by lyophilization, and sterilizing the microparticles, optionally by ionizing radiation, such as by y-irradiation.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 illustrates the preparation of drug-loaded microparticles as described herein.

[0043] FIG. 2 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 1 (a 1 -month triptorelin formulation) to rats as described in Example 1.

[0044] FIG. 3 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 2 (a 1 -month triptorelin formulation) to rats as described in Example 1.

[0045] FIG. 4 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 3 (a 1 -month triptorelin formulation) to rats as described in Example 1.

[0046] FIG. 5 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 9 (a 1 -month triptorelin formulation) to rats as described in Example 2.

[0047] FIG. 6 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 12 (a 1 -month triptorelin formulation) to rats as described in Example 2.

[0048] FIG. 7 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 16 (a 1 -month triptorelin formulation) to rats as described in Example 2.

[0049] FIG. 8 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 19 (a 1 -month triptorelin formulation) to rats as described in Example 2.

[0050] FIG. 9 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 28 (a 3-month triptorelin formulation) to rats as described in Example 3.

[0051] FIG. 10 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 29 (a 3-month triptorelin formulation) to rats as described in Example 3.

[0052] FIG. 11 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 37 (a 3-month triptorelin formulation) to rats as described in Example 4.

[0053] FIG. 12 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 43 (a 6-month triptorelin formulation) to rats as described in Example 5.

[0054] FIG. 13 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 45 (a 3-month rFSH formulation) to rats as described in Example 6.

[0055] FIG. 14 shows the mean plasma concentration-time profile after subcutaneous dosing of Formulation 51 (a 3-month rFSH formulation) to rats as described in Example 6.

[0056] FIG. 15 shows the mean plasma concentration-time profile for FSH after subcutaneous dosing to rats of 3 -month FSH microparticles as described herein formulated in a hyaluronate carrier or a dextran carrier, as described in Example 7.

[0057] FIG. 16 illustrates the IL-4 response after subcutaneous dosing to rats of 3 -month FSH microparticles as described herein formulated in a hyaluronate carrier or a dextran carrier, as described in Example 7.DETAILED DESCRIPTION

[0058] Described are extended release drug-loaded microparticles, pharmaceutical compositions comprising them, methods of making them, and therapeutic methods using them.Definitions

[0059] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined.

[0060] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0061] As used herein, the term “about” when qualifying a number or range means that the number or range is not limited to the exact number or range set forth, but encompass values around the stated number or range as will be understood by persons of ordinary skill in the art depending on the context in which the number or range is used. When not otherwise apparent from the context or convention in the art, “about” means up to plus or minus 10% of the particular term (± 10%).

[0062] The terms “administer,” “administration,” or “administering” as used herein refer to providing, giving, dosing and / or prescribing, such as by either a health professional or his or her authorized agent or under his or her direction, and putting into, taking or consuming, such as by a health professional or the subject or patient.

[0063] The terms “subject” and “patient” as used herein refer to any mammal, including, but not limited to, humans, pets and laboratory animals (e.g., dogs, cats, rodents, rabbits, guinea pigs, primates, etc.), and farm animals and livestock (e.g., horses, camels, donkeys, cattle, sheep, pigs, goats, etc.).

[0064] As used herein, the phrase “therapeutically effective amount” refers to a dose that provides or has been determined to provide the specific pharmacological effect for which the drug is administered in a subject in need of such treatment, such as relief of pain. However, a “therapeutically effective amount” may not always be effective in treating the condition in a given subject, even though such dose is deemed to be a therapeutically effective amount by those of skill in the art. Exemplary doses and therapeutically effective amounts are provided below with reference to adult human subjects. Those skilled in the art can adjust such amounts in accordance with standard practices as needed to treat a specific subject and / or condition.Extended Release Drug-loaded Microparticles

[0065] Described herein are extended release drug-loaded microparticles and pharmaceutical compositions comprising them that can release therapeutically effective amounts of a drug over an extended period of time after administration, such as for at least one week, at least 1 month, at least 6 weeks, at least 3 months, or at least 6 months after injection, e.g., subcutaneous or intramuscular injection. The microparticles are made by a process as described herein, comprising mixing a solution of a polymer in an organic solvent with a solution of the drug in an aqueous solvent to obtain a dispersion of the drug in the polymer solution and adding a surfactant in an aqueous solution (an aqueous surfactant solution) to the dispersion of the drug in the polymer solution. The aqueous surfactant solution acts as an extraction medium for the organic solvent and as the continuous phase for the suspension of microparticles that forms immediately due to precipitation of the polymer onto the drug. While not wanting to be bound by theory, it is believed addition of the aqueous surfactant solution induces a phase transition from the organic phase to the aqueous phase with immediate formation of a microparticle suspension. Thus, this method does not result in the formation of a double emulsion (W / O / W) from which solvent must beeliminated prior to formation of microparticles. It was surprisingly found that by using the polymers, solvents, and other excipients described herein, microparticles could be obtained that release therapeutically effective amounts of drug over an extended period of time after administration, such as for at least one week, at least 1 month, at least 6 weeks, at least 3 months, or at least 6 months after injection.

[0066] The type of drug that can be formulated in accordance with the present disclosure is not particularly limited, and includes small molecule drugs, peptide drugs, and protein drugs. In specific embodiments, the drug formulated as described herein is one or more selected from a peptide or protein drug. In further specific embodiments, the drug formulated as described herein is one or more selected from triptorelin, follicle stimulating hormone (FSH), leuteinizing hormone (LH), human chorionic gonadotrophin (hCG), atosiban, insulin, abiraterone, barusiban, degarelix, desmopressin, ganirelix (e.g., ganirelix acetate), gemcitabine, gonadorelin (e.g., gonadorelin acetate), menotropin or human menopausal gonadotropin (hMG), merotocin, progesterone, quinagolide, somatostatin, somatropin, and tolterodine. In further specific embodiments, the drug formulated as described herein is triptorelin. In further specific embodiments, the drug formulated as described herein is FSH. In further specific embodiments, the drug formulated as described herein is LH. In further specific embodiments, the drug formulated as described herein is hCG. In further specific embodiments, the drug formulated as described herein is atosiban. In further specific embodiments, the drug formulated as described herein is insulin. In further specific embodiments, the drug formulated as described herein is abiraterone. In further specific embodiments, the drug formulated as described herein is barusiban. In further specific embodiments, the drug formulated as described herein is degarelix. In further specific embodiments, the drug formulated as described herein is desmopressin. In further specific embodiments, the drug formulated as described herein is ganirelix (e.g., ganirelix acetate). In further specific embodiments, the drug formulated as described herein is gemcitabine. In further specific embodiments, the drug formulated as described herein is gonadorelin (e.g., gonadorelin acetate). In further specific embodiments, the drug formulated as described herein is menotropin or human menopausal gonadotropin (hMG). In further specific embodiments, the drug formulatedas described herein is merotocin. In further specific embodiments, the drug formulated as described herein is progesterone. In further specific embodiments, the drug formulated as described herein is quinagolide. In further specific embodiments, the drug formulated as described herein is somatostatin. In further specific embodiments, the drug formulated as described herein is somatropin. In further specific embodiments, the drug formulated as described herein is and tolterodine.

[0067] In any embodiments where the drug is triptorelin, the triptorelin may be triptorelin acetate.

[0068] In any embodiments where the drug is FSH, the FSH may be human FSH, including isolated human FSH or recombinant human FSH. In any embodiments where the drug is FSH, the FSH may be recombinant human FSH selected from follitropin alfa, follitropin beta, and follitropin delta. In specific embodiments, the FSH is follitropin delta. Follitropin delta is a human cell line- derived recombinant FSH approved as REKOVELLE® (follitropin delta), that can be produced by methods disclosed in WO 2009 / 127826.

[0069] In any embodiments, the drug may comprise two or more drugs. In specific embodiments, the drug comprises FSH (e.g., follitropin delta) and LH and / or hCG.

[0070] As discussed below, the approach described herein can be adopted to formulate different drugs. Parameters that typically will be tailored to a given drug include any solvents used, including the solvent for the drug solution, the concentration of the drug used to prepare the drug solution (if applicable), polymers used for the polymer component, the solvent for the polymer component, and the excipients used (e.g., the surfactant(s), pH adjusting agents (if any), stabilizing agents (if any), etc. For example, all solvents used should be compatible with the drug and, for example, not denature the drug such as may be a concern for protein drugs. Additionally or alternatively, process parameters may be tailored to a given drug, such as the time and stirring rate for the dispersing step and phase inversion step, and the composition of the solutions used for phase inversion and washing step(s), the nature of the drying step (e.g., by lyophilization) and excipients used for drying, and the nature of the sterilizing step. For instance, the solution(s) usedfor washing may be tailored to reduce the solubility of the drug in order to increase encapsulation efficiency, which could be done by, for example, modulating the pH and / or using excipients that reduce solubility of the drug in the solution. On the other hand, as discussed below, the polymer(s) may be selected to achieve a target extended release profile, e.g., of 1 week, 1 month, 6 weeks, 3 months, 6 months, or longer. Other parameters that may be selected and controlled to achieve various objections are highlighted in the following discussion.

[0071] The approach of the present disclosure is described below with reference to drugs that are very different from a physicochemical perspective: triptorelin (a 1.3 kDa decapeptide) and FSH (a 35 kDa glycoprotein dimer). Those skilled in the art will appreciate that the approach described herein can be applied to other drugs, including other protein and peptide drugs, including LH, hCG, atosiban, insulin, degarelix, desmopressin, ganirelix (e.g., ganirelix acetate), gonadorelin (e.g., gonadorelin acetate), hMG, merotocin, progesterone, somatostatin, and somatropin, as well as non-protein drugs such as abiraterone, barusiban, gemcitabine, quinagolide, and tolterodine.Triptorelin

[0072] In some embodiments, the drug is triptorelin or a pharmaceutically acceptable salt thereof, such as a water soluble salt, such as the acetate salt. Triptorelin is a decapeptide having the formula: pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2. The systematic (1UPAC) name is L- Pyroglutamyl-L-histidyl-Ltryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L- prolyl-glycinamide, or [D-Trp6]GnRH. Triptorelin has the molecular formula C64H82N18O13 and a molecular weight of 1311.5 g / mol. It is registered under CAS Registry Number 57773-63-4. As used herein, “triptorelin” refers to the triptorelin decapeptide and pharmaceutically acceptable salts thereof, including water soluble salts thereof, such as triptorelin acetate. As used herein, “triptorelin decapeptide” refers to the triptorelin decapeptide in particular.

[0073] Triptorelin is an agonist analog of gonadotropin releasing hormone (GnRH) and reversibly represses luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Triptorelin may be used in the treatment of hormone-responsive cancers, such as breast cancer or prostate cancer; inthe management of endometriosis, female infertility and uterine fibroids; and in treatment of precocious puberty. Injectable formulations of triptorelin are approved for use in treating prostate cancer (TRELSTAR®, Verity Pharmaceuticals) and central precocious puberty (TRIPTODUR®, Arbor Pharmaceuticals, LLC).Solvent for Triptorelin Solution

[0074] The solvent used for the triptorelin solution includes water, and may be an aqueous acidic solution, such as an acetic acid solution, a succinic acid solution, a lactic acid solution, a citric acid solution, or a phosphoric acid solution, optionally including a salt of the corresponding acid anion to increase or provide buffering capacity, such as an acetate salt, succinate salt, lactate salt, citrate salt or phosphate salt. Thus, the solvent used for the triptorelin solution may comprise an acid, such as acetic acid or succinic acid or lactic acid or citric acid or phosphoric acid (and, optionally a buffering salt), that may be present in any amount that provides a triptorelin solution having the desired pH, such as a target pH of about 4, as discussed in more detail below.

[0075] The triptorelin solution may have a pH of from about 3.5 to about 5.0, or from about 4.0 to about 4.8, including about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, or about 4.8. Lor example, the triptorelin solution may comprise an acid at from about 0.1% v / v to about 30% v / v. The triptorelin solution may have a pH of from about 3.9 to about 4.9, including a pH from 3.9 to 4.9, including a pH of from 3.9 to 4.8, including a pH of about 4, including a pH of 4. The pH of the triptorelin solution was found to influence core loading, encapsulation efficiency, and triptorelin release of the triptorelin-loaded microparticles.

[0076] In specific embodiments, the solvent used for the triptorelin solution is an aqueous acidic solution that provides a triptorelin solution having a pH of from 3.9 to 4.9, including a pH of from 3.9 to 4.8, including a pH of about 4, including a pH of 4. Lor example, the solvent may be an aqueous acetic acid solution that may comprise from about 2% to about 20% v / v, including about 9% v / v, including about 5.3% v / v, including about 4.4% v / v, including about 3.5% v / v of aceticacid, or an equivalent amount of a different acid. As another example, the solvent may be an aqueous acetate buffer solution having an acetate concentration of from about 10 mM to about 200 mM (e.g., as needed to achieve the target pH), or an equivalent amount of another aqueous acidic buffer.

[0077] In embodiments where the triptorelin is triptorelin acetate, the total molar concentration of acetate is the sum of acetate contributed by triptorelin acetate and acetate present in the aqueous acidic acetate buffer. In embodiments where the triptorelin is triptorelin acetate and the solvent used for the triptorelin solution is an aqueous acidic acetate buffer solution, it was determined that the total molar concentration of acetate had a significant effect on in vitro triptorelin release of the triptorelin-loaded microparticles, with a higher molar concentration of acetate ions being associated with slower in vitro triptorelin release. A similar effect on in vivo release is expected. Additionally, a similar effect is expected with other buffers.

[0078] The volume of the solvent used for the triptorelin solution may affect encapsulation efficiency and in vitro triptorelin release of the triptorelin-loaded microparticles. In particular, it was determined that reducing the volume of solvent led to decreased encapsulation efficiency and slower in vitro triptorelin release. A similar effect on in vivo release is expected. It also was found in some experiments that avoiding the use of sodium ions in the triptorelin solution was advantageous and led to, e.g., higher encapsulation efficiency, higher core loading, and slower in vitro triptorelin release of the triptorelin-loaded microparticles. Thus, in some embodiments, the solvent used for the triptorelin solution does not include sodium ions.

[0079] The concentration of triptorelin (e.g., triptorelin acetate) in the triptorelin solution may range from about 1% to about 60% w / v, including any value in between, such as from about 10% to about 40% w / v, from about 20% to about 60% w / v, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, and about 55% w / v. The concentration of the triptorelin in the triptorelin solution impacts microparticle properties and, therefore, may affect triptorelin release of the triptorelin-loaded microparticles. In some embodiments, when preparing microparticles that provide triptorelin release over a 1 -monthperiod, the concentration of triptorelin (e.g., triptorelin acetate) in the triptorelin solution may be from about 10% to about 30 % w / v. In some embodiments, when preparing microparticles that provide triptorelin release over a 1 -month period, the concentration of triptorelin (e.g., triptorelin acetate) in the triptorelin solution may be about 50 mM to about 250 mM. In some embodiments, when preparing microparticles that provide triptorelin release over a 3 -month period, the concentration of triptorelin (e.g., triptorelin acetate) in the triptorelin solution may be from about 15% to about 30 % w / v. In some embodiments, when preparing microparticles that provide triptorelin release over a 3 -month period, the concentration of triptorelin (e.g., triptorelin acetate) in the triptorelin solution may be about 100 mM to about 250 mM. In some embodiments, when preparing microparticles that provide triptorelin release over a 6-month period, the concentration of triptorelin (e.g., triptorelin acetate) in the triptorelin solution may be from about 15% to about 40% w / v. In some embodiments, when preparing microparticles that provide triptorelin release over a 6-month period, the concentration of triptorelin (e.g., triptorelin acetate) in the triptorelin solution may be about 100 mM to about 300 mM.

[0080] The triptorelin solution may include a viscosity enhancer, such as one or more of a polyol, e.g., glucose, fructose, lactose, sucrose, mannitol, dextran, or polyethylene glycol (PEG). Adding a viscosity enhancer, such as mannitol, to the triptorelin solution was found to affect in vitro triptorelin release of the triptorelin-loaded microparticles. It was expected that increasing the viscosity of the triptorelin solution would lead to slower in vitro triptorelin release; however, the opposite effect was observed. Specifically, it was found that adding mannitol (e.g., 20% w / v) led to a burst release of triptorelin followed by a faster in vitro triptorelin release. A similar high burst effect is expected in vivo. Without being bound by theory, it is postulated that mannitol was encapsulated in the microparticles, providing a very porous structure that led to faster in vitro triptorelin release. Additionally or alternatively, encapsulated mannitol could create an osmotic pressure gradient that promotes triptorelin release.FSH

[0081] As noted above, in some embodiments, the drug formulated as described herein is FSH. FSH is a 35.5 kDa glycoprotein heterodimer, consisting of two polypeptide units, alpha and beta. In some embodiments the FSH is human FSH. In some embodiments the FSH is human FSH isolated from a human donor. In some embodiments the FSH is recombinant FSH. In some embodiments the recombinant FSH has been produced / expressed in a mammalian cell line. In some embodiments the recombinant FSH has been produced / expressed in a Chinese Hamster Ovary cell. In some embodiments the recombinant FSH has been produced / expressed in a human cell line. Thus, the FSH formulated as described herein may be human FSH, including isolated human FSH (e.g., human urinary-derived FSH) or recombinant human FSH. In any embodiments where the drug is FSH, the FSH may be recombinant human FSH selected from follitropin alfa, follitropin beta, and follitropin delta. In specific embodiments, the FSH is follitropin delta. Follitropin delta is a human cell line-derived recombinant FSH approved as REKOVELLE® (follitropin delta), that can be produced by methods disclosed in WO 2009 / 127826.

[0082] It was surprisingly found that FSH can be formulated as described herein (e.g. encapsulated in microparticles as described herein) to obtain FSH-loaded microparticles and pharmaceutical compositions comprising them that achieve extended release of FSH in a biologically active form. This is particularly surprising for FSH, because it is known to be a “fragile” molecule, e.g., as a non-covalent dimer. It also was surprisingly found that FSH formulated as described herein exhibits low antigenicity. This is in contrast to un-encapsulated FSH, which has been reported to induce antibodies in mice.Solvent for FSH solution

[0083] Suitable solvents for FSH include solvents in which FSH is stable and solvents that stabilize FSH, physically and / or chemically. The solvent used for the FSH solution includes water, and may be an aqueous buffer solution including an aqueous buffer solution having a slightly acidic pH, such as a buffer solution comprising phosphoric acid, acetic acid, succinic acid, citricacid, or lactic acid, optionally including a salt of the corresponding acid anion to increase or provide buffering capacity, such as a phosphate salt, a citrate salt, an acetate salt, a succinate salt, or a lactate salt. Thus, the solvent used for the FSH solution may comprise an acid, such as phosphoric acid or citric acid or acetic acid or succinic acid or lactic acid or (and, optionally a buffering salt), that may be present in any amount that provides a FSH solution having the desired pH, such as a target pH of about 6.75, as discussed in more detail below.

[0084] The FSH solution may have a pH of from about 4.5 to about 9, including about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 6.75, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0, or any value therebetween. FSH dissociates relatively quickly below pH 4.5. In some embodiments, the FSH solution has a pH between 6 and 8, such as a pH from 6.5 to 7.0. In some embodiments, the FSH solution has a pH of about 6.75, including a pH of 6.75.

[0085] The FSH solution optionally may comprise one or more additional components, such as one or more additional components that may stabilize the FSH. In some embodiments the FSH solution comprises an antioxidant (such as methionine). Thus, in some embodiments, the FSH solution includes methionine, such as 0.5 mg / mL methionine. In some embodiments, the mass ratio of FSH to antioxidant (such as methionine) is about 1: 1. Additionally or alternatively, in some embodiments the FSH solution includes sodium sulfate, which may stabilize FSH.

[0086] The FSH solution may include a viscosity enhancer, such as one or more of a polyol, e.g., glucose, fructose, lactose, sucrose, mannitol, dextran, polyethylene glycol (PEG). As discussed above with reference to triptorelin microparticles, adding a viscosity enhancer, such as mannitol, to the drug solution may affect in vitro drug release from the drug-loaded microparticles.

[0087] The FSH solution may include a surfactant, such as one or more of Polysorbate 20, Polysorbate 80, and / or a poloxamer (e.g., Poloxamer 188). In some embodiments, the FSH solution includes Polysorbate 20 and / or Polysorbate 80 at a concentration of from about 0.0005 mg / mL to about 0.1 mg / mL. In some embodiments, the FSH solution includes a poloxamer (e.g., Poloxamer188) at a concentration of from about 0.0005 mg / mL to about 0.1 mg / mL. In other embodiments, the FSH solution includes functionally equivalent amounts of one or more other surfactant(s).

[0088] In some embodiments, the FSH solution comprises water, phosphoric acid (e.g., Na2HPO4 • 2H2O), methionine, and Polysorbate 20. In some embodiments, the FSH solution comprises water, phosphoric acid (e.g., Na2HPO4 • 2H2O), methionine, and Poloxamer 188.Solvent for Other Drugs

[0089] The solvent used to prepare the drug solution for other drugs typically will include water, and may be an aqueous buffer solution, including an aqueous buffer solution having a slightly acidic or acidic pH or slightly basic or basic pH, depending on the solubility and stability properties of the drug.

[0090] The drug solution may have any pH suitable for the drug, e.g., a pH at which the drug is suitably stable and soluble. As noted above, the pH of the drug solution may influence core loading, encapsulation efficiency, and drug release from the microparticles.

[0091] The volume of the solvent used for the drug solution may affect encapsulation efficiency and in vitro drug release. In particular, reducing the volume of solvent may lead to decreased encapsulation efficiency and slower in vitro drug release. A similar effect on in vivo release is expected.

[0092] The concentration of drug in the drug solution may be any suitable amount that achieves target microparticle properties. For example, the concentration of drug in the drug solution may range from about 0.01% to about 60% w / v, including any value in between, such as from about 1% to about 60% w / v, from about 10% to about 40% w / v, from about 20% to about 60% w / v, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, and about 55% w / v. In some embodiments, when preparing microparticles that provide drug release over a 1-week period, the concentration of drug in the drug solution may be from about 0.01% to about 55 % w / v, orabout 0.05 mM to about 500 mM. In some embodiments, when preparing microparticles that provide drug release over a 1 -month period, the concentration of drug in the drug solution may be from about 10% to about 30% w / v, or about 50 mM to about 250 mM. In some embodiments, when preparing microparticles that provide drug release over a 3 -month period, the concentration of drug in the drug solution may be from about 15% to about 30 % w / v, or about 100 mM to about 250 mM. In some embodiments, when preparing microparticles that provide drug release over a 6-month period, the concentration of drug in the drug solution may be from about 15% to about 40% w / v, or about 100 mM to about 300 mM.

[0093] The drug solution may include a viscosity enhancer such as one or more of a polyol, e.g., glucose, fructose, lactose, sucrose, mannitol, dextran, or polyethylene glycol (PEG). Adding a viscosity enhancer, such as mannitol, to the drug solution may affect in vitro drug release from the drug-loaded microparticles, as discussed above with reference to triptorelin and / or FSH.Polymers

[0094] Polymers suitable for forming microparticles as described herein include biodegradable polymers, such as biodegradable lactide / glycolide polymers, including polylactide (PLA), poly(D,L-lactide-co-glycolide) (PLGA) polymers, and mixtures thereof. Such polymers include RESOMER® polymers from Evonik Industries, EXPANSORB® polymers from Seqens Pharmaceuticals, PURASORB® polymers from Corbion, and VIATEL™ polymers from Ashland. Optionally, the polymers used to prepare microparticles as described herein are “purified” polymers, where “purified” refers to a reduced level of residual monomer content (e.g., less than about 0.11% of glycolide and less than about 0.04% of lactide, including less than 0.11% of glycolide and less than 0.04% of lactide).

[0095] Illustrative polymers, include but are not limited to, RESOMER® 502H, RESOMER® 502, RESOMER® Select 5050 DLG 2E-P, RESOMER® 503H, RESOMER® 503, RESOMER® Select 5050 DLG 3.8E-P, RESOMER® Select 4852 DLG 3.2E-P, RESOMER® Select 4852 DLG 4.4E-P, RESOMER® Select 5248 DLG 3.2E-P, RESOMER® Select 5248DLG 4.4E-P, RESOMER® 504H, RESOMER® 504, RESOMER® Select 5050 DLG 5E-P, RESOMER® RG752H, RESOMER® RG752S, RESOMER® RG753H, RESOMER® RG753S, RESOMER® Select 7525 DLG 3.8 E-P, RESOMER® Select 7723 DLG 3E-P, RESOMER® Select 7723 DLG 4.5E-P, RESOMER® Select 7327 DLG 3E-P, RESOMER® Select 7327 DLG 4.5E-P, RESOMER® RG756S, RESOMER® R202H, RESOMER® R202S, RESOMER® R203H, RESOMER® R203S, RESOMER® R205S, EXPANSORB® DLG 85-7A, EXPANSORB® DLG 85-7E, EXPANSORB® DL 100-5 A, EXPANSORB® DL 100-7A, VIATEL™ DLG 5002A, VIATEL™ DLG 5002E, VIATEL™ DLG 5003A, VIATEL™ DLG 5003E, VIATEL™ DLG 5005A, VIATEL™ DLG 5005 E, VIATEL™ DLG 7502A, VIATEL™ DLG 7502E, VIATEL™ DLG 7503A, VIATEL™ DLG 7503E, VIATEL™ DLG 7505A, VIATEL™ DLG 7505E, VIATEL™ Ultrapure DLG 5002A, VIATEL™ Ultrapure DLG 5002 E, VIATEL™ Ultrapure DLG 5003 A, VIATEL™ Ultrapure DLG 5003E, VIATEL™ Ultrapure DLG 5005A, VIATEL™ Ultrapure DLG 5005E, VIATEL™ Ultrapure DLG 7502A, VIATEL™ Ultrapure DLG 7502E, VIATEL™ Ultrapure DLG 7503A, VIATEL™ Ultrapure DLG 7503E, VIATEL™ Ultrapure DLG 7505A, VIATEL™ Ultrapure DLG 7505E, VIATEL™ DL 02A, VIATEL™ DL 02E, VIATEL™ DL 03A, VIATEL™ DL 03E, VIATEL™ DL 05A, VIATEL™ DLG 05 E, VIATEL™ Ultrapure DL 02A, VIATEL™ Ultrapure DL 02E, VIATEL™ Ultrapure DL 03A, VIATEL™ Ultrapure DL 03E, VIATEL™ Ultrapure DL 05A, and VIATEL™ Ultrapure DLG 05 E. The properties of the illustrative polymers are shown in the below table.Table A* Inherent viscosity was determined at a concentration of 0.5% polymer in CHCh at 25°C.

[0096] The polymer may be an ester-terminated PLA or PLGA polymer, a carboxyl-terminated PLA or PLGA polymer, or a polyethylene glycol (PEG)-terminated PLA or PLGA polymer. The polymer may have a lactide / glycolide ratio of from about 100:0 to about 50:50, including about 100:0, about 90:10, about 85:15, about 75:25, about 65:35, about 55:45, or about 50:50.

[0097] One or more PLGA polymers having a lactide:glycolide ratio of about 50:50, such as ester- terminated PLGA polymers, may be used. In some embodiments a single PLGA polymer is used, such as a single ester-terminated PLGA polymer, such as RG502, RG503 or RG504. In some embodiments, a mixture of two or more different PLGA polymers is used, including a mixture of two different ester-terminated PLGA polymers. An ester-terminated PLGA polymer, such as RG502, may particularly be used to prepare microparticles that provide drug release over a 1-week period. An ester-terminated PLGA polymer, such as RG503, may particularly be used to prepare microparticles that provide drug release over a 1 -month period. In some embodiments, the ester- terminated PLGA polymer used to prepare microparticles that provide drug release over a 1 weekperiod has a lower molecular weight than the ester-terminated PLGA polymer used to prepare microparticles that provide drug release over a 1 month period (such as RG502 versus RG503). Alternatively, a mixture of two or more ester-terminated PLGA polymers, such as RG503 and RG504, RG502 and RG503, and RG502 and RG504, may be used to prepare microparticles that provide drug release (e.g., triptorelin or FSH release) over a 1-month period, as illustrated in the examples below. Similar polymers may be used to prepare microparticles that provide drug release (e.g., triptorelin or FSH release) over a 6 week period. For any mixture of two ester-terminated PLGA polymers (e.g., a mixture of RG503 and RG504, RG502 and RG503, or RG502 and RG504), a ratio of the lower molecular weight polymer to the higher molecular weight polymer may be from about from about 10:90 to about 90:10, or any value in between, including about 80:20, about 75:25, about 70:30, about 60:40, about 50:50, about 40:60, or about 20:80. In some embodiments, a mixture of ester-terminated PLGA polymer RG503 and ester-terminated PLGA polymer RG504 is used in a ratio of from about 10:90 to about 90:10. In some embodiments, a mixture of ester-terminated PLGA polymer RG503 and ester-terminated PLGA polymer RG504 is used in a ratio of from about 85:15 to about 30:60, or any value in between, including about 80:20, about 60:40, about 50:50, or about 40:60, is used. In some embodiments, a mixture of ester- terminated PLGA polymer RG502 and ester-terminated PLGA polymer RG503 is used in a ratio of from about 10:90 to about 90:10. In some embodiments, a mixture of ester-terminated PLGA polymer RG502 and ester-terminated PLGA polymer RG503 is used in a ratio of from about 85: 15 to about 30:60, or any value in between, including about 80:20, about 60:40, about 50:50, or about 40:60, is used. In specific embodiments, a mixture of ester-terminated PLGA polymer RG503 and ester-terminated PLGA polymer RG504 is used in a ratio of from about 99: 1 to about 51 :49, or any value in between, including about 99:1, about 90: 10, about 80:20, about 70:30, about 60:40, about 55:45 and about 51:49. It was found that the residual monomer content of the polymer impacts the formation of drug-related impurities. Thus, in some embodiments, polymers having a low residual monomer content are used.

[0098] In some embodiments, such as for microparticles that provide triptorelin release over a 1- month period, the weight ratio of triptorelin to ester-terminated PLGA polymer(s) is from about4 / 100 to about 10 / 100, or higher. In some embodiments, such as for microparticles that provide FSH release over a 1 -week or a 1 -month period, the weight ratio of FSH to ester-terminated PLGA polymer(s) is from about 5 / 1000 to about 5 / 100, or higher.

[0099] The polymer may be an ester-terminated PLGA polymer with a lactide / glycolide ratio of from about 75:25. In some embodiments, the polymer is RG753S. In other embodiments the polymer is RG753H. In other embodiments the polymer is RG752S. An ester-terminated PLGA polymer with a lactide / glycolide ratio of from about 75:25, such as RG753S, may be used to prepare microparticles that provide drug release (e.g., triptorelin or FSH release) over a 3-month period, as illustrated in the examples below. In other embodiments the polymer for microparticles that provide drug release (e.g., triptorelin or FSH release) over a 3-month period is a mixture of RG753S and RG752S. In some embodiments, such as for microparticles that provide triptorelin release over a 3-month period, the ratio of triptorelin to ester-terminated PGLA polymer(s) is from about 5 / 100 to about 10 / 100, or higher. In some embodiments, such as for microparticles that provide FSH release over a 3-month period, the ratio of FSH to ester-terminated PGLA polymer(s) is from about 5 / 1000 to about 5 / 100, or higher.

[0100] A mixture of two or more PLA or PLGA polymers may be used, including a mixture of two PLA or PLGA polymers is used. In some embodiments, a mixture of two or more PLA polymers is used, including a mixture of two ester-terminated PLA polymers. The ratio of the lower molecular weight polymer to the higher molecular weight polymer may be from about 10:90 to about 90:10, or any value in between, including about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, or about 50:50. In some embodiments, a mixture of ester- terminated PLA polymer R203S and ester-terminated PLA polymer R205S is used in a ratio of from about 10:90 to about 90: 10. In some embodiments, a mixture of ester-terminated PLA polymer R203S and ester-terminated PLA polymer R205S is used in a ratio of from about 85: 15 to about 70:30, or any value in between, including about 85: 15, about 80:20, about 75:25, or about 70:30, is used. A mixture of two or more ester-terminated PLA polymers, such as R203S and R205S, may be used to prepare microparticles that provide drug release (e.g., triptorelin or FSH release) over a 6-month period, as illustrated in the examples below. In some embodiments, toprepare microparticles that provide drug release over a 6-month period, the mixture of two or more ester-terminated PLA polymers, such as R203S and R205S, may be used in a ratio of from about 85: 15 to about 70:30, or any value in between, including about 85:15, about 80:20, about 75:25, or about 70:30, is used. In some embodiments, such as for microparticles that provide triptorelin release over a 6-month period, the ratio of triptorelin to ester-terminated PLA polymers is from about 5 / 100 to about 15 / 100, or higher. In some embodiments, such as for microparticles that provide FSH release over a 6-month period, the ratio of FSH to ester-terminated PLA polymers is from about 5 / 1000 to about 10 / 100, or higher.

[0101] The in vivo degradation rate of the microparticles described herein is impacted by the ratio of lactide to glycolide in the polymer(s) and the molecular weight of the polymer(s). While polymers with a higher molecular weight generally exhibit lower degradation rates, it was surprisingly found that polymers with a lower molecular weight can be used to make microparticles as described herein that release a therapeutically effective amounts of drug over an extended period of time after administration, such as for at least 1 week, at least 1 month, at least 6 weeks, at least 3 months, or at least 6 months after injection.Solvent for Polymer(s)

[0102] The solvent for the polymer solution is a halogen-free organic solvent or solvent mixture that is partially miscible with water. The organic solvent or solvent mixture may have a solubility in water, or in the aqueous surfactant phase, with or without buffers, of between 1.5% w / w and 40% w / w at 20°C. Solvents with a water solubility greater than 40% w / w may be used in admixture with another solvent with a lower water solubility, such that the water solubility of the solvent mixture as a whole is less than 40% w / w. The solvent for the polymer solution may be acetone, ethyl formate (EtFo), ethyl acetate (EtAc), methyl acetate (MetAc), or methyl ethyl ketone (MEK). In some embodiments, the solvent for the polymer solution is ethyl acetate or acetone. In some embodiments, the solvent for the polymer solution is ethyl acetate. It was found that the solvent for the polymer solution could affect microparticle properties, including microparticle size, core loading, drug release, etc.

[0103] For embodiments, where the drug is a non-co valent multimer, such as a non-co valent dimer, such as FSH, the solvent for the polymer solution advantageously is one that does not denature or dissociate the non-covalent multimer. Examples of potentially denaturing solvents that likely would not be suitable include acetonitrile, dichloromethane (DCM), dimethyl sulfoxide (DMSO), and N-methyl-2-pyrrolidone (NMP). Butanone and ethyl formate also may be detrimental for fragile proteins, such as FSH. In some embodiments, such as where the drug is a non-covalent multimer, such as a non-covalent dimer, such as FSH, the solvent for the polymer solution is ethyl acetate or acetone.

[0104] For one week embodiments, a typical polymer solution may contain from about 10% w / v to about 30% w / v of dissolved polymer(s), such about 20% w / v polymer. For other embodiments (e.g., one month, three month, and six month embodiments), a typical polymer solution may contain from about 20% w / v to about 60% w / v of dissolved polymer(s), such as from about 30% w / v to about 50% w / v polymer, or from about 35% w / v to about 45% w / v polymer.

[0105] In some embodiments, such as to prepare microparticles that provide drug release over a 1-week period, the polymer solution contains from about 20% w / v to about 50% w / v of dissolved polymer(s), including about 20% w / v, about 25% w / v, about 30% w / v, about 35% w / v, about 40% w / v, about 45% w / v or about 50% w / v, and including any amount in between. In some embodiments, such as to prepare microparticles that provide drug release over a 1 -month period, the polymer solution contains from about 20% w / v to about 45% w / v of dissolved polymer(s), including about 20% w / v, about 25% w / v, about 30% w / v, about 35% w / v, about 40% w / v, including and any amount in between. In some embodiments, to prepare microparticles that provide drug release over a 1 -month period, the polymer solution contains about 30% w / v of dissolved polymer(s). In some embodiments, to prepare microparticles that provide drug release over a 3-month period, the polymer solution contains from about 30% w / v to about 50% w / v of dissolved polymer(s), including about 30% w / v, about 35% w / v, about 40% w / v, or about 45% w / v, including and any amount in between. In some embodiments, to prepare microparticles that provide drug release over a 3-month period, the polymer solution contains about 40% w / v of dissolved polymer(s). In some embodiments, to prepare microparticles that provide drug releaseover a 6-month period, the polymer solution contains from about 30% w / v to about 50% w / v of dissolved polymer(s), including about 30% w / v, about 35% w / v, about 40% w / v, or about 45% w / v, including and any amount in between. In some embodiments, to prepare microparticles that provide drug release over a 6-month period, the polymer solution contains about 40% w / v of dissolved polymer(s).Aqueous Surfactant Solution

[0106] The aqueous surfactant solution used for phase inversion may comprise a cationic, anionic, or non-ionic surfactant. The surfactant may be a poloxamer surfactant (e.g., Poloxamer 188). Additionally or alternatively, the surfactant may be a polyvinylalcohol (e.g., MOWIOL®) surfactant. Additionally or alternatively, the surfactant may be a POLOXAMNINE® polyalkoxylated symmetrical block polymer of ethylene diamine surfactant, a polyethylene glycol alkyl ether, a polysorbate (e.g., TWEEN® or SPAN®), a sucrose ester (e.g., SISTERNA® or Ryoto Sugar Ester), a fatty alcohol polyglycoside, 3-[(3-cholamidopropyl)dimethylammonio]-l- propanesulfonate (CHAPS), 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-l- propanesulfonate (CHAPSO), decyl-P-D-glycopyranoside, decyl-P-D maltopyranoside, dodecyl- P-D-maltopyranoside, sodium oleate, polyvinyl alcohol, polyoxylated fatty acid ethers (e.g., BRIJ®), polyethylene glycol tert-octylphenyl ether (e.g., Triton® X-l 00), and mixtures of any two or more thereof.

[0107] As noted above, the surfactant may be or comprise a poloxamer, which is a block copolymer of polyoxyethylene (also referred to as poly(ethylene oxide) or poly(ethylene glycol) blocks) and poly oxypropylene (also referred to as polypropylene oxide) or polypropylene glycol) blocks). A poloxamer typically has a central polyoxypropylene block flanked on each side by polyoxyethylene blocks. In accordance with conventional nomenclature for a poloxamer, the first two numbers x 100 correspond to the approximate molecular mass of the polyoxypropylene block, while the last number x 10 corresponds to the relative content of polyoxyethylene. Thus, Poloxamer 188 is a poly oxy ethylene-polyoxypropylene-poly oxy ethylene tri-block polymer with an approximate poly oxypropylene molecular mass of 1800 g / mol and a 80% polyoxyethylenecontent. In some embodiments, the surfactant is or comprises Poloxamer 188. In some embodiments, the aqueous surfactant solution used for phase inversion contains about 1% w / v to about 10% w / v of a poloxamer, such as Poloxamer 188, or a functionally equivalent amount of a different poloxamer, or of a mixture of, e.g., Poloxamer 188 and a different poloxamer. In some embodiments, to prepare microparticles that provide drug release over a 3 -month period, the aqueous surfactant solution used for phase inversion contains about 3% w / v of a poloxamer, such as Poloxamer 188, or a functionally equivalent amount of a different poloxamer, or a mixture of, e.g., Poloxamer 188 and a different poloxamer. It was found that the amount of surfactant used could affect microparticle properties, including size, core loading, drug release, etc.

[0108] As noted above, the surfactant may or comprise be a polyvinylalcohol. Suitable examples of polyvinylalcohols include those having a molecular weight of about 130,000, such as MOWIOL® 18-88. In some embodiments, the aqueous surfactant solution used for phase inversion contains about 0.5% w / v to about 5% w / v of a polyvinyl alcohol, such as MOWIOL® 18-88. In some embodiments, to prepare microparticles that provide triptorelin release over a 1- month period, the aqueous surfactant solution used for phase inversion contains about 1% w / v or about 2% w / v of a polyvinyl alcohol, such as MOWIOL® 18-88.

[0109] The aqueous surfactant solution used for phase inversion may comprise a viscosity modifier, such as one or more of a polyol, e.g., glucose, fructose, lactose, sucrose, mannitol, dextran, or polyethylene glycol. The aqueous surfactant may comprise polyethylene glycol, such as those having a molecular weight of about 6000 g / mol or less, including about 6000 g / mol, about 5000 g / mol, about 4000 g / mol, about 3000 g / mol, about 2000 g / mol, and about 1000 g / mol. Thus, the aqueous surfactant solution may contain from about 0% w / v to about 50% w / v, including from about 1% w / v to about 50% w / v, such as from about 10% w / v to about 40% w / v, or from about 20% w / v to about 30% w / v viscosity modifier. In some embodiments, the surfactant solution used for phase inversion contains about 10% w / v, about 15% w / v, about 20% w / v, about 25% w / v, about 30% w / v, about 35% w / v, about 40% w / v, about 45% w / v, or about 50% w / v, sucrose, or any amount there between, or an equivalent amount of another viscosity modifier. In some embodiments the surfactant solution used for phase inversion contains about 25% w / v sucrose,including 25% w / v sucrose, or an equivalent amount of another viscosity modifier. In some embodiments, the surfactant solution used for phase inversion contains about 5% w / v, about 10% w / v, about 15% w / v, about 20% w / v, about 25% w / w, or about 30% w / v PEG (such as PEG3000, PEG3350, PEG4000 or PEG6000), or any amount there between, or an equivalent amount of another viscosity modifier. In some embodiments, the surfactant solution used for phase inversion contains about 25% w / v PEG (such as PEG3000, PEG3350, PEG4000 or PEG6000), including 25% w / v PEG (such as PEG3000, PEG3350, PEG4000 or PEG6000) or any amount there between, or an equivalent amount of another viscosity modifier. In some embodiments, the aqueous surfactant solution does not comprise a viscosity modifier.

[0110] Additionally or alternatively, the aqueous surfactant solution may comprise a salt, such as an alkaline salt (e.g., a salt of an alkali metal such a sodium chloride). Thus, the aqueous surfactant solution may contain from about 0% w / v to about 20% w / v, including from about 1% w / v to about 20% w / v, or from about 5% w / v to about 15% w / v an alkaline salt (e.g., sodium chloride). In some embodiments, the surfactant solution used for phase inversion contains about 2% w / v, about 5% w / v, about 10% w / v, or about 15% w / v, sodium chloride, or any amount there between, or an equivalent amount of another salt. In some embodiments the surfactant solution used for phase inversion contains about 10% w / v sodium chloride, including 10% w / v sodium chloride, or an equivalent amount of another salt. In some embodiments the surfactant solution used for phase inversion contains about 5% w / v sodium chloride, including 5% w / v sodium chloride, or an equivalent amount of another salt. In some embodiments the surfactant solution used for phase inversion contains about 15% w / v sodium chloride, including 15% w / v sodium chloride, or an equivalent amount of another salt. In some embodiments the surfactant solution used for phase inversion contains about 2% w / v sodium chloride, including 2% w / v sodium chloride, or an equivalent amount of another salt. It was found that adding sodium chloride in an appropriate amount (including, e.g., about 5% w / v) to the aqueous surfactant solution used for phase inversion increased the mean particle size of the microparticles without affecting particle morphology. It also was found that adding sodium chloride in an appropriate amount (including, e.g., about 5% w / v) led to slower in vitro triptorelin release of the triptorelin microparticles. In someembodiments, the aqueous surfactant solution does not comprise a salt. In some embodiments, the aqueous surfactant solution comprises about 2% w / v sodium chloride and optionally, 25% w / v sucrose.

[0111] The aqueous surfactant solution may comprise a co-solvent that is water miscible and a solvent for the solvent of the polymer solution, but not a solvent for the polymer(s). When a cosolvent is used, the aqueous surfactant may extract more of the polymer solvent from the polymer solution compared to an equivalent volume of aqueous surfactant solution, which may reduce the volume fraction of aqueous surfactant required to form the microparticle suspension, thus reducing the amount of surfactant to be removed from the microparticle suspension. The amount of cosolvent used can be selected based on the polymer and polymer solvent. Typically, about 1-40 w / w % co-solvent may be added. Suitable co-solvents include, but are not limited to, alcohols such as ethanol.

[0112] Additionally or alternatively, the aqueous surfactant solution may comprise an aqueous buffer, such as a citrate buffer, at a specific pH (e.g., pH 6.5). The buffer concentration may alter the solubility of the polymer solvent in the surfactant solution used for phase inversion. Thus, it may have impact on the drug release performance of the drug-loaded microparticles. Moreover, the buffer concentration may have an impact on the solubility of non-encapsulated drug molecules (e.g., triptorelin or FSH) which may be present in the surfactant solution after phase inversion. Additionally or alternatively, the solubility of the drug may be a function of pH, which would could be exploited to change the solubility of the drug in the surfactant solution by altering the pH. As one specific example, in the context of encapsulated FSH, the pH of the surfactant solution used for phase inversion may be adjusted to pH 4.5 in order to decrease the solubility of FSH in the surfactant solution.

[0113] It was determined that the surfactant phase composition may impact properties of the microparticles that impact drug release. For example, in the production of triptorelin microparticles, it was determined that adding a viscosity modifier such as sucrose to the aqueous surfactant solution used for phase inversion at 50% w / v decreased the mean particle size of thetriptorelin-loaded microparticles. When no viscosity modifier was added, a lower viscosity was obtained and sponge-like microparticles as well as microcapsules were formed. In the production of FSH microparticles, it was determined that adding a viscosity modifier such as polyethylene glycol to the aqueous surfactant solution used for phase inversion at 25% w / v decreased the mean particle size of the FSH-loaded microparticles. When a lower concentration of polyethylene glycol was used, a lower viscosity was obtained, and sponge-like microparticles and / or microcapsules were formed. Moreover, the solubility of FSH in aqueous solutions was studied, and it was found that polyethylene glycol significantly decreased the solubility of FSH, which could reduce or eliminate leakage of FSH from microparticles during solvent removal by solvent evaporation.Other Components

[0114] One or more or all of the drug solution, polymer solution and aqueous surfactant solution may include one or more additional components suitable for use in a pharmaceutical composition, such as bulking agents, buffering agents, chelating agents, preservatives antioxidants, co-solvents, and cryoprotectants. Such additional components may be used in amounts that do not unduly negatively impact target properties of the microparticle, such as, for example, drug loading and release properties.Method of Manufacture

[0115] Drug-loaded microparticles as described herein can be prepared by any suitable method. Drug-loaded microparticles as described herein can be prepared by combining a drug solution as described herein with a polymer solution as described herein, and then adding an aqueous surfactant solution as described herein to trigger phase inversion. As depicted in FIG. 1 , a drug solution may be prepared, e.g., by dissolving drug in an aqueous buffer solution as described herein. A polymer solution may be prepared, e.g., by dissolving polymer(s) in a halogen-free organic solvent or solvent mixture that is partially miscible with water as described herein. As depicted in FIG. 1, the drug solution is added to the polymer solution in a vessel with mixing to obtain a dispersion (e.g., emulsion) of the drug solution in the polymer solution. Then, a phaseinversion solution comprising a surfactant is added to the vessel with mixing to induce a phase inversion which in turn leads to the formation of a suspension of polymer microparticles comprising drug in the aqueous surfactant solution. Although FIG. 1 refers to a triptorelin solution, other drug-loaded microparticles, including FSH-loaded microparticles, can be prepared by the same process.

[0116] Provided herein is a method of making drug-loaded microparticles comprising:(a) adding (i) a drug solution comprising a drug in an aqueous solvent to (ii) a polymer solution comprising one or polymers selected from ester-terminated poly(D,L-lactide-co- glycolide) (PLGA) polymers having a lactide: glycolide ratio of about 50:50, ester-terminated PLGA polymers having a lactide:glycolide ratio of about 75:25, and ester-terminated polylactide (PLA) polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the drug solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of drug- loaded microparticles comprising drug and the one or more polymers, e.g., the one or more polymers selected from ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50, ester-terminated PLGA polymers having a lactide: glycolide ratio of about 75:25, and ester- terminated PLA polymers.

[0117] Provided in one aspect are methods of making triptorelin-loaded microparticles that provide triptorelin release over a 1-week period. For an example, a method for making triptorelin-loaded microparticles may comprise:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous buffer solution to (ii) a polymer solution comprising a mixture of one or more ester-terminated PLGA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of a viscosity modifier (such as polyethylene glycol) and an buffer salt(such as sodium citrate), and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the one or more PLGA polymers.

[0118] Provided in another aspect are methods of making triptorelin-loaded microparticles that provide triptorelin release over a 1-week period. For an example, a method for making triptorelin- loaded microparticles may comprise:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising an ester-terminated PLGA polymer in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLGA polymer.

[0119] Provided in another aspect are methods of making triptorelin-loaded microparticles that provide triptorelin release over a 1 -month period. For an example, a method for making triptorelin-loaded microparticles may comprise:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising a mixture of one or more ester-terminated PLGA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of a viscosity modifier (such as sucrose) and an alkaline salt (such as sodium chloride), and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the one or more PLGA polymers.

[0120] Provided in another aspect are methods of making triptorelin-loaded microparticles that provide triptorelin release over a 1 -month period. For an example, a method for making triptorelin- loaded microparticles may comprise:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising a mixture of two different ester-terminated PLGA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLGA polymers.

[0121] Provided in another aspect are methods of making triptorelin-loaded microparticles that provide triptorelin release over a 1 -month period. For an example, a method for making triptorelin- loaded microparticles may comprise:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising an ester-terminated PLGA polymer in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of a viscosity modifier (such as sucrose and polyethylene glycol) and an alkaline salt (such as sodium chloride), and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLGA polymer.

[0122] Provided in another aspect are methods of making triptorelin-loaded microparticles that provide triptorelin release over a 3 -month period. For an example, a method for making triptorelin- loaded microparticles may comprise:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising an ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25 in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of a viscosity modifier (such as sucrose) and an alkaline salt (such as sodiumchloride), and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLGA polymer.

[0123] Provided in another aspect are methods of making triptorelin-loaded microparticles that provide triptorelin release over a 6-month period. For an example, a method for making triptorelin- loaded microparticles may comprise:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising a mixture of two different ester-terminated PLA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of a viscosity modifier (such as sucrose) and an alkaline salt (such as sodium chloride) to the dispersion, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLA polymers.

[0124] Provided in another aspect are methods of making FSH-loaded microparticles that provide FSH release over a 1-week period. For an example, a method for making FSH-loaded microparticles may comprise:(a) adding (i) a FSH solution comprising FSH in an aqueous buffer solution to (ii) a polymer solution comprising a mixture of one or more ester-terminated PLGA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of a viscosity modifier (such as polyethylene glycol) and an buffer salt (such as sodium citrate), and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the one or more PLGA polymers.

[0125] Provided in another aspect are methods of making FSH-loaded microparticles that provide FSH release over a 1-week period. For an example, a method for making FSH-loaded microparticles may comprise:(a) adding (i) a FSH solution comprising FSH in an aqueous acidic solvent to (ii) a polymer solution comprising an ester-terminated PLGA polymer in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLGA polymer.

[0126] Provided in another aspect are methods of making FSH-loaded microparticles that provide FSH release over a 1 -month period. For an example, a method for making FSH-loaded microparticles may comprise:(a) adding (i) a FSH solution comprising FSH in an aqueous buffer solution to (ii) a polymer solution comprising a mixture of one or more ester-terminated PLGA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of a viscosity modifier (such as polyethylene glycol) and an buffer salt (such as sodium citrate), and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the one or more PLGA polymers.

[0127] Provided in another aspect are methods of making FSH-loaded microparticles that provide FSH release over a 1 -month period. For an example, a method for making FSH-loaded microparticles may comprise:(a) adding (i) a FSH solution comprising FSH in an aqueous buffer solution to (ii) a polymer solution comprising a mixture of two different ester-terminated PLGA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLGA polymers.

[0128] Provided in another aspect are methods of making FSH-loaded microparticles that provide FSH release over a 1 -month period. For an example, a method for making FSH-loaded microparticles may comprise:(a) adding (i) a FSH solution comprising FSH in an aqueous acidic solvent to (ii) a polymer solution comprising an ester-terminated PLGA polymer in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLGA polymer.

[0129] Provided in another aspect are methods of making FSH-loaded microparticles that provide FSH release over a 3-month period. For an example, a method for making FSH-loaded microparticles may comprise:(a) adding (i) a FSH solution comprising FSH in an aqueous buffer solution solvent to (ii) a polymer solution comprising an ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25 in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of a viscosity modifier (such as polyethylene glycol) and a buffer salt (such as sodium citrate), and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLGA polymer.

[0130] Provided in another aspect are methods of making FSH-loaded microparticles that provide FSH release over a 6-month period. For an example, a method for making FSH-loaded microparticles may comprise:(a) adding (i) a FSH solution comprising FSH in an aqueous buffer solution to (ii) a polymer solution comprising a mixture of two different ester-terminated PLA polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution (dispersion step); and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of a viscosity modifier (such as polyethylene glycol) and an buffer salt (such as sodium citrate) to the dispersion, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLA polymers.

[0131] For all methods provided herein, the volume fractions of each of the polymer solution and the drug solution are selected such that a suspension of microparticles is formed immediately upon combining them with the aqueous surfactant solution.

[0132] Several process parameter were found to impact properties of the microparticles (such as microparticle size and release properties), including temperature at dispersion step (a) (which is generally carried out at ambient temperatures, e.g., at about 20-25°C), mixing time and mixing speed at dispersion step (a), and phase inversion solution transfer rate in step (b). The specific impact of these parameters may vary from drug to drug, but the principles discussed herein are believed to be applicable across different drugs.

[0133] As described above, there are two mixing times - the mixing time of dispersion step (a) to provide a dispersion of the drug solution in the polymer solution, and the mixing time of step (b) to obtain a suspension of drug-loaded microparticles comprising drug and at least one polymer. The mixing time of step (a) may be selected and adjusted as needed to disperse the drug in the polymer solution and partially evaporate the solvent from the polymer solution. The mixing time of step (b) may be selected and adjusted as needed to obtain a suspension of drug-loaded microparticles, and typically is shorter than the mixing time of step (a).

[0134] Step (a) (e.g., preparing a dispersion of the drug solution in the polymer solution), may be performed under a nitrogen stream (or under a stream of any other inert gas). It was determined that increasing the nitrogen gas flow rate led to the formation of larger microparticles with a less open structure that have higher core loadings and slower in vitro release rates. A similar impact on in vivo release is expected.

[0135] The stirring speed for the mixing of step (a) and step (b) may be selected independently, but may conveniently be the same speed, and may be any suitable speed, including from about 500 rpm to about 8000 rpm, from about 800 rpm to about 2400 rpm, or from about 4000 rpm to about 8000, and may vary with the scale of production.

[0136] In general, increasing the stirring speed for the mixing of at least one of step (a) and step (b) may lead to the formation of smaller microparticles, which in turn may lead to faster in vitro and in vivo drug release. It also was determined that increasing the stirring speed may lead to higher core loading and improved encapsulation efficiency. It also was determined that adjusting the stirring speed and mixing time may lead to an increase in the mean particle size of the microparticles, and, consequently slower in vitro and in vivo drug release.

[0137] The phase inversion solution transfer rate may be any suitable rate to achieve the intended phase transfer and microparticle formation, and may vary with the scale of production. It was determined that increasing the phase inversion solution transfer rate led to the formation of larger microparticles that have higher core loadings and slower in vitro release rates. A similar effect on in vivo release rate is expected.

[0138] To remove excess excipients after microparticle formation, a wash phase can be added to the microparticle suspension after step (b) (e.g., after obtaining the suspension of drug-loaded microparticles). A wash phase typically is an aqueous solution, and may comprise an aqueous buffer, such as an aqueous acidic buffer (such as a citrate buffer at pH 4.5 to pH 6.5) or an aqueous buffer with a pH greater than 7.0 (such as a TRIS buffer at pH 7.4), a surfactant, such as a polyethylene-polypropylene glycol surfactant (e.g., a poloxamer such as POLOXAMER® 188) or polyvinylalcohol, and a salt (e.g., sodium chloride). In some embodiments, the wash phase includes a viscosity modifier, such as PEG, as described above. Thus, in some embodiments, after step (b), an aqueous wash phase is added. Without being bound by theory, it is believed that the wash phase may act as a colloidal stabilizer for the microparticles and also as an additional extraction medium for any residual organic polymer solvent still present in the microparticles.

[0139] The organic solvent component of the polymer solvent may be removed, e.g., by vacuum and / or elevated temperature. In general, the evaporation of organic solvents is increased by decreasing vacuum pressure and elevated temperature. In some instances, the formation of peptide related impurities is dependent on temperature. Solvent evaporation under vacuum conditions increases the transition of the organic solvent (e.g., ethyl acetate) from the microparticles into the continuous phase of the suspension, leading to complete hardening of the microparticles. In some embodiments, the microparticle suspension is agitated by means of vibrational mixing during solvent evaporation under vacuum conditions. In some embodiments, after step (b), organic solvent (e.g., ethyl acetate) is removed by vacuum.

[0140] The microparticles may be removed from the suspension by any suitable means, such as centrifugation and / or tangential flow / cross flow filtration. For instance, in some embodiments, after step (b), the microparticles are separated from the suspension, such as by centrifugation or filtration. In some embodiments, the microparticles are separated from the suspension after solvent removal by vacuum. In some embodiments, tangential flow / cross flow filtration is used to concentrate the microparticles in the process chamber and to subsequently wash the microparticles by water to remove excess surfactant from the suspension.

[0141] The microparticles can be subject to further processing, such as freeze-drying or spray drying or drying in a fluidized bed. In some embodiments, the microparticles are lyophilized, typically with protective excipients, such as one or more of ascorbic acid, an amino acid, mannitol, and tocopherol acetate.

[0142] The microparticles may be sterilized by any suitable means, such as by ionizing radiation, such as y-irradiation. In some embodiments, the microparticles may be sterilized by y-irradiation at a dose of about 15 kGy to 45 kGy, including 20 kGy ± 10% (e.g., 18 kGy - 22 kGy), 25 kGy ± 10% (e.g., 22.5 kGy - 27.5 kGy), 27.75 kGy ± 10% (e.g., 25 kGy - 30.5 kGy), 35 kGy ± 10% (e.g., 31.5 kGy - 38.5 kGy), 40 kGy ± 10% (e.g., 36 kGy - 44 kGy), and 45 kGy ± 10% (e.g., 41.5 kGy - 49.5 kGy). In some embodiments the microparticles may be sterilized by e-beam (13- irradiation). It was surprisingly found that drug-loaded microparticles as described herein that havebeen subjected to gamma irradiation are stable, e.g., against degradation of the drug. This was particularly surprising for FSH-loaded microparticles, because FSH is generally unstable and subject to degradation. Thus, y-irradiatied FSH-loaded microparticles as described herein may advantageously exhibit greater stability of the FSH and represent an advantageous embodiment. Nevertheless, in some embodiments the microparticles may be manufactured aseptically, i.e., without final sterilization by ionizing radiation.

[0143] The encapsulation efficiency of the method described herein typically is at least 80% by weight, including from 90 to 95% by weight, based on the weight of triptorelin.Microparticles Characteristics

[0144] Provided in one aspect is a drug-loaded microparticle comprising:(a) a drug;(b) a polymer component comprising one or more polymers selected from ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymers having a lactide: glycolide ratio of about 50:50, ester-terminated PLGA polymers having a lactide: glycolide ratio of about 75:25, and ester- terminated polylactide (PLA) polymers;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 1 week after administration by subcutaneous or intramuscular injection. In some embodiments, the microparticle releases the drug in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection. In some embodiments, the microparticle releases the drug in vivo for a period of at least 3 months after administration by subcutaneous or intramuscular injection. In some embodiments, the microparticle releases the drug in vivo for a period of at least 6 months after administration by subcutaneous or intramuscular injection.

[0145] In some embodiments, the drug is one or more selected from a small molecule drug, a peptide drug, and a protein drug. In some embodiments, the drug is one or more selected from triptorelin, follicle stimulating hormone (FSH), leuteinizing hormone (LH), human chorionicgonadotrophin (hCG), atosiban, insulin, abiraterone, barusiban, degarelix, desmopressin, ganirelix (e.g., ganirelix acetate), gemcitabine, gonadorelin (e.g., gonadorelin acetate), menotropin or human menopausal gonadotropin (hMG), merotocin, progesterone, quinagolide, somatostatin, somatropin, and tolterodine. In some embodiments, the drug is triptorelin. In some embodiments, the drug is follicle stimulating hormone (FSH). In some embodiments, the drug comprises two or more drugs. In some embodiments, the drug comprises FSH and LH and / or hCG.

[0146] Provided in aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin FSH in vivo for a period of at least 1 week after administration by subcutaneous or intramuscular injection.

[0147] Provided in one aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) one or more polymers selected from ester-terminated PLGA polymers having a lactide: glycolide ratio of about 50:50, an ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25, and two different ester-terminated PLA polymers;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0148] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0149] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50, such as RG503;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0150] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50, such as RG503; and(c) a poloxamer, such as Poloxamer 188, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0151] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50, such as RG503; and(c) a poloxamer, such as Poloxamer 188; and(d) one or both of sodium chloride and polyethylene glycol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0152] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50, such as RG503; and(c) a polyvinylalcohol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0153] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50, such as RG503; and(c) a polyvinylalcohol; and(d) one or both of sodium chloride and polyethylene glycol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0154] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) a mixture of two different ester terminated PLGA polymers having a lactide:glycolide ratio of about 50:50, such as RG504 and RG503;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0155] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) a mixture of two different ester terminated PLGA polymers having a lactide:glycolide ratio of about 50:50, wherein the two different ester-terminated polylactide PLGA polymers are RG504 and RG503; and(c) a poloxamer, such as Poloxamer 188; and(d) one or both of sodium chloride and a polyethylene glycol; wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0156] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) a mixture of two different ester terminated PLGA polymers having a lactide:glycolide ratio of about 50:50, wherein the two different ester-terminated polylactide PLGA polymers are RG504 and RG503; and(c) a polyvinylalcohol; and(d) one or both of sodium chloride and polyethylene glycol; wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0157] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) a mixture of three different ester-terminated PLGA polymers having a lactide: glycolide ratio of about 50:50, such as RG504 and RG503 and RG502;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0158] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) a mixture of three different ester terminated PLGA polymers having a lactide: glycolide ratio of about 50:50, wherein the three different ester-terminated poly lactide PLGA polymers are RG504 and RG503 and RG502; and(c) a poloxamer, such as Poloxamer 188; wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0159] Provided in one aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) an ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 3 months after administration by subcutaneous or intramuscular injection.

[0160] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin acetate;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 75:25, such as RG753S;(c) a poloxamer, such as Poloxamer 188; and(d) one or both of sodium chloride and sucrose, wherein the microparticle releases triptorelin in vivo for a period of at least 3 months after administration by subcutaneous or intramuscular injection.

[0161] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin;(b) two different ester-terminated PLA polymers;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol,wherein the microparticle releases triptorelin in vivo for a period of at least 6 months after administration by subcutaneous or intramuscular injection.

[0162] Provided in another aspect are triptorelin-loaded microparticles comprising:(a) triptorelin acetate;(b) two different ester-terminated polylactide (PLA) polymers, wherein the two different ester-terminated polylactide PLA polymers are R203S and R205S;(c) a poloxamer, such as Poloxamer 188; and(d) one or both of sodium chloride and sucrose, wherein the microparticle releases triptorelin in vivo for a period of at least 6 months after administration by subcutaneous or intramuscular injection.

[0163] Provided in one aspect are FSH-loaded microparticles comprising:(a) FSH;(b) one or more polymers selected from ester-terminated PLGA polymers having a lactide: glycolide ratio of about 50:50, an ester-terminated PLGA polymers having a lactide: glycolide ratio of about 75:25, and two different ester-terminated PLA polymers;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 1 week after administration by subcutaneous or intramuscular injection.

[0164] Provided in one aspect are FSH-loaded microparticles comprising:(a) FSH;(b) one or more polymers selected from ester-terminated PLGA polymers having a lactide: glycolide ratio of about 50:50, an ester-terminated PLGA polymers having a lactide: glycolide ratio of about 75:25, and two different ester-terminated PLA polymers;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol,wherein the microparticle releases FSH in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection, optionally wherein the microparticle releases FSH in vivo for a period of at least 6 weeks after administration by subcutaneous or intramuscular injection.

[0165] Provided in another aspect are FSH-loaded microparticles comprising:(a) FSH;(b) one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0166] Provided in another aspect are FSH-loaded microparticles comprising:(a) FSH;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50, such as RG503;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 1 month after administration by subcutaneous or intramuscular injection.

[0167] Provided in another aspect are FSH-loaded microparticles comprising:(a) FSH;(b) one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol,wherein the microparticle releases FSH in vivo for a period of at least 6 weeks after administration by subcutaneous or intramuscular injection.

[0168] Provided in another aspect are FSH-loaded microparticles comprising:(a) FSH;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50, such as RG502;(c) a surfactant; and(d) optionally one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 6 weeks after administration by subcutaneous or intramuscular injection.

[0169] Provided in one aspect are FSH-loaded microparticles comprising:(a) FSH;(b) an ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 3 months after administration by subcutaneous or intramuscular injection.

[0170] Provided in another aspect are FSH-loaded microparticles comprising:(a) FSH;(b) an ester-terminated PLGA polymer having a lactide:glycolide ratio of about 75:25, such as RG753S;(c) a poloxamer, such as Poloxamer 188; and(d) one or both of sodium chloride and polyethylene glycol, wherein the microparticle releases FSH in vivo for a period of at least 3 months after administration by subcutaneous or intramuscular injection.

[0171] Provided in another aspect are FSH-loaded microparticles comprising:(a) FSH;(b) two different ester-terminated PLA polymers;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 6 months after administration by subcutaneous or intramuscular injection.

[0172] Provided in another aspect are FSH-loaded microparticles comprising:(a) FSH;(b) two different ester-terminated PLA polymers, wherein the two different ester- terminated polylactide PLA polymers are R203S and R205S;(c) a poloxamer, such as Poloxamer 188; and(d) one or both of sodium chloride and polyethylene glycol, wherein the microparticle releases FSH in vivo for a period of at least 6 months after administration by subcutaneous or intramuscular injection.

[0173] Residual Content: The microparticles described herein may have a residual content of one or more of the components used to make the microparticles, one or more of the components present in one or more of the polymer solution, the drug solution, the phase inversion solution, and the wash phase, such as solvent of the polymer solution; acid and / or buffering salt of the aqueous acid solution used in the triptorelin solution; solvent, surfactant, alkaline salt, viscosity modifier, etc., of the phase inversion solution; surfactant or buffering salt of the wash phase, etc. For example, microparticles as described herein may have a residual content of one or more of (i) organic solvent of the polymer solution, from about 50 ppm to about 20000 ppm; (ii) acid and / or buffering salt of the aqueous acid solution used in the drug solution, from about 0.01% w / w to about 1.0% w / w; (iii) viscosity modifier, from about 0.1% w / w to about 5% w / w; (iv) surfactant, from about 0% w / w to about 4% w / w; (v) alkaline salt, from about 0% to about 0.5% w / w; (vi) buffering salt of the phase inversion / surfactant solution or wash phase, from about 0.01% w / w to about 1.0% w / w. For instance, microparticles, prepared using the components described in the examples may have a residual content of one or more or each of ethyl acetate, acetic acid / acetate,sucrose, NaCl, citric acid / citrate, a poloxamer (e.g., Poloxamer 188) or polyvinylalcohol, and polyethylene glycol. For example, microparticles prepared using the components described in the examples may have a residual acetic acid content of from about 0.01% w / w to about 1.0% w / w; a residual sucrose content of from about 0.20% w / wto about 5.0% w / w; a residual citric acid content of from about 0.01% w / w to about 1.0% w / w; a residual ethyl acetate content of from about 50 ppm to about 20000 ppm; a residual surfactant content of from about 0% w / w to about 4% w / w; a residual polyethylene glycol content from about 0.20% w / w to about 5% w / w; and / or a residual NaCl content of about < 0.5% w / w.

[0174] Microparticle size: As discussed above, the size of the microparticles can be adjusted by adjusting various process parameters. For instance, microparticles as described herein may have a mean particle size of from about 20 pm to about 180 pm, including from about 30 pm to about 150 pm, such as from about 30 pm to about 120 pm, including from about 30 pm to about 80 pm, including from 20 pm to 180 pm, from 30 pm to 150 pm, from 30 pm to 120 pm, and from 30 pm to 80 pm.

[0175] Target Dose: The microparticles as described herein may be formulated in a composition as described herein to deliver a target dose of the drug being formulated over a 6-month period, over a 3 -month period, over a 1 -month period, over a 1-week period. For triptorelin, the 6-month target dose for a typical human patient may be 22.5 mg; the 3-month target dose for a typical human patient may be 11.25 mg; the 1-month target dose for a typical human patient may be 3.75 mg, and the 1-week target dose for a typical human patient may be about 1 mg. For FSH, e.g., follitropin delta, a target dose of the drug being formulated over a 6-month period, over a 3-month period, over a 6- week period, over a 1-month period, over a 1-week period may be a dose that delivers about 12 pg per day. For FSH, e.g., follitropin delta, the 6-month target dose for a typical human patient (e.g., a typical male patient) may be about 2 mg or more; the 3-month target dose for a typical human patient may be about 1 mg or more; the 6-week target dose for a typical human patient may be about 0.5 mg or more; the 1-month target dose for a typical human patient may be about 0.35 mg or more; and the 1-week target dose for a typical human patient may be about 85 pg or more. For example, to account for potential burst release, a target dose for a 1 month productmay be from about 1 mg to about 5 mg, while a target dose for a 6 month product may be from about 2 mg to about 10 mg. Target doses for products of other duration (e.g., 6 weeks) can be adjusted analogously.

[0176] In vivo Release: As discussed above, the microparticles described herein are extended release drug-loaded microparticles. For example, as discussed above, depending on the polymer(s) used, the microparticles as described herein may provide in vivo release of drug over an extended period, such as a 1-week period, a 1 -month period, a 3-month period, or a 6-month period. The microparticles may be prepared to provide therapeutically effective plasma levels of the drug over an extended period, such as a 1-week period, a 1 -month period, a 3-month period, or a 6-month period. For triptorelin, the microparticles may be prepared to provide a mean plasma concentration of triptorelin of at least 0.1 ng / mL in a typical human patient over an extended period (e.g., a 1- month period, a 3-month period, or a 6-month period). For FSH, the microparticles may be prepared to provide a mean plasma concentration of FSH, e.g., follitropin delta, of at least 14 mIU / ml in a typical human patient (e.g., a typical male patient) over an extended period (e.g., a 1- week period, a 1 -month period, a 3-month period, or a 6-month period).Pharmaceutical Compositions

[0177] The microparticles described herein may be formulated in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, diluents, and / or excipients, including bulking agents, buffering agents, chelating agents, antioxidants, preservatives, cosolvents, etc.

[0178] Examples of suitable excipients include polyethylene glycol (PEG), polyols such as trehalose or mannitol, preservatives such as phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzalkonium chloride, and benzethonium chloride.

[0179] The microparticles described herein may be lyophilized and then reconstituted prior to use.

[0180] In specific embodiments, the microparticles are formulated for subcutaneous or intramuscular injection. In some embodiments, drug-loaded microparticles as described herein are formulated in a carrier comprising carboxymethylcellulose. In some embodiments, drug-loaded microparticles as described herein are formulated in a carrier comprising dextran. In some embodiments, drug-loaded microparticles as described herein are formulated in a carrier comprising hyaluronic acid or a salt thereof (e.g., sodium hyaluronate). As discussed below and illustrated in the examples, it was surprisingly found that formulating drug-loaded microparticles as described herein with hyaluronic acid or a salt thereof may offer several advantages.

[0181] As used herein, hyaluronic acid refers to a polymer of D-glucuronic acid and N-acetyl-D- glucosamine disaccharides, which may be linked via alternating 0-(l — >4) and 0-(l — 3) glycosidic bonds. As used herein, salts of hyaluronic acid (also referred to as hyaluronic salts or hyaluronates) includes metal salts of hyaluronic acid, such as sodium salts, potassium salts, and the like. In the discussion that follows hyaluronic acid and salts thereof are referred to collectively as “HA”.

[0182] HA used as a carrier / diluent in a pharmaceutical composition as described herein may have an average molecular weight of greater than about 1 MDa, or greater than about 3 MDa, such as an average molecular weight from about 1 MDa to about 5 MDa, or from about 2 MDa to about 4 MDa, including about 1.5 MDa, about 2.0 MDa, about 2.5 MDa, about 3.0 MDa, about 3.5 MDa, about 4 MDa, or about 5 MDa,. In specific embodiments, the HA has a molecular weight of about 3 MDa. In further specific embodiments, the HA has a molecular weight of 3.2 ± 0.6 g / mol, or greater.

[0183] When HA is used as a carrier / diluent in a pharmaceutical composition as described herein, it may be used in any suitable amount to achieve a composition with desired properties. For example, HA may be used in an amount from about 0.01% (w / v) to about 5% (w / v), based on the volume of the pharmaceutical composition, including from about 1% (w / v) to about 3 % (w / v). In some embodiments, HA is used in an amount of about 1.5 mg / mL pharmaceutical composition. In further specific embodiments, HA having a molecular weight of 3.2 ± 0.6 g / mol is used in an amount of about 1.5 mg / mL pharmaceutical composition.

[0184] As noted above, formulating drug-loaded microparticles as described herein with HA may offer several advantages. For example, formulating drug-loaded microparticles as described herein in a carrier comprising HA (e.g., comprising an HA diluent) was found to provide advantageous shear thinning / viscoelastic behavior, providing higher microparticle suspension stability and better injectability as compared to formulations prepared with dextran (for example). In particular, it was found a carrier comprising HA (e.g., comprising an HA diluent) provides high viscosity at low shear rates, which delays sedimentation of the microparticles, while also exhibiting low viscosity at a high shear rate, i.e., while being injected, permitting facile injection through a syringe and needle. Additionally, formulating drug-loaded microparticles as described herein with HA may increase the zeta potential. Thus, formulating drug-loaded microparticles as described herein in a composition comprising HA may provide a composition with good physicochemical stability and good injectability.

[0185] Additionally, it was surprisingly found that formulating drug-loaded microparticles as described herein with HA may reduce the immunogenicity of the composition, as reflected in one or both of (i) a reduced local immune response (e.g., reduced local inflammatory response) to the composition at the she of injection and (ii) a reduced systemic immune response, as may be reflected in an increased IL-4 (anti-inflammatory) response and / or reduced level of neutralizing anti-drug antibodies, e.g., as compared to a composition formulated without HA, such as a composition formulated with dextran, as illustrated in Example 7. Without being bound by theory, it is believed that formulation with HA may reduce plasma protein adsorption to the microparticles. Plasma proteins are known to act as complement with potential pro-inflammatory action; thus, such a reduction in plasma protein adsorption to the microparticles could play a role in the reduced immunogenicity of microparticle formulations formulated with HA. Thus, formulating drug- loaded microparticles as described herein in a composition comprising HA may provide a composition with reduced (e.g., low) immunogenicity.

[0186] Additionally, for some drugs, it was surprisingly found that formulating drug-loaded microparticles as described herein with HA may impact pharmacokinetic profiles. For example, as illustrated in Example 7 below, formulating FSH-loaded microparticles as described herein withHA may increase the plasma concentration-time profile of FSH after injection as compared to formulation with dextran. On the other hand, formulating triptorelin-loaded microparticles as described herein with HA was not found to impact the plasma concentration-time profile of triptorelin after injection as compared to formulation with dextran.

[0187] In the discussion that follows, it should be understood that any pharmaceutical composition as described herein may comprise a carrier as discussed above, even if not specifically mentioned.

[0188] Provided in some aspects is a composition comprising drug-loaded microparticles formulated to contain an amount of drug-loaded microparticles that provides a target dose of the drug effective for 1 week, 1 month, 6 weeks, 3 months, or 6 months, such as an amount that provides therapeutically effective plasma levels over the target period, in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising drug-loaded microparticles that additionally or alternatively provides a therapeutically effective mean plasma concentration of the drug in a typical human patient for a period of at least 1 week after administration of a single dose by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising drug-loaded microparticles that additionally or alternatively provides a therapeutically effective mean plasma concentration of the drug in a typical human patient for a period of at least 1 month after administration of a single dose by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising drug-loaded microparticles that additionally or alternatively provides a therapeutically effective mean plasma concentration of the drug in a typical human patient for a period of at least 6 weeks after administration of a single dose by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising drug-loaded microparticles that additionally or alternatively provides a mean plasma concentration of the drug in a typical human patient for a period of at least 3 months after administration of a single dose by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising drug-loaded microparticles that additionally or alternatively provides a mean plasma concentration of the drug in a typical human patient for a period of at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

[0189] Thus, provided in one aspect is a pharmaceutical composition comprising triptorelin- loaded microparticles formulated to contain an amount of the triptorelin-loaded microparticles that provides about 1 mg of triptorelin in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising triptorelin-loaded microparticles that additionally or alternatively provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL in a typical human patient for a period of at least 1 week after administration of a single dose by subcutaneous or intramuscular injection.

[0190] Provided in another aspect is a pharmaceutical composition comprising triptorelin-loaded microparticles formulated to contain an amount of the triptorelin-loaded microparticles that provides 3.75 mg of triptorelin in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising triptorelin-loaded microparticles that additionally or alternatively provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL in a typical human patient for a period of at least 1 month after administration of a single dose by subcutaneous or intramuscular injection.

[0191] Provided in another aspect is a pharmaceutical composition comprising triptorelin-loaded microparticles formulated to contain an amount of the triptorelin-loaded microparticles that provides 11.25 mg of triptorelin in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising triptorelin-loaded microparticles that additionally or alternatively provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL in a typical human patient for a period of at least 3 months after administration of a single dose by subcutaneous or intramuscular injection.

[0192] Provided in another aspect is a pharmaceutical composition comprising triptorelin-loaded microparticles that are formulated to contain an amount of the triptorelin-loaded microparticles that provides 22.5 mg of triptorelin in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising triptorelin-loaded microparticles that additionally or alternatively provides a mean plasma concentration oftriptorelin of at least 0.1 ng / mL in a typical human patient for a period of at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

[0193] Provided in another aspect is a pharmaceutical composition comprising FSH-loaded microparticles formulated to contain an amount of the FSH-loaded microparticles that provides about 12 pg per day of FSH in a single dose, e.g., by subcutaneous or intramuscular injection.

[0194] Provided in another aspect is a pharmaceutical composition comprising FSH-loaded microparticles formulated to contain an amount of the FSH-loaded microparticles that provides about 85 pg of FSH (e.g., follitropin delta) or more in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising FSH-loaded microparticles that additionally or alternatively provides a mean plasma concentration of FSH of at least 14 mlU / mL in a typical human patient (e.g., typical male patient) for a period of at least 1 week after administration of a single dose by subcutaneous or intramuscular injection.

[0195] Provided in another aspect is a pharmaceutical composition comprising FSH-loaded microparticles formulated to contain an amount of the FSH-loaded microparticles that provides about 0.35 mg of FSH (e.g., follitropin delta) or more in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising FSH-loaded microparticles that additionally or alternatively provides a mean plasma concentration of FSH of at least 14 mlU / mL in a typical human patient (e.g., typical male patient) for a period of at least 1 month after administration of a single dose by subcutaneous or intramuscular injection.

[0196] Provided in another aspect is a pharmaceutical composition comprising FSH-loaded microparticles formulated to contain an amount of the FSH-loaded microparticles that provides about 0.5 mg of FSH (e.g., follitropin delta) or more in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising FSH-loaded microparticles that additionally or alternatively provides a mean plasmaconcentration of FSH of at least 14 mlU / mL in a typical human patient (e.g., typical male patient) for a period of at least 6 weeks after administration of a single dose by subcutaneous or intramuscular injection.

[0197] Provided in another aspect is a pharmaceutical composition comprising FSH-loaded microparticles formulated to contain an amount of the FSH-loaded microparticles that provides about 1 mg of FSH (e.g., follitropin delta) or more in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising FSH-loaded microparticles that additionally or alternatively provides a mean plasma concentration of FSH of at least 14 mlU / mL in a typical human patient (e.g., typical male patient) for a period of at least 3 months after administration of a single dose by subcutaneous or intramuscular injection.

[0198] Provided in another aspect is a pharmaceutical composition comprising FSH-loaded microparticles that are formulated to contain an amount of the FSH-loaded microparticles that provides about 2 mg of FSH (e.g., follitropin delta) or more in a single dose, e.g., by subcutaneous or intramuscular injection. Also provided is a pharmaceutical composition comprising FSH-loaded microparticles that additionally or alternatively provides a mean plasma concentration of FSH of at least 14 mlU / mL in a typical human patient (e.g., typical male patient) for a period of at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

[0199] As noted above, the described FSH doses may be adjusted (e.g., increased) to account for any burst release of FSH, to still provide sustained release of FSH over the intended period (e.g., 1 week, 1 month, 6 weeks, 3 months, 6 months, etc.).

[0200] Also provided is a kit comprising (a) a container containing drug-loaded microparticles as described herein in lyophilized form, in an amount that provides a therapeutically effective amount of the drug, and (b) instructions for preparation of a pharmaceutical composition comprising the microparticles and a pharmaceutically acceptable carrier, e.g., suitable for administration by subcutaneous or intramuscular injection.

[0201] Also provided is a kit comprising (a) a container containing triptorelin-loaded microparticles as described herein in lyophilized form, in an amount that provides 3.75 mg triptorelin, 11.25 mg triptorelin, or 22.5 mg triptorelin, and (b) instructions for preparation of a pharmaceutical composition comprising the microparticles and a pharmaceutically acceptable carrier, e.g., suitable for administration by subcutaneous or intramuscular injection.

[0202] Also provided is a kit comprising (a) a container containing FSH-loaded microparticles as described herein (e.g., follitropin delta-loaded microparticles) in lyophilized form, in an amount that provides, e.g., 85 pg FSH, 0.35 mg FSH, 0.5 mg FSH, 1 mg FSH, or 2 mg FSH, or more and (b) instructions for preparation of a pharmaceutical composition comprising the microparticles and a pharmaceutically acceptable carrier, e.g., suitable for administration by subcutaneous or intramuscular injection.Therapeutic Methods

[0203] Also provided herein are therapeutic methods using a pharmaceutical composition as described herein. The methods may comprise administering a pharmaceutical composition as described herein to a subject in need thereof, such as by injecting (administering to) a subject in need thereof with a therapeutically effective amount of a composition as described herein.

[0204] The amount of drug-loaded microparticle composition administered may be any amount effective to provide a target dose of drug, such as an amount that provides a target dose of drug effective for 1 week, 1 month, 6 weeks, 3 months, or 6 months, such as an amount that provides therapeutically effective plasma levels of drug over the target period, such as a 1 week period, a 1 -month period, a 3-month period, or a 6-month period, such as discussed above for each of triptorelin and FSH.

[0205] For triptorelin compositions, the subject may be suffering from and / or in need of treatment for one or more conditions selected from a hormone-responsive cancer, such as breast cancer or prostate cancer, endometriosis, female infertility, uterine fibroids, or precocious puberty. Inspecific embodiments, the subject being treated with triptorelin-loaded microparticles as disclosed herein is suffering from hormone-sensitive prostate cancer or hormone-sensitive breast cancer.

[0206] The amount of composition comprising triptorelin-loaded microparticles as disclosed herein administered may be any amount effective to provide a target dose of triptorelin, such as an amount that provides a target dose of triptorelin effective for 1 week, 1 month, 6 weeks, 3 months, or 6 months, such as an amount that provides therapeutically effective plasma levels of triptorelin over the target period, such as a 1-week period, a 1 -month period, a 6- week period, a 3 -month period, or a 6-month period, such as a mean plasma concentration of triptorelin of at least 0.1 ng / mL over an extended period (e.g., a 1-week period, a 1-month period, a 6-week period, a 3- month period, or a 6-month period). A typical volume of composition for subcutaneous injection is on the order of 0.5 to 2 mL. A typical volume of composition for intramuscular injection is on the order of 0.5 to 3 mL, and in some embodiments, 0.5 to 5 mL.

[0207] Thus, provided herein are methods of treatment comprising administering a pharmaceutical composition comprising triptorelin-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides about 1 mg of triptorelin. In some embodiments, the method provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 1 week after subcutaneous or intramuscular injection of the single dose.

[0208] Also provided herein are methods of treatment comprising administering a pharmaceutical composition comprising triptorelin-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides 3.75 mg of triptorelin. In some embodiments, the method provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 1 month after subcutaneous or intramuscular injection of the single dose.

[0209] Also provided herein are methods of treatment comprising administering a pharmaceutical composition comprising triptorelin-loaded microparticles as disclosed herein to a human subjectin need thereof by subcutaneous or intramuscular injection of a single dose that provides 11.25 mg of triptorelin. In some embodiments, the method provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 3 months after subcutaneous or intramuscular injection of the single dose.

[0210] Also provided herein are methods of treatment comprising administering a pharmaceutical composition comprising triptorelin-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides 22.5 mg of triptorelin. In some embodiments, the method provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 6 months after subcutaneous or intramuscular injection of the single dose.

[0211] For FSH-loaded microparticle compositions, the subject may be suffering from and / or in need of treatment for female infertility or male infertility.

[0212] The amount of FSH composition administered may be any amount effective to provide a target dose of FSH, such as an amount that provides a target dose of FSH effective for 1 week, 1 month, 6 weeks, 3 months, or 6 months, such as an amount that provides therapeutically effective plasma levels of FSH over the target period, such as a 1-week, 1 -month, 6- week, 3 -month, or 6-month period, such as a mean plasma concentration of FSH of at least 14 mlU / mL over an extended period (e.g., a 1-week period, a 1 -month period, a 6- week period, a 3 -month period, or a 6-month period). A typical volume of composition for subcutaneous injection is on the order of 0.1 to 2 mL. A typical volume of composition for intramuscular injection is on the order of 0.1 to 3 mL, and in some embodiments, 0.1 to 3 mL.

[0213] Thus, provided herein are methods of treatment comprising administering a pharmaceutical composition comprising FSH-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides about 85 pg of FSH (e.g., follitropin delta) or more. In some embodiments, the method provides a mean plasmaconcentration of FSH of at least 14 mlU / mL for a period of at least 1 week after subcutaneous or intramuscular injection of the single dose.

[0214] Also provided herein are methods of treatment comprising administering a pharmaceutical composition comprising FSH-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides about 0.35 mg of FSH (e.g., follitropin delta) or more. In some embodiments, the method provides a mean plasma concentration of FSH of at least 14 mlU / mL for a period of at least 1 month after subcutaneous or intramuscular injection of the single dose.

[0215] Also provided herein are methods of treatment comprising administering a pharmaceutical composition comprising FSH-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides about 0.5 mg of FSH (e.g., follitropin delta) or more. In some embodiments, the method provides a mean plasma concentration of FSH of at least 14 mlU / mL for a period of at least 6 weeks after subcutaneous or intramuscular injection of the single dose.

[0216] Also provided herein are methods of treatment comprising administering a pharmaceutical composition comprising FSH-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides about 1 mg of FSH (e.g., follitropin delta) or more. In some embodiments, the method provides a mean plasma concentration of FSH of at least 14 mlU / mL for a period of at least 3 months after subcutaneous or intramuscular injection of the single dose.

[0217] Also provided herein are methods of treatment comprising administering a pharmaceutical composition comprising FSH-loaded microparticles as disclosed herein to a human subject in need thereof by subcutaneous or intramuscular injection of a single dose that provides about 2 mg of FSH (e.g., follitropin delta) or more. In some embodiments, the method provides a mean plasma concentration of FSH of at least 14 mlU / mL for a period of at least 6 months after subcutaneous injection of the single dose.EXAMPLES

[0218] The following specific examples are included as illustrative of the invention described herein. These examples are in no way intended to limit the scope of the invention.Example 1 : 1 -Month Triptorelin Microparticles (laboratory scale)

[0219] 1 -month triptorelin microparticle formulations were prepared at laboratory scale (3 g) by the following general process:Step 1 : Preparation of feed solutions for the microparticle formation step;Step 2: Microparticle formation;Step 3: Removal of ethyl acetate from the polymeric microparticles by evaporation;Step 4: Separation of microparticles from the suspension and washing; andStep 5: Lyophilization.

[0220] Prepared polymer solution and drug solution of known quantity were successively transferred into a dispersing vessel. Subsequently, the drug solution was dispersed in the polymer solution at a certain mixing speed. After a dispersing time, the surfactant solution containing Poloxamer 188 was transferred into the dispersing vessel. This action induced a phase inversion, which in turn led to the formation of triptorelin-loaded microparticles. As discussed above, the dispersion step temperature, dispersion step mixing speed, dispersion step mixing time, and phase inversion transfer rate can affect particle size and triptorelin release rates, and were selected and adjusted to prepare the triptorelin-loaded microparticles described in this example. The drug- loaded microparticle suspensions contain excess organic polymer solvent as well as nonencapsulated drug and excipients, which were each removed in the subsequent solvent evaporation and washing steps, respectively. The wash phase solution comprising 16% Poloxamer 188 in citrate buffer pH 6.5 was added to stabilize the microparticles. The excess polymer solvent was removed by application of vacuum and elevated temperature during continuous agitation by magnetic agitation. The ethyl acetate was removed by means of evaporation, whereby increasingthe transition of ethyl acetate from the microparticles into the aqueous surfactant solution (continuous phase) causes a complete hardening of the polymeric microparticles.

[0221] For this lab scale process, the separation of the microparticles from the continuous phase was by centrifugation, wherein the supernatant containing the residual solvent and excipients was discarded, the microparticle pellet was resuspended with ultrapure water and centrifuged again, the supernatant was discarded, and subsequently, the microparticle pellet was resuspended with a surfactant solution (0.25% Poloxamer 188) and centrifuged again. Aliquots of the microparticle suspension were transferred to into freeze-drying vials and vials containing the microparticle suspension were lyophilized under appropriate conditions to provide triptorelin-loaded microparticle formulation. The triptorelin-loaded microparticle formulations were sterilized by y- irradiation at a dose of 25 kGy ± 10%.

[0222] The following tables set forth the components used to prepare the 1 -month formulations at laboratory scale and properties of the microparticles.Table 1. 1 Month Triptorelin Formulation (Lab Scale; 3 g scale)F68 = Poloxamer 188 (F68) (KOLLIPHOR® Pl 88), Ph. Eur. from BASF / BTC (Europe, Geismar, USA). n.d. = not determinedFormulation 1 (Laboratory Scale!Formulation 2 (Laboratory Scale)Formulation 3 (Laboratory Scale)Formulation 4 (Laboratory Scale)Formulation 5 (Laboratory Scale)Formulation 6 (Laboratory Scale)Formulation 7 (Laboratory Scale)Formulation 8 (Laboratory Scale)In vitro Release of 1-Month Triptorelin Microparticles (Laboratory Scale)

[0223] The in vitro release of the triptorelin-loaded microparticle formulations prepared as described above was assessed as follows. Ten aliquots of 10 mg microparticles were weighed into polypropylene screw vessels and 1.5 mL aqueous buffer was added as a release medium to each aliquot. The samples were placed on an overhead shaker and agitated at 37°C. At appropriate sampling times (0 hours, 4 hours, 24 hours, 3 days and 7 days), two samples were centrifuged to separate the microparticles from the release medium. The release medium was filtered through a syringe pre-filter, and the triptorelin content was determined by HPLC analysis. Formulations 1, 2, and 3 showed different drug release profiles over seven days in vitro.PK Studies of 1-Month Triptorelin Microparticles (Laboratory Scale)

[0224] For in vivo assessment, the 1 -month triptorelin-loaded microparticles described above (Formulations 1-8) were administered in a single subcutaneous (s.c.) injection to Sprague Dawley (SD) rats to provide a dose of 1 mg triptorelin, with 8 rats per treatment group. The duration of the study was up to three (3) months. Blood samples for bioanalysis and PK evaluation were collected at the following timepoints: 1, 3, 5, 7, 14, 28, 42, 56, 70 and 84 days post-dose. For the first four blood sampling time points, blood was sampled from 4 animals per group allowing for staggered blood sampling. Body weights were determined at the day of dosing and then again on days 28, 56, and 84. Injection sites were carefully monitored.

[0225] Results are shown in FIGS. 2-4 for Formulations 1 (polymer RG503), 2 (polymer RG504), and 3 (polymers RG503 / RG502), respectively. All test groups achieved mean triptorelin plasma concentrations above 100 pg / mL (the target minimum concentration) at 1 month (672 hours). Two months after triptorelin administration, the mean triptorelin plasma concentrations were close to or below the 100 pg / mL threshold, with PK Test Group 8 / Formulation 8 having a meanconcentration well above the 100 pg / mL threshold (not shown). All mean plasma triptorelin concentrations were below the 100 pg / mL threshold 3 months after triptorelin administration.Stability Studies of 1 -Month Triptorelin Microparticles (Laboratory Scale)

[0226] The storage stability of selected 1 -month triptorelin-loaded microparticles of this example was assessed by monitoring the evolution of main impurities over time by HPLC analysis. The main focus of the stability study was on the evolution of the following main individual impurities having the largest relative peak areas at the following relative retention times (RRT), which are relative to the retention time of the triptorelin peak: at RRT 1.09 / 1.10; RRT 1.13 / 1.14; RRT 1.16 / 1.17; RRT 1.18 / 1.19; and RRT 1.22 / 1.23. The evolution of these individual impurities over a storage time of 6 months (TO, Tl, T3, T6 months) and exposure to different conditions (5°C ± 3°C, 25°C ± 2°C / 60% RH, 30°C ± 2°C / 65% RH) were examined. According to ICH Q3B guideline, for an amount of 3.75 mg of drug substance administered per day, the identification threshold is 0.5% and the qualification threshold is 1.0%. Therefore, it was important that one individual impurity does not exceed 1.0%.

[0227] All formulations prepared with ester-terminated polymers (RG504, RG503, RG502, or mixtures thereof) exhibited good stability, with individual impurities remaining far below the qualification threshold of 1.0% (e.g., Formulation 2 (RG504) and Formulation 3 (RG503 / RG502)). In addition, the following observations were made comparing individual impurity results of different formulations:■ The individual impurities at RRT 1.09 / 1.10 and RRT 1.13 / 1.14 are most relevant, as they are growing over time (only slowly and still far below the qualification threshold of 1.0%).■ The individual impurity at RRT 1.09 / 1.10 is larger for lower molecular weight polymers.■ The individual impurity at RRT 1.09 / 1.10 is larger for higher polymer / drug ratio.■ y-irradiation seems to have no significant effect on the individual impurity at RRT 1.09 / 1.10.■ y-irradiation had a strong effect on the individual impurity at RRT 1.13 / 1.14; this impurity is not seen for the non-irradiated material; i.e., < 0.1%.Example 2: 1 -Month Triptorelin Microparticles (Intermediate Scale)

[0228] 1 -month triptorelin microparticle formulations were prepared at intermediate scale (51 g scale) in a manner similar to the process described in Example 1 using the components and process parameters noted in the tables below. Different from Example 1, the following steps were used for the separation of the microparticles from the suspension: Following the evaporation step, the microparticle suspension was concentrated by means of tangential flow / cross flow filtration in the process chamber. Water was used to wash the microparticle suspension in the process chamber to wash out excess surfactant and non-encapsulated API. Lyophilization and sterilization was performed in a manner similar to the process described in Example 1.

[0229] The following tables set forth the components used to prepare the 1 -month formulations at intermediate scale and properties of the microparticles.Table 2. 1 -Month Triptorelin Microparticles (Intermediate Scale; 51 g scale)n.d. = not determined.Poloxamer 188 (EMPROVE® Expert), Ph. Eur. from Merck.PEG 6000, Polyethylene glycol 6000, Ph. Eur. From Carl Roth. MOWIOL® 18-88, Polyvinyl alcohol from Aldrich / Merck.Formulation 9 (Intermediate Scale)Formulation 10 (Intermediate Scale)Formulation 11 (Intermediate Scale)Formulation 12 (Intermediate Scale)Formulation 13 (Intermediate Scale)Formulation 14 (Intermediate Scale)Formulation 15 (Intermediate Scale)Formulation 16 (Intermediate Scale)Formulation 17 (Intermediate Scale)Formulation 18 (Intermediate Scale)Formulation 19 (Intermediate Scale)Formulation 20 (Intermediate Scale)Formulation 21 (Intermediate Scale)Formulation 22 (Intermediate Scale)Formulation 23 (Intermediate Scale)Formulation 24 (Intermediate Scale)Formulation 25 (Intermediate Scale)Formulation 26 (Intermediate Scale)Formulation 27 (Intermediate Scale)In Vitro Release of 1-Month Triptorelin Microparticles (Intermediate Scale)

[0230] The in vitro release of triptorelin-loaded microparticle formulations prepared at intermediate scale was assessed as described in Example 1. Formulations 9 to 27 (before y- irradiation) showed substantial drug release over seven days in vitro.PK Studies of 1-Month Triptorelin Microparticles (Intermediate Scale)

[0231] For in vivo assessment, the 1 -month triptorelin-loaded microparticles formulations prepared at intermediate scale as described above (Formulations 9-20) were administered in a single subcutaneous (s.c.) injection to Sprague Dawley (SD) rats to provide a dose of 1 mg triptorelin, with 8 rats per treatment group. The duration of the study was up to three (3) months. Blood samples for bioanalysis and PK evaluation were collected at the following timepoints: 1, 3, 5, 7, 14, 28, 42, 56, 70 and 84 days post dose. For the first four blood sampling time points, blood was sampled from 4 animals per group allowing for staggered blood sampling. Body weights were determined at the day of dosing and then again on days 28, 56, and 84. Injection sites were carefully monitored.

[0232] Results are shown in in FIGS. 5-8 for Formulation 9 (RG503 (purified), PEG6000, F68), Formulation 12 (RG503 (purified) / RG502, PEG6000, F68), Formulation 16 (RG 503 (purified),PEG4000, polyvinylalcohol), and Formulation 19 (RG503, PEG3350, F68), respectively. All test groups achieved mean triptorelin plasma concentrations above 100 pg / mL at 1 month (672 hours). Two months after triptorelin administration, the mean triptorelin plasma concentrations in all test groups were below the 100 pg / mL threshold.Stability Studies of 1-Month Triptorelin Microparticles (Intermediate Scale)

[0233] The storage stability of selected 1 -month triptorelin-loaded microparticles of this example was assessed by monitoring the evolution of main impurities over time by HPLC analysis. The main focus of the stability study was on the evolution of the following main individual impurities having the largest relative peak areas at the following relative retention times (RRT), which are relative to the retention time of the triptorelin peak: at RRT 1.09 / 1.10; RRT 1.13 / 1.14; RRT 1.16 / 1.17; RRT 1.18 / 1.19; and RRT 1.22 / 1.23. The evolution of these individual impurities over a storage time of 12 months (TO, Tl, T3, T6, T12 months) and exposure to different conditions (5°C ± 3°C, 25°C ± 2°C / 60% RH, 30°C ± 2°C / 75% RH) were examined. According to ICH Q3B guidelines, for an amount of 3.75 mg of drug substance administered per day, the identification threshold is 0.5% and the qualification threshold is 1.0%. Therefore, it was important that one individual impurity does not exceed 1.0%.

[0234] In summary, the following results were observed:Core Loading: Following an initial decrease as a result of y-irradiation, the core loadings showed good stability over 12 months, almost independent of the storage condition. PEG molecular weight had no impact on core loading showing identical profiles at all storage conditions over 12 months (e.g. Formulation 14 (PEG 6000) and Formulation 16 (PEG4000)).Total Impurity: Following a strong increase as a result of y-irradiation, the total impurities of the samples stored at 5°C were in most cases slightly lower than those of the samples stored at 25°C / 60% RH or 30°C / 65% RH, respectively.Largest Single Impurities: All formulations prepared with ester-terminated polymers (RG503 or mixtures of RG503 and RG502) exhibited good stability, with individual impurities remainingbelow the qualification threshold of 1.0%. In addition, the following observations were made comparing individual impurity results of different formulations:■ The individual impurity at RRT 1.09 / 1.10 is dependent on the storage conditions above 25°C.■ The individual impurity at RRT 1.09 / 1.10 is larger using polymer blend (e.g. Formulation 12 (RG503 / RG502).■ y-irradiation seems to have no significant effect on the individual impurity at RRT 1.09 / 1.10.■ y-irradiation had a strong effect on the individual impurity at RRT 1.17 / 1.18; this impurity is not seen for the non- irradiated material; i.e., < 0.1%.■ The individual impurity at RRT 1.17 / 1.18 is larger using PVA (e.g., Formulation 14 and Formulation 16).Residual Ethyl Acetate Content: Following an initial decrease as a result of y -irradiation, the ethyl acetate content was very consistent and showed a trend of continuous decrease over 12 months at 25°C and above. At 5°C ± 3°C a slight increase in residual EtAc was observed over 12 months storage.Residual Water Content: Almost identical values were observed at TO and T12; it was concluded that no water has entered the vials over time.In-Vitro Release Profiles: All in-vitro release profiles exhibited a very similar shape. A significant deceleration of the IVR profiles is caused by y-irradiation. The IVR profile of the samples stored over 12 months at 25°C / 60% RH or 30°C / 75% was slightly accelerated but still below the IVR profile before irradiation.Example 3: 3 -Month Triptorelin Microparticles (Intermediate Scale)

[0235] 3 -month triptorelin microparticles were prepared at intermediate scale (51 g) by the following general process:Step 1 : Preparation of feed solutions for the microparticle formation step;Step 2: Microparticle formation;Step 3: Removal of ethyl acetate from the polymeric microparticles by evaporation;Step 4: Separation of microparticles from the suspension and washing; andStep 5: Lyophilization.

[0236] Prepared polymer solution and drug solution of known quantity were successively transferred into a dispersing vessel. Subsequently, the drug solution was dispersed in the polymer solution under a nitrogen gas flow. After a dispersing time, the surfactant solution containing Poloxamerl 88 and sucrose was transferred into the dispersing vessel. This action induced a phase inversion, which in turn led to the formation of triptorelin-loaded microparticles. As discussed above, the dispersion step temperature, dispersion step mixing speed, dispersion step mixing time, nitrogen flow rate, and phase inversion transfer rate can affect particle size and triptorelin release rates, and were selected and adjusted to prepare the triptorelin-loaded microparticles described in this example. The drug-loaded microparticle suspensions contain excess organic polymer solvent as well as non-encapsulated drug and excipients, which were each removed in the subsequent solvent evaporation and washing steps, respectively. The wash phase solution comprising 16% Poloxamer 188) in citrate buffer pH 6.5 was added to stabilize the microparticles. The excess polymer solvent was removed by application of vacuum and elevated temperature during continuous agitation by vibration. The ethyl acetate was removed by means of evaporation, whereby increasing the transition of ethyl acetate from the microparticles into the aqueous surfactant solution (continuous phase) causes a complete hardening of the polymeric microparticles. Following the evaporation step, the microparticle suspension was concentrated by means of tangential flow / cross flow filtration in the process chamber. Water was used to wash the microparticle suspension in the process chamber to wash out excess surfactant and nonencapsulated API. Aliquots of the microparticle suspension were transferred to into freeze-drying vials and vials containing the microparticle suspension were then lyophilized under appropriate conditions to provide triptorelin-loaded microparticle formulation. The triptorelin-loaded microparticle formulation was sterilized by y-irradiation at a dose of 28 kGy ± 10%.

[0237] The components used to prepare 3 -month triptorelin formulations at intermediate scale and properties of the microparticles are shown in the following tables.Table 3. 3-Month Triptorelin Microparticles Formulations (Intermediate Scale)Geismar, USA)*Formulation 29 was tested in-vivo after reconstitution in different diluents.**The microparticles of Formulation 30 were divided during manufacturing: Formulation 31 was prepared by subjecting one portion of the Formulation 30 microparticles to an additional 140 pm sieving step.***The microparticles of Formulation 34 were divided during manufacturing: Formulation 35 was prepared by subjecting one portion of the Formulation 34 microparticles to sterilization by y-irradiation.Formulation 28 (Intermediate Scale)Formulation 29 (Intermediate Scale)Formulation 30 (Intermediate Scale)Formulation 31 (Intermediate Scale)Example 32 (Intermediate Scale)Formulation 33 (Intermediate Scale)Formulation 34 (Intermediate Scale)Formulation 35 (Intermediate Scale)Example 36 (Intermediate Scale)In Vitro Release of 3-Month Triptorelin Microparticles (Intermediate Scale)

[0238] The in vitro release of 3 -month triptorelin-loaded microparticle formulations prepared at intermediate scale (prior to y-irradiation) was assessed as described in Example 1. All formulations prepared at intermediate scale (Formulation 28-36) showed substantial drug release over seven days in vitro.PK Studies of 3-Month Triptorelin Microparticles (Intermediate Scale)

[0239] For in vivo assessment, 3-month triptorelin-loaded microparticles formulations prepared at intermediate scale as described above (Formulations 28-36) were administered in a single subcutaneous (s.c.) injection to Sprague Dawley (SD) rats to provide a dose of 3 mg triptorelin, with 8 rats per treatment group. (Formulation 35 was sterilized by y-irradiation prior to injection.) The duration of the study was a period of 112 days (4 months). The following table summarizes the PK study groups in this study. Blood samples for bioanalysis and PK evaluation were collected at the following timepoints: 1, 3, 5, 7, 14, 28, 42, 56, 84, and 112 days post dose. For the first fourblood sampling time points, blood was sampled from 4 animals per group allowing for staggered blood sampling. Body weights were determined at the day of dosing and then again on days 28, 84, and 112. Injection sites were carefully monitored.

[0240] Results for Formulation 28 are shown in FIG. 9. All test groups achieved mean triptorelin plasma concentrations above 100 pg / mL, for at least 3 months (2016 hours).

[0241] The 3 -month triptorelin-loaded microparticles of Formulation 29 were formulated in two different carriers / diluents for injection: (i) Dextran 70 / Polysorbate 80 diluent or (ii) Sodium Hyaluronate / Polysorbate 80 diluent. PK results for these different compositions comprising the Formulation 29 microparticles are shown in FIG. 10.Lons Term Stability Studies of 3-Month Triptorelin Microparticles (Intermediate Scale)

[0242] The long term stability of the triptorelin microparticles of Formulations 28-36 was evaluated over a 24 month period under the following three storage conditions: 5°C ± 3°C, 25°C ± 2°C / 60% RH ± 5% RH; 30°C ± 2°C / 65% RH ± 5% RH. The samples were analyzed after y-irradiation (TO) and at the following time points: 1 month (Tl), 3 months (T3), 6 months (T6), 9 months (T9), 12 months (T12), 18 months (T18), and 24 months (T24). The following attributes were assessed: core loading, purity, in vitro release, particle-size distribution, morphology, appearance, residual water, residual solvent, molecular weight distribution. In summary, the following results were observed:Core Loading: Following an initial decrease as a result of y-irradiation, the core loadings fluctuated around the TO value, with a slight downward trend over time, which was less pronounced for the samples stored at 5°C. However, these differences were only marginal.Total Impurity: Following a strong increase as a result of y -irradiation, the total impurities of the samples stored at 5°C were in most cases slightly lower than those of the samples stored at 25°C / 60% RH or 30°C / 65%RH, respectively.Largest Single Impurity: The largest single impurity in all y-irradiated batches was found at RRT 1.08 / 1.09. The peak signals each increased with storage time (up to about 12 months) and theneither remained at a constant level or even decreased slightly. Moreover, both a temperature effect (more pronounced increase for higher storage temperature) and a dose effect (more pronounced increase for higher irradiation dose) were observed.Residual Ethyl Acetate Content: Following an initial decrease as a result of y -irradiation, the ethyl acetate content remained generally constant in the samples stored at 5°C. With increasing storage temperature and storage time, however, a more pronounced ethyl acetate reduction was observed. Residual Water Content: Almost identical values were observed at TO, T12, and T24; it was concluded that no water has entered the vials over time.Molecular Weight of Polymer: Based on just a few data points (T6 and T12 values), it was observed that the molecular weight of the polymer slightly decreased over time. This decrease was more pronounced with higher storage temperature and higher y -irradiation dose.In-Vitro Release Profiles: All in-vitro release profiles exhibited a very similar shape. Starting with very low initial burst, the fluctuation range increased with the storage time, but remained within a reasonable limit. In general, it was observed that y -irradiation causes a slower release.Example 4: 3-Month Triptorelin Microparticles (Commercial Scale; 510 g)

[0243] 3 -month triptorelin microparticles were prepared at commercial scale (510 g) by the following general process:Step 1 : Preparation of feed solutions for the microparticle formation step;Step 2: Microparticle formation;Step 3: Removal of ethyl acetate from the polymeric microparticles by evaporation;Step 4: Separation of microparticles from the suspension and washing; andStep 5: Lyophilization.

[0244] Prepared polymer solution and drug solution of known quantity were transferred into a dispersing vessel, and the drug solution was dispersed in the polymer solution under a nitrogen gas flow. After a dispersing time, the surfactant solution containing Poloxamer 188, and sucrose was transferred into the dispersing vessel. This induced a phase inversion, which in turn led to the formation of triptorelin-loaded microparticles. As discussed above, the dispersion steptemperature, dispersion step mixing speed, dispersion step mixing time, nitrogen flow rate, and phase inversion transfer rate can affect particle size and triptorelin release rates, and were selected and adjusted to prepare the triptorelin-loaded microparticles described in this Example. The drug- loaded microparticle suspensions contain excess organic polymer solvent as well as nonencapsulated drug and excipients, which were each removed in subsequent solvent evaporation and washing steps, respectively. The wash phase solution comprising 16% Poloxamer 188 in citrate buffer pH 6.5 was added to stabilize the microparticles. The excess polymer solvent was removed by application of vacuum and elevated temperature during continuous agitation by vibration. The ethyl acetate was removed by means of evaporation, whereby increasing the transition of ethyl acetate from the microparticles into the aqueous surfactant solution (continuous phase) causes a complete hardening of the polymeric microparticles. Following the evaporation step, the microparticle suspension was concentrated by means of tangential flow / cross flow filtration in the process chamber. Water was used to wash the microparticle suspension in the process chamber to wash out excess surfactant and non-encapsulated drug. Aliquots of the microparticle suspension were transferred into freeze-drying vials and vials containing the microparticle suspension were lyophilized under appropriate conditions to provide a triptorelin- loaded microparticle formulation. The triptorelin-loaded microparticle formulation was sterilized by y-irradiation at a dose of 25 kGy ± 10%.

[0245] The components used to prepare the 3 -month triptorelin formulations at commercial scale and properties of the microparticles are shown in the following tables.Table 4. 3-Month Microparticles Formulations (Commercial Scale; 510 g scale)F68 = Poloxamer 188 (F68) (KOLLIPHOR® Pl 88), Ph. Eur. from BASF / BTC (Europe, Geismar, USA) or Poloxamer 188 from ThermoFisher Scientific (formerly Alfa Aesar).Formulation 37 (Commercial Scale)Formulation 38 (Commercial Scale)Formulation 39 (Commercial Scale)Formulation 40 (Commercial Scale)Formulation 41 (Commercial Scale)Formulation 42 (Commercial Scale)In Vitro Release of 3-Month Triptorelin Microparticles (Commercial Scale)

[0246] The in vitro release of the 3-month triptorelin-loaded microparticles of Formulation 37 and 38 (before and after y-irradiation) was assessed as described in Example 1, and showed release of a substantial amount of triptorelin over a seven day period.PK Studies of 3-Month Triptorelin Microparticle (Commercial Scale)

[0247] A first commercial scale study investigated the release profile of a 3-month triptorelin microparticle formulation prepared at commercial scale (Formulation 37) by assessing resulting plasma concentrations after dosing as single subcutaneous (s.c.) injections to Sprague Dawley (SD) rats. The study was performed in 8 male Sprague Dawley rats. The duration of the study was up to 6 months.

[0248] A second commercial scale study compared the release profile of a 3-month triptorelin microparticle formulation prepared at commercial scale (Formulation 38) with that of an approved reference formulation with a release over 3 months (Decapeptyl SR, 11.25 mg), by assessing resulting plasma concentrations after dosing as single subcutaneous (s.c.) injections to Sprague Dawley (SD) rats. The study was performed in 16 male Sprague Dawley rats, divided into 2 treatment groups of 8 rats. The duration of the study was up to 6 months.

[0249] In both studies, blood samples for bioanalysis and PK evaluation were collected at the following timepoints: 1, 3, 5, 7, 14, 28, 42, 56, 70, 84, 112, 140, and 168 days post dose. For thefirst four blood sampling time points, blood was sampled from 4 animals per group allowing for staggered blood sampling.

[0250] In the first study, the test formulation (Formulation 37) had a confirmed sustained release profile with individual and mean triptorelin plasma concentrations above the clinically relevant 100 pg / mL threshold for at least 4 months (FIG. 11) and thereafter for up to 6 months concentration-time profiles steadily decreased to around 100 pg / mL or below. In the second study, the test formulation (Formulation 38) had a confirmed sustained release profile with individual and mean triptorelin plasma concentrations above the clinically relevant 100 pg / mL threshold for at least 4 months and thereafter for up to 6 months concentration-time profiles steadily decreased to around 100 pg / mL or below (data not shown). The profiles for the reference formulation (Decapeptyl SR) were more variable up until 3 months, but at 4 months and beyond all individual measurements were below the detection limit.PK Studies of 3-Month Triptorelin Microparticles (Commercial Scale) After Storage

[0251] To investigate the impact of potential degradation during storage at 12 months and 22 months at 25°C on the pharmacokinetics of the triptorelin 3-month microparticle formulations prepared at commercial scale, resulting plasma concentrations after dosing as single subcutaneous (s.c.) injections to Sprague Dawley (SD) rats were assessed. Specifically, this example evaluated the release profiles of a 3-month triptorelin microparticle formulation prepared at commercial scale (Formulation 38; PK Group 4) after storage for 12 months at 25°C and a 3-month triptorelin microparticle formulation prepared at the intermediate scale (Formulation 28; PK Group 5) after storage for 22 months at 25°C. Each treatment group consisted of 8 rats, and comprised administration of 3.0 mg triptorelin. The duration of the study was up to 6 months.

[0252] Blood samples for bioanalysis and PK evaluation were collected at the following timepoints: 1, 3, 5, 7, 14, 28, 42, 56, 70, 84, 112, 140, and 168 days post dose. For the first four blood sampling time points, blood was sampled from 4 animals per group allowing for staggered blood sampling. The test groups achieved mean triptorelin plasma concentrations above 100pg / mL for at least 3 months (2016 hours). At 6 months, the mean concentrations of the test groups declined to values below 100 pg / mL.Example 5: 6-Month Triptorelin Microparticles (Laboratory scale)

[0253] The formulation comprising triptorelin-loaded microparticles prepared in this example provided drug release above the minimum targeted level (e.g., 0.1 ng / mL) for over 6 months after subcutaneous injection (S.C.) into rats. The components used to prepare the six-month formulation comprising triptorelin-loaded microparticles are shown below.Table 5.R203S (Evonik): lactide / glycolide ratio of 100:0 with ester terminal groups.R205S (Evonik): lactide / glycolide ratio of 100:0 with ester terminal groups.F68 = Poloxamer 188 (F68) (KOLLIPHOR® Pl 88), Ph. Eur. from BASF / BTC (Europe, Geismar, USA).

[0254] The microparticle manufacturing process used to prepare these triptorelin formulations is similar to the process described in Example 1. The microparticle formation and removal of solvent is performed in a manner similar to the process described in Example 1. The separation of the microparticles from the continuous phase in this example was by centrifugation. The supernatant containing the residual solvent and excipients was discarded. The microparticle pellet was resuspended with ultrapure water and centrifuged again. The supernatant was discarded and subsequently, the microparticle pellet was resuspended with a surfactant solution (0.25%Poloxamer 188) and centrifuged again. Lyophilization and sterilization were performed in a manner similar to the process described in Example 1.

[0255] The following table summarizes the physical properties of the triptorelin-loaded microparticle formulation prepared in this example.

[0256] 6-month triptorelin-loaded microparticles were administered in a single subcutaneous (s.c.) injection to Sprague Dawley (SD) rats, and plasma levels of triptorelin were monitored. FIG. 12 shows the mean pharmacokinetic profile with standard deviation. All individual animals had triptorelin plasma concentrations above 100 pg / mL for at least 6 months after dosing (FIG. 12).Example 6: 6-Month Triptorelin Microparticles (Intermediate Scale)

[0257] A formulation comprising triptorelin-loaded microparticles designed to provide drug release over 6 months was prepared in intermediate scale. The components used to prepare this six-month formulation is shown in the following table.Table 6.R205S (Evonik): lactide / glycolide ratio of 100:0 with ester terminal groups.F68 = Poloxamer 188 (F68) (KOLLIPHOR® Pl 88), Ph. Eur. from BASF / BTC (Europe, Geismar, USA).

[0258] The microparticle manufacturing process used to prepare these triptorelin formulations is similar to the process described in Example 1. The microparticle formation and removal of solvent was performed in a manner similar to the process described in Example 3. Lyophilization and sterilization were performed in a manner similar to the process described in Example 3.

[0259] The table below summarizes the physical properties of the triptorelin-loaded microparticle formulation prepared in this example.

[0260] The in vitro release of the 6-month triptorelin-loaded microparticle formulations prepared at intermediate scale as described above was assessed as described in Example 1 , and exhibited substantial drug release within 7 days in vitro. The results showed that this example, which was prepared at intermediate scale using 51 g of polymer, reproduced the in vitro release profile of Example 5, which was prepared on a smaller scale.Example 6: 3 -Month FSH Microparticles (Laboratory Scale; 3 g)

[0261] FSH-loaded microparticles designed to provide drug release over at least 3 months were prepared using a process similar to that described in Example 1 , using follitropin delta as the drug (API) instead of triptorelin.

[0262] Follitropin delta (FSH) microparticles were prepared from the follitropin delta drug substance, which is provided as a buffer solution with a concentration of 0.6 mg / mL. Prior to microparticle formulation, the follitropin delta drug substance solution was concentrated by ultracentrifugation.

[0263] The microparticle formation and removal of solvent were performed in a manner similar to the process described in Example 1 but using the components and process parameters noted in the below table. The separation of the microparticles from the continuous phase in this example was by centrifugation, wherein the supernatant containing the residual solvent and excipients was discarded. The microparticle pellet was resuspended with ultrapure water and centrifuged again and the supernatant was discarded and the microparticle pellet was resuspended with a surfactant solution (0.25% Poloxamer 188) and centrifuged again. Lyophilization and sterilization were performed in a manner similar to the process described in Example 1.

[0264] The compositions used to prepare the formulations and properties of the FSH microparticles are shown in the following tables.Table 7. 3 Month FSH Microparticles Formulations (Laboratory Scale)Formulation 45 (Laboratory Scale)Formulation 46 (Laboratory Scale)Formulation 47 (Laboratory Scale)Formulation 48 (Laboratory Scale)Formulation 49 (Laboratory Scale)Formulation 50 (Laboratory Scale)Formulation 51 (Laboratory Scale)

[0265] The in vitro release of the 3-month follitropin delta-loaded microparticle formulations was assessed as follows. For each sampling point, an aliquot of 30 mg microparticles were weighed into polypropylene screw vessels and 1.5 mL aqueous buffer was added as a release medium to each aliquot. The samples were placed on an overhead shaker and agitated at 37°C. At appropriate sampling times (0 hours, 4 hours, 24 hours, 3 days, 7 days and 10 days), two samples were centrifuged to separate the microparticles from the release medium. The release medium was filtered through a syringe pre-filter, and the FSH content was determined by HPLC analysis. The in vitro release of the 3-month follitropin delta-loaded microparticles of Formulation 45 exhibited substantial release of FSH within 7 days in vitro. Size exclusion chromatography of Formulations 45 and 46 confirmed that FSH released in the in vitro studies were structurally intact. The volume of the aqueous drug phase (e.g., drug volume loading) was found to have an influence on core loading and in vitro release profile.PK Studies of 3-Month FSH Microparticles

[0266] The 3-month follitropin delta-loaded microparticles prepared as described above were administered in a single subcutaneous (s.c.) injection (0.218 mg / 0.6 mL) to Sprague Dawley (SD) rats (8 rats per group), and plasma levels of follitropin delta were monitored. FIG. 13 and FIG. 14 show the mean FSH plasma concentration over 12 weeks for Formulations 45 and 51. Formulations 45, 46, and 51 exhibited prolonged exposure over 12 weeks with a release profile that was comparable to that of the triptorelin-loaded microparticles.Example 7: Diluent Studies

[0267] Studies were conducted to assess formulations having a dextran diluent or a sodium hyaluronate diluent.Viscosity StudyFormulations were prepared having the components set forth below to compare viscosity properties of formulations having a dextran diluent or a sodium hyaluronate diluent.Formulations

[0268] Viscosity testing was performed on a research rheometer (DHR2, TA Instruments) fitted with a 40 mm 1° cone measuring system and the testing gap set to 29 pm. A solvent trap cover was employed to minimize drying of the sample at the exposed edges. Following a 30 second equilibration time at 25°C, the samples were exposed to a 30 second pre-shear at a rate of 0.1s-l. This led immediately into a shear rate sweep, 0.1s-l to 10000s- 1, logarithmically scaled, 8 points per decade of shear rate, shear applied for 30 seconds at each rate with viscosity calculated over the final 5 seconds of each step.

[0269] An important parameter that can be derived from the viscosity data is the degree of viscoelasticity, i.e., the ratio of viscosity at low shear ("zero shear viscosity") to that at high shear. It was found that HA provides high viscosity at low shear rates, so that it can serve as a nonflowing support delaying sedimentation of drug-loaded microparticles. However, for injectable formulations, the diluent advantageously also exhibits low viscosity at a high shear rate, i.e., while it is being injected, so that injection through the syringe and needle can be reasonably effortless. The below data presents viscoelastic data calculated as the ratio of the viscosity at a shear rate of 0.1 sec-1 (close to zero shear) to the viscosity at a shear rate of 1000 sec-1, and shows the advantageous properties of an HA diluent.Injectability Study

[0270] To assess injectability, approximately 235 mg of lyophilized 3-month follitropin delta- loaded microparticles (having mean volume diameter of 49 pm) were weighed into vials. Ten vials were prepared and then closed with a stopper and encapsulated with an aluminum cap. Then, 2 ml syringes were filled with 1.2 ml diluent (having the components set forth above), which was transferred to each vial via a vial adapter, and the microparticles were reconstituted by gently swirling and tapping the vial. Following reconstitution, the resulting microparticle suspension was withdrawn back into the syringe and a 13 mm 27G ultra-thin wall needle was mounted on the syringe. Testing was performed on a TA.XTplusC texture analyzer from Stable Micro Systems operating at a fixed plunger speed of 2.5 mm / second. The formulation with the HA diluent exhibited better injectability as determined by the maximum force required to expel the syringe contents.Plasma Protein Adsorption

[0271] Adsorption of human plasma proteins to 3-month follitropin delta-loaded microparticles was assessed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining under reducing conditions. For the study, duplicate samples were prepared by incubating 10 mg of lyophilized FSH-loaded microparticles with 0.7 ml of a solution comprising either (A) 10 mg / ml Tween 80, 10 mM sodium phosphate, pH 7.2, (B) dextran based diluent (see above) or (C) 10 mg / ml Tween 80, 10 mM sodium phosphate, 15 mg sodium hyaluronate (MW 3.2M g / mol), for 30 minutes at ambient temperature. Then 0.3 mL human plasma was added to each vial and further incubated for 1 hour at 37°C, after which themicroparticles were washed 3 times with purified water (by centrifugation at 3000 rpm and resuspension in 1 mL purified water three times). The resulting microparticle pellets were incubated with SDS buffer under reducing conditions and extracted protein was applied to a 4-12% BisTris gel with molecular weight markers in lane 1.

[0272] The results (not shown) revealed significantly more plasma protein adsorption to microparticles preincubated with the dextran-based diluent, with the microparticles preincubated with the HA-based diluent showing the lowest degree of plasma protein adsorption. As discussed above, this may have implications for an immune response, because plasma proteins are known to act as complement with potential pro-inflammatory action.PK Studies

[0273] 3 -month follitropin delta-loaded microparticles prepared as described above and formulated with dextran-based or HA-based diluent (as described above) were administered in a single subcutaneous (s.c.) injection (0.218 mg / 0.6 mL) to Sprague Dawley (SD) rats (8 rats per group), and plasma levels of follitropin delta were monitored. FIG. 15 shows the mean FSH plasma concentration over 12 weeks. As shown in the figure, the diluent had a surprising impact on FSH plasma concentration, with greater plasma levels being sustained for a longer period of time with the HA-based formulation. As noted above, a similar study was conducted with 3 -month triptorelin-loaded microparticles. FIG. 10 shows the mean triptorelin plasma concentration over 12 weeks. Unlike the results achieved with the follitropin delta-loaded microparticles, the plasma concentration-time profile for the triptorelin-loaded microparticles was not impacted by the carrier (e.g., the results achieve with both formulations were about the same).Immune Response Studies

[0274] Immune responses of rats to s.c. injection of follitropin delta-loaded microparticles prepared as described above formulated with dextran-based or HA-based diluent as described above also were assessed. It was found that the HA formulation had anti-inflammatory properties and reduced the local inflammatory response to follitropin delta-loaded microparticles towards infiltrating neutrophils, monocytes, and macrophages via interaction with the CD44 receptor onthese cell types (data not shown). While levels of IFN-y, TNF-a, and IL-6 were similar in both groups, levels of IL-4 were higher in the HA group, indicating an anti-inflammatory response (FIG. 16). Additionally, while the total antibody signal was similar for the two groups, because the plasma concentration of FSH is higher in the HA group, that indicates the amount of neutralizing antibodies is lower in HA group as compared to the dextran group, further supporting the reduced immunogenicity of formulation in an HA-based diluent.

Claims

WHAT IS CLAIMED IS:

1. A drug-loaded microparticle comprising:(a) a drug;(b) a polymer component comprising one or more polymers selected from ester- terminated poly(D,L-lactide-co-glycolide) (PLGA) polymers having a lactide:glycolide ratio of about 50:50, ester-terminated PLGA polymers having a lactide:glycolide ratio of about 75:25, and ester-terminated polylactide (PLA) polymers;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases the drug in vivo for a period of at least 1 week after administration by subcutaneous injection.

2. The drug-loaded microparticle of claim 1 , wherein the drug is one or more selected from a small molecule drug, a peptide drug, and a protein drug.

3. The drug-loaded microparticle of any one of claims 1-2, wherein the drug is one or more selected from triptorelin, follicle stimulating hormone (FSH), leuteinizing hormone (LH), human chorionic gonadotrophin (hCG), atosiban, insulin, abiraterone, barusiban, degarelix, desmopressin, ganirelix (e.g., ganirelix acetate), gemcitabine, gonadorelin (e.g., gonadorelin acetate), menotropin or human menopausal gonadotropin (hMG), merotocin, progesterone, quinagolide, somatostatin, somatropin, and tolterodine.

4. The drug-loaded microparticle of claim 1, wherein the drug comprises two or more drugs, optionally wherein the two or more drugs comprise FSH and LH and / or hCG.

5. The drug-loaded microparticle of any one of claims 1-4, wherein the mean particle size of the drug-loaded microparticle is from about 2 pm to about 180 pm.

6. The drug-loaded microparticle of any one of claims 1-5, wherein the drug is triptorelin.

7. The drug-loaded microparticle of claim 6, wherein the triptorelin is triptorelin acetate.

8. The drug-loaded microparticle of any one of claims 6-7, comprising:(a) triptorelin;(b) one or more ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 1 month after administration by subcutaneous injection.

9. The drug-loaded microparticle of claim 8, wherein the one or more ester-terminated PLGA polymers having a lactide:glycolide ratio of about 50:50 comprise one or more selected from RG502, RG503, and RG504.

10. The drug-loaded microparticle of claim 8 or claim 9, wherein the one or more ester- terminated PLGA polymers having a lactide: glycolide ratio of about 50:50 is one ester- terminated PLGA polymer having a lactide:glycolide ratio of about 50:50.

11. The drug-loaded microparticle of claim 9, wherein the one ester-terminated PLGA polymer having a lactide:glycolide ratio of about 50:50 is RG503.

12. The drug-loaded microparticle of claim 8 or claim 9, wherein the one or more ester- terminated PLGA polymers having a lactide: glycolide ratio of about 50:50 comprises two different ester-terminated PLGA polymers having a lactide: glycolide ratio of about 50:50.

13. The drug-loaded microparticle of claim 12, wherein the two different ester-terminated PLGA polymers are present in a weight ratio of lower molecular weight PLGA to higher molecular weight PLGA of from about 99: 1 to about 51 :49, such as a ratio of about 99: 1, about 90: 10, about 80:20, about 70:30, about 60:40, about 55:45, or about 51:49.

14. The drug-loaded microparticle of claim 12 or claim 13, wherein the two different ester- terminated PLGA polymers having a lactide: glycolide ratio of about 50:50 are RG504 and RG503.

15. The drug-loaded microparticle of any one of claims 5-14, wherein the weight ratio of triptorelin to one or more ester-terminated PGLA-polymers is about 4 / 100 to about 10 / 100.

16. The drug-loaded microparticle of any one of claims 6-7, comprising:(a) triptorelin;(b) an ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymer having a lactide: glycolide ratio of about 75:25;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 3 months after administration by subcutaneous injection.

17. The drug-loaded microparticle of claim 16, wherein the ester-terminated PLGA polymer is RG753S.

18. The drug-loaded microparticle of any one of claims 16-17, wherein the weight ratio of triptorelin to ester-terminated PGLA-polymer is about 5 / 100 to about 10 / 100.

19. The drug-loaded microparticle according to any one of claims 6-7, comprising:(a) triptorelin;(b) two different ester-terminated polylactide (PLA) polymers;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases triptorelin in vivo for a period of at least 6 months after administration by subcutaneous injection.

20. The drug-loaded microparticle of claim 19, wherein the two different ester-terminated PLA polymers are present in a weight ratio of lower molecular weight PLA to higher molecular weight PLA of from about 90: 10 to about 60:40, such as a ratio of about 90: 10, about 85: 15, about 80:20, about 75:25, about 70:30, about 65:35, or about 60:40.

21. The drug-loaded microparticle of claim 19 or claim 20, wherein the two different ester- terminated polylactide PLA polymers are R203S and R205S.

22. The drug-loaded microparticle of any one of claims 19-21, wherein the weight ratio of triptorelin to PLA polymers is about 5 / 100 to about 15 / 100.

23. The drug-loaded microparticle of any one of claims 1-5, wherein the drug is FSH, optionally wherein the FSH is recombinant FSH, optionally wherein the FSH is selected from follitropin alfa, follitropin beta, and follitropin delta, further optionally wherein the FSH is follitropin delta.

24. The drug-loaded microparticle of claim 23, comprising:(a) FSH;(b) one or more ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol,wherein the microparticle releases FSH in vivo for a period of at least 1 month after administration by subcutaneous injection.

25. The drug-loaded microparticle of claim 23, comprising:(a) FSH;(b) one or more ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymers having a lactide:glycolide ratio of about 50:50;(c) a surfactant; and(d) optionally, one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 6 weeks after administration by subcutaneous injection.

26. The drug-loaded microparticle of claim 24 or claim 25, wherein the one or more ester- terminated PLGA polymers having a lactide:glycolide ratio of about 50:50 comprise one or more selected from RG502, RG503, and RG504.

27. The drug-loaded microparticle of claim 23, comprising:(a) FSH;(b) an ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymer having a lactide: glycolide ratio of about 75:25;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 3 months after administration by subcutaneous injection.

28. The drug-loaded microparticle of claim 27, wherein the ester-terminated PLGA polymer is RG753S.

29. The drug-loaded microparticle of any one of claims 27-28, wherein the weight ratio of FSH to ester-terminated PGLA-polymer is about 5 / 100 to about 10 / 100.

30. The drug-loaded microparticle of claim 23, comprising:(a) FSH;(b) two different ester-terminated polylactide (PLA) polymers;(c) a surfactant; and(d) one or both of an alkaline salt and a polyol, wherein the microparticle releases FSH in vivo for a period of at least 6 months after administration by subcutaneous injection.

31. The drug-loaded microparticle of claim 30, wherein the two different ester-terminated PLA polymers are present in a weight ratio of lower molecular weight PLA to higher molecular weight PLA of from about 90: 10 to about 60:40, such as a ratio of about 90: 10, about 85: 15, about 80:20, about 75:25, about 70:30, about 65:35, or about 60:40.

32. The drug-loaded microparticle of any one of claims 30-31, wherein the two different ester-terminated polylactide PLA polymers are R203S and R205S.

33. The drug-loaded microparticle of any one of claims 30-32, wherein the weight ratio of FSH to PLA polymers is about 5 / 100 to about 15 / 100.

34. The drug-loaded microparticle of any one of claims 1-33, comprising Poloxamer 188 or polyvinyl alcohol as a surfactant.

35. The drug-loaded microparticle of any one of claims 1-34, comprising sodium chloride as an alkaline salt.

36. The drug-loaded microparticle of any one of claims 1-35, comprising sucrose as a polyol.

37. The drug-loaded microparticle of any one of claims 1-36, comprising one or more of: a polyol content of from about 0.1% w / w to about 5% w / w, such as a sucrose and / or polyethylene glycol content of from about 0.20% w / w to about 5% w / w; a residual content of acid, such as acetic acid, of from about 0.01% w / w to about 1.0% w / w; a residual content of a buffering salt, such as citric acid, of from about 0.01% w / w to about 1.0% w / w; a residual organic solvent content of about 50 ppm to about 20000 ppm, such as an ethyl acetate content of from about 50 ppm to about 20000 ppm; and a surfactant content, such as Poloxamer 188 and / or polyethylene glycol content, of from about 0% w / w to about 4% w / w.

38. A pharmaceutical composition comprising drug-loaded microparticles according to any one of claims 1-37.

39. The pharmaceutical composition of claim 38, formulated for subcutaneous injection.

40. The pharmaceutical composition of claim 39, formulated for intramuscular injection.

41. The pharmaceutical composition of any one of claims 38-40, comprising the drug-loaded microparticles and one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

42. The pharmaceutical composition of any one of claims 38-41, comprising the drug-loaded microparticles and a pharmaceutically acceptable diluent selected from carboxymethylcellulose, dextran, and hyaluronic acid and salts thereof.

43. The pharmaceutical composition of any one of claims 38-42, comprising the drug-loaded microparticles and a pharmaceutically acceptable diluent comprising hyaluronic acid and / or a salt thereof, optionally wherein the hyaluronic acid and / or salt thereof has an average molecularweight of from about 1 MDa to about 5 MDa, further optionally wherein the hyaluronic acid and / or salt thereof has an average molecular weight of about 3.2 MDa, further optionally wherein the composition comprises from about 0.01% (w / v) to about 5% (w / v) of the hyaluronic acid and / or salt thereof, based on the volume of the pharmaceutical composition, including from about 1% (w / v) to about 3 % (w / v), or about 1.5 mg hyaluronic acid and / or a salt thereof per mL pharmaceutical composition.

44. A pharmaceutical composition comprising triptorelin-loaded microparticles according to any one of claims 1-7 or 34-37, formulated to contain an amount of the triptorelin-loaded microparticles that provides about 1 mg of triptorelin in a single dose, and / or that provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 1 week after administration of a single dose by subcutaneous or intramuscular injection.

45. A pharmaceutical composition comprising triptorelin-loaded microparticles according to any one of claims 1-15 or 34-37, formulated to contain an amount of the triptorelin-loaded microparticles that provides 3.75 mg of triptorelin in a single dose, and / or that provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 1 month after administration of a single dose by subcutaneous or intramuscular injection.

46. A pharmaceutical composition comprising triptorelin-loaded microparticles according to any one of claims 16-18 or 34-37, formulated to contain an amount of the triptorelin-loaded microparticles that provides 11.25 mg of triptorelin in a single dose, and / or that provides a mean plasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 3 months after administration of a single dose by subcutaneous or intramuscular injection.

47. A pharmaceutical composition comprising triptorelin-loaded microparticles according to any one of claims 19-22 or 34-37, formulated to contain an amount of the triptorelin-loaded microparticles that provides 22.5 mg of triptorelin in a single dose, and / or that provides a meanplasma concentration of triptorelin of at least 0.1 ng / mL for a period of at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

48. The pharmaceutical composition of any one of claims 44-47, comprising the drug-loaded microparticles and one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

49. The pharmaceutical composition of any one of claims 44-47, comprising the drug-loaded microparticles and a pharmaceutically acceptable diluent selected from carboxymethylcellulose, dextran, and hyaluronic acid and salts thereof.

50. The pharmaceutical composition of any one of claims 44-47, comprising the drug-loaded microparticles and a pharmaceutically acceptable diluent comprising hyaluronic acid and / or a salt thereof, optionally wherein the hyaluronic acid and / or salt thereof has an average molecular weight of from about 1 MDa to about 5 MDa, further optionally wherein the hyaluronic acid and / or salt thereof has an average molecular weight of about 3.2 MDa, further optionally wherein the composition comprises from about 0.01% (w / v) to about 5% (w / v) of the hyaluronic acid and / or salt thereof, based on the volume of the pharmaceutical composition, including from about 1% (w / v) to about 3 % (w / v), or about 1.5 mg hyaluronic acid and / or a salt thereof per mL pharmaceutical composition.

51. A pharmaceutical composition comprising FSH-loaded microparticles according to any one of claims 23, 24, 26 or 34-37, formulated to contain an amount of the FSH-loaded microparticles that provides about 0.35 mg FSH (e.g., follitropin delta) or more in a single dose, and / or that provides a mean plasma concentration of FSH of at least 14 mlU / mL for a period of at least 1 month after administration of a single dose by subcutaneous or intramuscular injection.

52. A pharmaceutical composition comprising FSH-loaded microparticles according to any one of claims 23-26 or 34-37, formulated to contain an amount of the FSH-loaded microparticles that provides about 0.5 mg FSH (e.g., follitropin delta) or more in a single dose, and / or thatprovides a mean plasma concentration of FSH of at least 14 mlU / mL for a period of at least 6 weeks after administration of a single dose by subcutaneous or intramuscular injection.

53. A pharmaceutical composition comprising FSH-loaded microparticles according to any one of claims 27-29 or 34-37, formulated to contain an amount of the FSH-loaded microparticles that provides about 1-5 mg of FSH (e.g., follitropin delta) in a single dose, and / or that provides a mean plasma concentration of FSH of at least 14 mlU / mL for a period of at least 3 months after administration of a single dose by subcutaneous or intramuscular injection.

54. A pharmaceutical composition comprising FSH-loaded microparticles according to any one of claims 30-37, formulated to contain an amount of the FSH-loaded microparticles that provides about 2-10 mg of FSH (e.g., follitropin delta) in a single dose, and / or that provides a mean plasma concentration of FSH of at least 14 mlU / ml for a period of at least 6 months after administration of a single dose by subcutaneous or intramuscular injection.

55. The pharmaceutical composition of any one of claims 51-54, comprising the drug-loaded microparticles and one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

56. The pharmaceutical composition of any one of claims 51-54, comprising the drug-loaded microparticles and a pharmaceutically acceptable diluent selected from carboxymethylcellulose, dextran, and hyaluronic acid and salts thereof.

57. The pharmaceutical composition of any one of claims 51-54, comprising the drug-loaded microparticles and a pharmaceutically acceptable diluent comprising hyaluronic acid and / or a salt thereof, optionally wherein the hyaluronic acid and / or salt thereof has an average molecular weight of from about 1 MDa to about 5 MDa, further optionally wherein the hyaluronic acid and / or salt thereof has an average molecular weight of about 3.2 MDa, further optionally wherein the composition comprises from about 0.01% (w / v) to about 5% (w / v) of the hyaluronic acid and / or salt thereof, based on the volume of the pharmaceutical composition, including fromabout 1% (w / v) to about 3 % (w / v), or about 1.5 mg hyaluronic acid and / or a salt thereof per mL pharmaceutical composition.

58. A method of treatment, comprising administering a pharmaceutical composition according to any one of claims 38-57 to a subject in need thereof by subcutaneous or intramuscular injection.

59. A method of administering triptorelin to a subject in need thereof, comprising administering a triptorelin-loaded microparticle according to any one of claims 1-22 or 34-37 or a composition comprising triptorelin-loaded microparticles according to any one of claims 38-50, to a subject in need thereof by subcutaneous or intramuscular injection, optionally wherein the subject is in need of treatment of one or more of hormone-responsive cancer, endometriosis, female infertility, uterine fibroids, and central precocious puberty.

60. A method of administering FSH to a subject in need thereof, comprising administering a FSH-loaded microparticle according to any one of claims 23-37 or a composition comprising FSH -loaded microparticles according to any one of claims 38-43 or 51-57 to a subject in need thereof by subcutaneous or intramuscular injection, optionally wherein the subject is in need of treatment for female infertility or male infertility.

61. A kit comprising (a) a container containing drug-loaded microparticles according to any one of claims 1-37 in lyophilized form and (b) instructions for preparation of a pharmaceutical composition comprising the microparticles and a pharmaceutically acceptable carrier.

62. A method of making drug-loaded microparticles comprising:(a) adding (i) a drug solution comprising a drug in an aqueous solvent to (ii) a polymer solution comprising one or polymers selected from ester-terminated poly(D,L-lactide-co- glycolide) (PLGA) polymers having a lactide: glycolide ratio of about 50:50, ester-terminated PLGA polymer having a lactide: glycolide ratio of about 75:25, and ester-terminated polylactide(PLA) polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the drug solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of drug-loaded microparticles comprising the drug and one or more polymers selected from ester- terminated PLGA polymers having a lactide:glycolide ratio of about 50:50, ester-terminated PLGA polymers having a lactide:glycolide ratio of about 75:25, and ester-terminated PLA polymers.

63. The method of claim 62, wherein the drug is one or more selected from a small molecule drug, a peptide drug, and a protein drug.

64. The method of claim 62 or claim 63, wherein the drug is one or more selected from triptorelin, follicle stimulating hormone (FSH), leuteinizing hormone (LH), human chorionic gonadotrophin (hCG), atosiban, insulin, abiraterone, barusiban, degarelix, desmopressin, ganirelix (e.g., ganirelix acetate), gemcitabine, gonadorelin (e.g., gonadorelin acetate), menotropin or human menopausal gonadotropin (hMG), merotocin, progesterone, quinagolide, somatostatin, somatropin, and tolterodine.

65. The method of any one of claims 62-64, wherein the drug is triptorelin, optionally wherein the triptorelin is triptorelin acetate.

66. The method of any one of claims 62-64, wherein the drug is FSH, optionally wherein the FSH is recombinant FSH, optionally wherein the FSH is selected from follitropin alfa, follitropin beta, and follitropin delta, further optionally wherein the FSH is follitropin delta.

67. The method of any one of claims 62-66, wherein the drug comprises two or more drugs, optionally wherein the drug comprises FSH and LH and / or hCG.

68. A method of making triptorelin-loaded microparticles comprising:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising a mixture of one or more ester-terminated poly(D,L-lactide- co-glycolide) (PLGA) polymers having a lactide: glycolide ratio of about 50:50 in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and optionally, one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the one or more PLGA polymers.

69. A method of making triptorelin-loaded microparticles comprising:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising an ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymer having a lactide:glycolide ratio of about 75:25 in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of triptorelin- loaded microparticles comprising triptorelin and the PLGA polymer.

70. A method of making triptorelin-loaded microparticles comprising:(a) adding (i) a triptorelin solution comprising triptorelin in an aqueous acidic solvent to (ii) a polymer solution comprising a mixture of two different ester-terminated polylactide (PLA) polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the triptorelin in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or more both of an alkaline salt and a polyol to the dispersion, and mixing to obtain a suspension of triptorelin-loaded microparticles comprising triptorelin and the PLA polymers.

71. The method of any one of claims 68-70, wherein the triptorelin is triptorelin acetate.

72. The method of any one of claims 68-71, wherein the triptorelin solution has a pH of about 4.

73. The method of any one of claims 68-72, wherein the aqueous acidic solvent is an aqueous acetic acid solvent.

74. A method of making FSH-loaded microparticles comprising:(a) adding (i) a FSH solution comprising FSH in an aqueous solvent to (ii) a polymer solution comprising an ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymer having a lactide: glycolide ratio of about 50:50 in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLGA polymer.

75. A method of making FSH-loaded microparticles comprising:(a) adding (i) a FSH solution comprising FSH in an aqueous solvent to (ii) a polymer solution comprising an ester-terminated poly(D,L-lactide-co-glycolide) (PLGA) polymer having a lactide: glycolide ratio of about 75:25 in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH solution in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or both of an alkaline salt and a polyol, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLGA polymer.

76. A method of making FSH-loaded microparticles comprising:(a) adding (i) a FSH solution comprising FSH in an aqueous solvent to (ii) a polymer solution comprising a mixture of two different ester-terminated polylactide (PLA) polymers in an ethyl acetate solution, and mixing to obtain a dispersion of the FSH in the polymer solution; and(b) adding to the dispersion (iii) a phase inversion solution comprising a surfactant and one or more both of an alkaline salt and a polyol to the dispersion, and mixing to obtain a suspension of FSH-loaded microparticles comprising FSH and the PLA polymers.

77. The method of any one of claims 74-76, wherein the phase inversion solution comprises poloxamer or polyvinylalcohol as a surfactant.

78. The method of any one of claims 74-77, wherein the phase inversion solution comprises sodium chloride as an alkaline salt.

79. The method of any one of claims 74-78, wherein the phase inversion solution comprises sucrose or polyethylene glycol as a polyol.

80. The method of any one of claims 62-79, further comprising, after step (b), adding a wash phase solution, optionally comprising an aqueous buffer and a surfactant.

81. The method of claim 80, wherein the wash phase solution comprises a citrate buffer and Poloxamer 188 and / or polyvinylalcohol.

82. The method of any one of claims 62-81, further comprising, after step (b), removing the solvent, optionally by vacuum.

83. The method of any one of claims 62-82, further comprising, after step (b), separating the microparticles from the suspension, optionally by centrifugation or filtration.

84. The method of claim 62-83, further comprising drying the microparticles, optionally by lyophilization.

85. The method of any one of claims62-84, further comprising sterilizing the microparticles by ionizing radiation, optionally by y-irradiation.

86. A triptorelin-loaded microparticle according to any one of claims 1-22 or 34-37 or a composition comprising triptorelin-loaded microparticles according to any one of claims 38-50, for use in treating one or more of hormone- responsive cancer, endometriosis, female infertility, uterine fibroids, and central precocious puberty.

87. A FSH-loaded microparticle according to any one of claims 23-37 or a composition comprising FSH -loaded microparticles according to any one of claims 38-43 or 51-57, for use in treating female infertility or male infertility.

88. Use of a triptorelin-loaded microparticle according to any one of claims 1-22 or 34-37 or a composition comprising triptorelin-loaded microparticles according to any one of claims 38-50, in the preparation of a medicament for treating one or more of hormone-responsive cancer, endometriosis, female infertility, uterine fibroids, and central precocious puberty.

89. Use of a FSH-loaded microparticle according to any one of claims 23-37 or a composition comprising FSH -loaded microparticles according to any one of claims 38-43 or 51-57, in the preparation of a medicament for treating female infertility or male infertility.

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