Microsphere-based injectable tirzepatide formulation

Abstract

This invention provides a biodegradable microsphere, wherein the microsphere (i) has a diameter of from 1 μm to 500 μm; (ii) comprises a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carries pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, releases tirzepatide for at least four weeks. This invention also provides related microsphere compositions, injectable formulations, therapeutic methods, and articles of manufacture.

Description

[0001] MICROSPHERE-BASED INJECTABLE TIRZEPATIDE FORMULATION

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 655,701 , filed June 4, 2024, the contents of which are incorporated herein by reference.

[0003] Throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

[0004] Field of the Invention

[0005] The present invention relates to methods for treating glucagon-like-peptide-1 -receptor (GLP-1 R)-related disorders via injection of tirzepatide-containing biodegradable microspheres.

[0006] Background of the Invention

[0007] GLP-1 Receptor Agonists

[0008] Type 2 diabetes is a disorder characterized by high blood glucose levels. Despite advances in the treatment of type 2 diabetes, optimal glycemic control is often not achieved. Glucagon-like peptide~1 (GLP-1) is a natural peptide of 30 amino acid residues secreted by intestinal cells after meals. It stimulates glucose-dependent insulin release and suppresses postprandial glucagon secretion. To mimic the biological function of GLP-1 , GLP-1 receptor agonists (GLP-1 RAs), such as semaglutide and tirzepatide, were developed for glycemic control in type 2 diabetes, risk reduction of major adverse cardiovascular events, weight loss, management of obesity-related indications (e.g., metabolic dysfunction-associated steatohepatitis, obstructive sleep apnea, osteoarthritis, chronic kidney disease, heart failure, and alcoholic fatty liver disease), treatment of neurodegenerative disorders (e.g., Alzheimer’s disease), and treatment of addictive disorders (e.g., alcohol use disorder, substance use disorder). In addition to GLP-1 receptor agonism, tirzepatide is also an agonist for the gastric-inhibitory-peptide (GIP) receptor, which further increases its effects on lowering blood glucose and weight loss.

[0009] Semaglutide (commercially known as Wegovy® and Ozempic®) and tirzepatide (commercially known as Mounjaro® and Zepbound®) are administered as once weekly subcutaneous injections. For chronic conditions such as diabetes and obesity, a lower injection frequency is associated with higher patient compliance and better disease control. To achieve sustained release of peptide drugs such as semaglutide and tirzepatide in a long-acting injectable formulation, encapsulation by biodegradable polymers is typically used.

[0010] PLGA Microspheres Generally

[0011] An important biodegradable material commonly used for extended-release drug delivery is polylactic co-glycolic acid copolymer (PLGA). PLGA is made of polylactic acid (PLA) units, polyglycolic acid (PGA) units, and typically both. As an FDA-approved polymer, it has been extensively investigated in many medical and pharmaceutical fields due to its biodegradability and biocompatibility. PLGA-containing microspheres have shown sustained release characteristics due to degradation and diffusion mechanisms. The drug release profile of a PLGA microsphere preparation is dependent on certain factors, such as the specific properties of the drug, the ratio of PLA to PGA, the type of end cap of the polymer (i.e. , ester or acid), the molecular weight and inherent viscosity of the polymer, the loading ratio of drug to the polymer, and the size of the microspheres.

[0012] Known Challenge of PLGA Microspheres

[0013] One major challenge for developing PLGA microspheres is achieving a high drug loading ratio that reduces the total injection burden to the patient. A microsphere drug with high drug loading is associated with fewer injection site reactions and less injection site pain. As discussed by Park et al., 2019,!i[t]he important properties of microparticles for clinical applications... include... high drug loading....” However, “(t]he obstacles hindering more widespread use of PLGA for producing sustained-release formulations for clinical use include low drug loading...” (Han et al., 2016). In particular, PLGA microspheres with encapsulated peptide drugs especially suffer from this problem of low drug loading. For example, Bydureon BCise has about 5% drug loading (calculated from the FDA label of Bydureon BCise). One-Month Lupron Depot has an 8.3% drug loading (Zhou et al., 2018). Sandostatin LAR has about 5% drug loading (Song et al., 2022), and Trelstar has drug loading in the range of 2.65%- 10.95% (calculated from the FDA label of Trelstar).

[0014] Increasing drug loading in PLGA microspheres is highly challenging. As discussed by Park et al. 2019, “[tjhe interactions among drug, PLGA, and solvent affect the microparticle properties in unpredictable ways, as their impacts are not linear... Due to each drug’s unique physicochemical properties, each drug formulation requires an ideal combination of PLGA type, solvent type, and microparticle formation conditions.” Thus, to increase drug loading in PLGA microspheres requires repeated experimentation, especially for peptide drugs such as semaglutide and tirzepatide.

[0015] There is a need for an injectable formulation of tirzepatide microspheres that are long- lasting and that have a high loading ratio.

[0016] Summary of the Invention

[0017] This invention provides a biodegradable microsphere, wherein the microsphere (i) has a diameter of from 1 μm to 500 pm; (ii) comprises a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carries pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, releases tirzepatide for at least four weeks.

[0018] This invention also provides a plurality of biodegradable microspheres, wherein the microspheres (i) have a dw value of at least 1 μm and a dgo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

[0019] This invention further provides an injectable formulation comprising (a) a pharmaceutically acceptable carrier and (b) a plurality of biodegradable microspheres wherein the microspheres (i) have a dw value of at least 1 μm and a dgo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

[0020] This invention still further provides a method for treating a subject afflicted with a GLP- 1 R-related disorder comprising administering biodegradable microspheres to the subject, wherein the microspheres (i) have a dw value of at least 1 μm and a dgo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

[0021] Finally, this invention provides an article of manufacture (kit) comprising, in separate compartments, (a) (I) a diluent and (ii) optionally, a label instructing the user to administer the biodegradable microspheres subcutaneously or intramuscularly and (b) a plurality of biodegradable microspheres, wherein the microspheres (i) have a dw value of at least 1 μm and a dgo value of 500 μm or less; (ii) comprise a polylactic-co- glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

[0022] Brief Description of the Figures

[0023] Figure 1

[0024] This figure shows a mixer (Model L5MA, Silverson, Massachusetts, US) with an in-line mixing chamber and general-purpose disintegrating head that was used to continuously create oil-in-water emulsions and generate microspheres.

[0025] Detailed Description of the Invention

[0026] This invention provides tirzepatide-containing biodegradable microspheres and methods for using them to treat GLP-1 R-related disorders. Definitions

[0027] In this application, certain terms are used which shall have the meanings set forth as fallows.

[0028] As used herein, “administering”, with respect to biodegradable microspheres, means delivering to a specified part of the body, such as subcutaneous tissue (e.g., in the abdomen, thigh, or upper arm) or intramuscular tissue (e.g., in the deltoid or buttocks). Methods of administering biodegradable microspheres to subcutaneous tissue are known and include, for example, injection with an auto-injector. See, e.g., the Bydureon BCise® label.

[0029] As used herein, a “biodegradable microsphere” comprises a polylactic-co-glycolic acid copolymer (PLGA) matrix, which matrix can include solely polylactic acid (PLA), solely polyglycolic acid (PGA), or a polymeric combination of lactic acid and glycolic acid units. In general, for certain lactic acid to glycolic acid ratios (e.g., 50:50 to 100:0), the higher a microsphere’s lactic acid content, the slower it degrades and, thus, the more stable it is. Conversely, for such ratios, the higher a microsphere’s glycolic acid content, the faster it degrades and the less stable it is. In one embodiment, the biodegradable microsphere contains a combination of lactic acid and glycolic acid units wherein the molar ratio of lactic acid to glycolic acid units (i.e., the “lactic acid to glycolic acid ratio”, or “LG ratio”) is 0:100, 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65,

[0030] 40:60, 45:55, 46:54, 47:53, 48:52, 49:51 , 50:50, 51 :49, 52:48, 53:47, 54:46, 55:45,

[0031] 60:40, 61 :39, 62:38, 63:37, 64:36, 65:35, 66:34, 67:33, 68:32, 69:31 , 70:30, 71 :29,

[0032] 72:28, 73:27, 74:26, 75:25, 76:24, 77:23, 78:22, 79:21 , 80:20, 85:15, 90:10, 95:5, or

[0033] 100:0. In another embodiment, the biodegradable microsphere contains a combination of lactic acid and glycolic acid units wherein the molar ratio of lactic acid to glycolic acid is from 5:95 to 20:80, from 20:80 to 40:60, from 40:60 to 50:50, from 40:60 to 60:40, from 50:50 to 60:40, from 60:40 to 70:30, from 70:30 to 80:20, from 80:20 to 90:10, from 90:10 to 100:0, from 50:50 to 70:30, from 60:40 to 80:20, from 70:30 to 90:10, from 80:20 to 100:0, from 40:60 to 70:30, from 50:50 to 80:20, from 60:40 tom 90:10, from 70:30 to 100:0, from 40:60 to 80:20, from 50:50 to 90:10, from 60:40 to 100:0, from 50:50 to 100:0, from 45:55 to 55:45, from 45:55 to 65:35, from 45:55 to 75:25, from 45:55 to 85:15, from 45:55 to 95:5, from 55:45 to 65:35, from 55:45 to 75:25, from 55:45 to 85:15, from 55:45 to 95:5, from 65:35 to 75:25, from 65:35 to 85:15, from 65:35 to 95:5, from 75:25 to 85:15, from 75:25 to 95:5, from 85:15 to 95:5, from 47:53 to 53:47, from 63:37 to 67:33, from 73:27 to 77:23, from 47:53 to 77:23, from 47:53 to 67:33, or from 63:37 to 77:23.. The population of biodegradable microspheres used in this invention can be homogeneous or heterogeneous with respect to the microspheres' molar ratio of lactic acid to glycolic acid. In one embodiment, the population of biodegradable microspheres is homogeneous with respect to the microspheres' molar ratio of lactic acid to glycolic acid (e.g., the population includes only microspheres wherein the molar ratio of lactic acid to glycolic acid is 50:50 or 65:35). In another embodiment, the population of biodegradable microspheres is heterogeneous (e.g., the population includes both (i) microspheres wherein the molar ratio of lactic acid to glycolic acid is 65:35, and (ii) microspheres wherein the molar ratio of lactic acid to glycolic acid is 75:25). In a preferred embodiment, the present microspheres contain PLGA having an inherent viscosity of 0.1 to 2.4 dl / g (e.g., 0.16 to 1.7 dl / g), and a molecular weight from 1 ,000 to 600,000 (e.g., from 7,000 to 240,000). In one embodiment, the biodegradable microsphere has a viscosity of 0.1 dl / g, 0.2 dl / g, 0.3 dl / g, 0.4 dl / g, 0.5 dl / g, 0.6 dl / g, 0.7 dl / g, 0.8 dl / g, 0.9 dl / g, 1 .0 dl / g, 1.1 dl / g, 1 .2 dl / g, 1 .3 dl / g, 1.4 dl / g, 1.5 dl / g, 1.6 dl / g, 1.7 dl / g, 1.8 dl / g, 1.9 dl / g, 2.0 dl / g, 2.1 dl / g, 2.2 dl / g, 2.3 dl / g, or 2.4 dl / g. In another embodiment, the biodegradable microsphere has a viscosity of from 0.1 dl / g to 0.2 dl / g, from 0.2 dl / g to 0.3 dl / g, from 0.3 dl / g to 0.4 dl / g, from 0.4 dl / g to 0.5 dl / g, from 0.5 dl / g to 0.6 dl / g, from 0.6 dl / g to 0.7 dl / g, from 0.7 dl / g to 0.8 dl / g, from 0.8 dl / g to 0.9 dl / g, from 0.9 dl / g to 1.0 dl / g, from 1.0 dl / g to 1.1 dl / g, from 1.1 dl / g to 1 .2 dl / g, from 1 .2 dl / g to 1 .3 dl / g, from 1 .3 dl / g to 1 .4 dl / g, from 1 .4 dl / g to 1 .5 dl / g, from 1 .5 dl / g to 1 .6 dl / g, from 1 .6 dl / g to 1 .7 dl / g, from 1 .7 dl / g to 1 .8 dl / g, from 1 .8 dl / g to 1 .9 dl / g, from 1 .9 dl / g to 2.0 dl / g, from 2.0 dl / g to 2.1 dl / g, from 2.1 dl / g to 2.2 dl / g, from 2.2 dl / g to 2.3 dl / g, or from 2.3 dl / g to 2.4 dl / g. In a further embodiment, the biodegradable microsphere has a viscosity of from 0.1 dl / g to 0.6 dl / g, from 0.2 dl / g to 0.6 dl / g, from 0.3 dl / g to 0.6 dl / g, from 0.6 dl / g to 1 .0 dl / g, from 1 .0 dl / g to 1 .5 dl / g, from 1 .5 dl / g to 2.0 dl / g, or from 2.0 dl / g to 2.4 dl / g. The inherent viscosity of PLGA used in the present invention is measured at a concentration of 0.1 % (w / v) in chloroform at 25°C using an Ubbelohde viscometer. Preferred embodiments of the present microspheres are (i) PLGA with L:G ratio from 47:53 to 53:47, viscosity of 0.3- 0.6 dl / g, acid-terminated; (ii) PLGA with LG ratio from 63:37 to 67:33, viscosity of 0.3- 0.5 dl / g, acid-terminated; and (iii) PLGA with LG ratio from 73:27 to 77:23, viscosity 0.3-0.5 dl / g, acid-terminated. In a further embodiment, the drug loading ratio for the present biodegradable microsphere is

[0034] (i) 10%, 10.5%, 11 %, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21 %, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31 %, 31.5%, 32%, 32.5%, 33%, 33.5%, 34%, 34.5%, 35%, 35.5%, 36%, 36.5%, 37%, 37.5%, 38%, 38.5%, 39%, 39.5%, 40%, 40.5%, 41 %, 41.5%, 42%, 42.5%, 43%, 43.5%, 44%, 44.5%, 45%, 45.5%, 46%, 46.5%, 47%, 47.5%, 48%, 48.5%, 49%, 49.5%, 50%, 50.5%, 51 %, 51.5%, 52%, 52.5%, 53%, 53.5%, 54%, 54.5%, 55%, 55.5%, 56%, 56.5%, 57%, 57.5%, 58%, 58.5%, 59%, 59.5%, or 60%;

[0035] (ii) from 10% to 10.5%, from 10.5% to 11 %, from 11 % to 11.5%, from 11.5% to 12%, from 12% to 12.5%, from 12.5% to 13%, from 13% to 13.5%, from 13.5% to 14%, from 14% to 14.5%, from 14.5% to 15%, from 15% to 15.5%, from 15.5% to 16%, from 16% to 16.5%, from 16.5% to 17%, from 17% to 17.5%, from 17.5% to 18%, from 18% to 18.5%, from 18.5% to 19%, from 19% to 19.5%, from 19.5% to 20%, from 20% to

[0036] 20.5%, from 20.5% to 21 %, from 21 % to 21 .5%, from 21 .5% to 22%, from 22% to

[0037] 22.5%, from 22.5% to 23%, from 23% to 23.5%, from 23.5% to 24%, from 24% to

[0038] 24.5%, from 24.5% to 25%, from 25% to 25.5%, from 25.5% to 26%, from 26% to

[0039] 26.5%, from 26.5% to 27%, from 27% to 27.5%, from 27.5% to 28%, from 28% to

[0040] 28.5%, from 28.5% to 29%, from 29% to 29.5%, from 29.5% to 30%, from 30% to

[0041] 30.5%, from 30.5% to 31 %, from 31 % to 31 .5%, from 31 .5% to 32%, from 32% to

[0042] 32.5%, from 32.5% to 33%, from 33% to 33.5%, from 33.5% to 34%, from 34% to

[0043] 34.5%, from 34.5% to 35%, from 35% to 35.5%, from 35.5% to 36%, from 36% to

[0044] 36.5%, from 36.5% to 37%, from 37% to 37.5%, from 37.5% to 38%, from 38% to

[0045] 38.5%, from 38.5% to 39%, from 39% to 39.5%, from 39.5% to 40%, from 40% to

[0046] 40.5%, from 40.5% to 41 %, from 41 % to 41 .5%, from 41 .5% to 42%, from 42% to

[0047] 42.5%, from 42.5% to 43%, from 43% to 43.5%, from 43.5% to 44%, from 44% to

[0048] 44.5%, from 44.5% to 45%, from 45% to 45.5%, from 45.5% to 46%, from 46% to

[0049] 46.5%, from 46.5% to 47%, from 47% to 47.5%, from 47.5% to 48%, from 48% to

[0050] 48.5%, from 48.5% to 49%, from 49% to 49.5%, from 49.5% to 50%, from 50% to

[0051] 50.5%, from 50.5% to 51 %, from 51 % to 51 .5%, from 51 .5% to 52%, from 52% to

[0052] 52.5%, from 52.5% to 53%, from 53% to 53.5%, from 53.5% to 54%, from 54% to

[0053] 54.5%, from 54.5% to 55%, from 55% to 55.5%, from 55.5% to 56%, from 56% to 56.5%, from 56.5% to 57%, from 57% to 57.5%, from 57.5% to 58%, from 58% to 58.5%, from 58.5% to 59%, from 59% to 59.5%, or from 59.5% to 60%;

[0054] (iii) from 10% to 11 %, from 11 % to 12%, from 12% to 13%, from 13% to 14%, from 14% to 15%, from 15% to 16%, from 16% to 17%, from 17% to 18%, from 18% to 19%, from 19% to 20%, from 20% to 21 %, from 21 % to 22%, from 22% to 23%, from 23% to 24%, from 24% to 25%, from 25% to 26%, from 26% to 27%, from 27% to 28%, from 28% to 29%, from 29% to 30%, from 30% to 31 %, from 31 % to 32%, from 32% to 33%, from 33% to 34%, from 34% to 35%, from 35% to 36%, from 36% to 37%, from 37% to 38%, from 38% to 39%, from 39% to 40%, from 40% to 41 %, from 41 % to 42%, from 42% to 43%, from 43% to 44%, from 44% to 45%, from 45% to 46%, from 46% to 47%, from 47% to 48%, from 48% to 49%, from 49% to 50%, from 50% to 51 %, from 51 % to 52%, from 52% to 53%, from 53% to 54%, from 54% to 55%, from 55% to 56%, from 56% to 57%, from 57% to 58%, from 58% to 59%, or from 59% to 60%;

[0055] (iv) from 10% to 12%, from 12% to 14%, from 14% to 16%, from 16% to 18%, from 18% to 20%, from 20% to 22%, from 22% to 24%, from 24% to 26%, from 26% to 28%, from 28% to 30%, from 30% to 32%, from 32% to 34%, from 34% to 36%, from 36% to 38%, from 38% to 40%, from 40% to 42%, from 42% to 44%, from 44% to 46%, from 46% to 48%, from 48% to 50%, from 50% to 52%, from 52% to 54%, from 54% to 56%, from 56% to 58%, or from 58% to 60%; or

[0056] (v) from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, 30% to 35%, from 35% to 40%, from 40% to 45%, from 45% to 50%, from 50% to 55%, from 55% to 60%, from 15% to 25%, from 15% to 30%, from 15% to 35%, from 15% to 40%, from 15% to 45%, from 15% to 50%, from 15% to 55%, from 15% to 60%, from 20% to 30%, from 20% to 35%, from 20% to 40%, from 20% to 45%, from 20% to 50%, from 20% to 55%, from 20% to 60%, from 25% to 35%, from 25% to 40%, from 25% to 45%, from 25% to 50%, from 25% to 55%, from 25% to 60%, from 30% to 40%, from 30% to 45%, from 30% to 50%, from 30% to 55%, from 30% to 60%, from 35% to 45%, from 35% to 50%, from 35% to 55%, from 35% to 60%, from 40% to 50%, from 40% to 55%, or from 40% to 60%.

[0057] The subject biodegradable microsphere (i) has a diameter from 1 μm to 500 pm, (ii) can carry a therapeutic agent (e.g., tirzepatide), and (iii) depending on its polymeric composition, degrades over a period lasting, for example, from four weeks to over six months when placed subcutaneously or intramuscularly. Microsphere diameters, set forth as ranges from dw to dso (as defined herein), include, for example, the following: from 1 μm to 20 pm, from 20 μm to 40 pm, from 40 μm to 60 pm, from 60 μm to 80 pm, from 80 μm to 100 pm, from 100 μm to 120 pm, from 120 μm to 140 pm, from 140 pm to 160 pm, from 160 μm to 180 pm, from 180 μm to 200 pm, from 200 μm to 250 pm, from 250 μm to 300 pm, from 300 μm to 350 pm, from 350 μm to 400 pm, from 400 pm to 450 pm, and from 450 μm to 500 pm. Microsphere diameters also include, for example, the following: 20 pm, 40 pm, 60 pm, 80 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, and 500 pm. Microsphere diameters, set forth as ranges from dw to d&o (as defined herein), also include, for example, the following: from 20 μm to 100 pm, from 20 μm to 150 pm, from 50 μm to 100 pm, and from 50 μm to 150 pm. In a further embodiment, the dw values for the present microsphere diameters include the following: (I) 5 pm, 10 pm, 15 pm, 20 pm, 25 pm, 30 pm, 35 pm, and 40 pm; (ii) from 5 μm to 10 pm, from 10 μm to 15 pm, from 15 μm to 20 pm, from 20 μm to 25 pm, from 25 μm to 30 pm, from 30 μm to 35 pm, and from 35 μm to 40 pm; (iii) from 5 μm to 15 μm, from 15 μm to 25 pm, and from 25 μm to 35 pm; and (iv) from 5 μm to 25 pm, from 25 μm to 40 pm, and from 5 μm to 40 pm.

[0058] In a further embodiment, the dgo values for the present microsphere diameters include the following: (i) 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 110 pm, and 120 pm; (ii) from 20 μm to 30 pm, from 30 μm to 40 pm, from 40 μm to 50 pm, from 50 μm to 60 pm, from 60 μm to 70 pm, from 70 μm to 80 pm, from 80 μm to 90 pm, from 90 μm to 100 pm, from 100 μm to 110 pm, and from 110 μm to 120 pm; (iii) from 20 μm to 40 pm, from 40 μm to 60 pm, from 60 μm to 80 pm, from 80 μm to 100 pm, or from 100 μm to 120 pm; and (iv) from 20 μm to 60 pm, from 60 μm to 100 pm, and from 20 μm to 120 pm.

[0059] The subject biodegradable microspheres can further comprise polyethylene glycol (PEG). Biodegradable PLGA microspheres (including homogeneous and heterogeneous populations thereof having defined molar ratios of lactic acid to glycolic acid units) are commercially available from, among other sources, Millipore-SIgma in the form of Degradex® products (Burlington, MA) and Evonik Industries in the form of Resomer® products (Essen, Germany). The subject biodegradable microspheres can be prepared by an “oil-in-water” single emulsion process, which consists of (a) preparing an “oil phase” by dispersing or dissolving tirzepatide or a pharmaceutically acceptable salt and biocompatible polymers (e.g., PLGA) in one or more organic solvents (e.g., dichloromethane, DMSO, or acetic acid); (b) adding the “oil phase" into a aqueous solution containing surfactants (e.g., polyvinyl alcohol); (c) emulsification by mixing, stirring, or sonication; and (d) drying to remove the organic solvents and aqueous solvents.

[0060] Alternatively, the subject biodegradable microspheres can be prepared by a “water-in- oil-in-water” double emulsion process, which consists of (a) preparing an aqueous dispersion or solution containing tirzepatide or a pharmaceutically acceptable salt; (b) adding the aqueous phase into an organic solution containing biocompatible polymers (e.g., dichloromethane solution containing PLGA); (c) primary emulsification by mixing, stirring, or sonication; (d) adding this emulsion into another aqueous solution containing surfactants (e.g., polyvinyl alcohol); (e) secondary emulsification by mixing, stirring, or sonication; and (f) drying to remove the organic solvents and aqueous solvents.

[0061] As used herein, the term “carry”, with respect to pharmaceutical tirzepatide and a biodegradable microsphere, means that the pharmaceutical tirzepatide is bound to, or otherwise contained in or on, the biodegradable microsphere in a manner permitting release from the microsphere during its biodegradation.

[0062] As used herein, the phrase “causing weight loss” in a subject includes, without limitation, (i) causing a loss of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least, 45%, at least 50%, at least 55%, or at least 60% of the subject’s body weight; (ii) causing a loss of from 5% to 10%, from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%, from 35% to 40%, from 40% to 50%, or from 50% to 60% of the subject’s body weight; and (ill) causing a loss of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55% or 60% of the subject’s body weight. Preferably, weight loss as exemplified above is measured over a time period of from one to six months (e.g., after one month, after two months, after three months, after four months, after five months, or after six months). In a further embodiment of this invention, the subject method, rather than causing weight loss, either stops weight gain or reduces its rate (e.g., by limiting weight gain over the above time period to below 1 %, 2%, 3%, 4% or 5% of the subject's body weight). As used herein, the term “diiuent” includes, without limitation, sodium chloride, carboxymethylcellulose sodium, polysorbate 80, mannitol (which can optionally be incorporated on and / or into the microspheres to improve suspendability), water, and medium chain fatty acids.

[0063] As used herein, the term “dgo value”, with respect to the present microspheres, means the 90thpercentile diameter in the microsphere population on a volume-weighted basis. The term “dso value" means the 50thpercentile diameter in the microsphere population on a volume-weighted basis. The term “dw value” means the 10ihpercentile diameter in the microsphere population on a volume-weighted basis.

[0064] An “GLP-1 R-related disorder" (i.e., a glucacon-like-peptide-1-receptor (GLP-1 R)-related disorder) includes, without limitation, obesity, diabetes (e.g., type 1 diabetes and type 2 diabetes), poor glycemic control (e.g., in type 2 diabetes), cardiovascular disease, heart failure, metabolic associated steatohepatitis, obstructive sleep apnea, osteoarthritis, chronic kidney disease, alcoholic fatty liver disease, neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Lou Gehrig’s disease, Creutzfeldt-Jakob disease, stroke, and multiple sclerosis) and addictive disorders (e.g., alcohol use disorder, substance use disorder).

[0065] As used herein, the term “pharmaceutical tirzepatide” includes, without limitation, tirzepatide free base, tirzepatide acetate salt, and other pharmaceutical salts and esters of tirzepatide. Among the pharmaceutically acceptable salts of tirzepatide, acid addition salts include, for example, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, genticinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, 2-hydroxyethanesulfonate, isethionate, nicotinate, 2-naphthalenesulfonate, oxalate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, thiocyanate, glutamate, or bicarbonate of tirzepatide. Among the pharmaceutically acceptable salts of tirzepatide, base addition salts include, for example, alkali metal salts, alkaline earth metal salts, and quaternary ammonium salts of tirzepatide. “Pharmaceutically acceptable carriers" are well known and include, without limitation, the diluents described herein.

[0066] As used herein, a biodegradable microsphere “releases” tirzepatide when some or all of the tirzepatide contained by the microsphere is freed into the microsphere’s surrounding milieu. In one embodiment, the tirzepatide release profile of the present plurality of tirzepatide-containing microspheres includes an extended period of tirzepatide release, which extended period is preferably continuous. In another embodiment, the tirzepatide release profile of the present plurality of tirzepatide- containing microspheres includes an initial tirzepatide “burst release” period followed by an extended period of tirzepatide release, which extended period is preferably continuous. By way of example, in one scenario, the tirzepatide release profile includes a five-day “burst release” period followed by a three-month continuous tirzepatide release period. In another scenario, the tirzepatide release profile includes only a three-month continuous tirzepatide release period.

[0067] With respect to the subject plurality of biodegradable microspheres, the term “burst release” means the portion (e.g., 10%) of tirzepatide released from the microspheres into a surrounding milieu (e.g., PBS solution or subcutaneous environment in an animal or a human) within the first several days after the microspheres are introduced into this milieu. In one embodiment, burst release means the portion of tirzepatide released from the microspheres into a surrounding milieu within the first one, two, three, four, five, six, or seven days after the microspheres are introduced into this milieu. In vitro, burst release can be measured, for example, by the amount of tirzepatide released and dissolved in the surrounding medium during a period of time divided by the total amount of encapsulated tirzepatide in the microspheres, or, alternatively, by the difference in amount of encapsulated tirzepatide in the microspheres at the beginning versus at the end of a measurement period. In vivo, burst release can be measured, for example, by the area-under-the-curve (AUC) of tirzepatide during the measurement period divided by the AUC from time 0 to time of complete tirzepatide release. The period of time to monitor the burst release can be, for example, within one day, within two days or within three days after the microspheres are introduced to the milieu for drug release. The burst release within one day includes the following, for example: less than 1 %, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, less than 10%, less than 15%, less than 20%, less than 25%, or less than 30%. The burst release within two days includes the following, for example: less than 1 %, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, less than 10%, less than 15%, less than 20%, less than 25%, or less than 30%. The burst release within three days includes the following, for example: less than 1 %, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, less than 10%, less than 15%, less than 20%, less than 25%, or less than 30%.

[0068] As an example of continuous tirzepatide release, in a plurality of tirzepatide-carrying biodegradable microspheres having an average release per day of X mg (after the optional burst release), the tirzepatide released per day (after the optional burst release) is, e.g., from 0.1X mg to 10X mg, from 0.2X mg to 10X mg, from 0.3X mg to 10X mg, from 0.4X mg to 10X mg, from 0.5X mg to 10X mg, from 0.1X mg to 9X mg, from 0.2X mg to 9X mg, from 0.3X mg to 9X mg, from 0.4X mg to 9X mg, from 0.5X mg to 9X mg, from 0.1X mg to 8X mg, from 0.2X mg to 8X mg, from 0.3X mg to 8X mg, from 0.4X mg to 8X mg, from 0.5X mg to 8X mg, from 0.1X mg to 7X mg, from 0.2X mg to 7X mg, from 0.3X mg to 7X mg, from 0.4X mg to 7X mg, from 0.5X mg to 7X mg, from 0.1X mg to 6X mg, from 0.2X mg to 6X mg, from 0.3X mg to 6X mg, from 0.4X mg to 6X mg, from 0.5X mg to 6X mg; from 0.1X mg to 5X mg, from 0.2X mg to 5X mg, from 0.3X mg to 5X mg, from 0.4X mg to 5X mg, from 0.5X mg to 5X mg, from 0.1X mg to 4X mg, from 0.2X mg to 4X mg, from 0.3X mg to 4X mg, from 0.4X mg to 4X mg, from 0.5X mg to 4X mg, from 0.1 X mg to 3X mg, from 0.2X mg to 3X mg, from 0.3X mg to 3X mg, from 0.4X mg to 3X mg, from 0.5X mg to 3X mg, from 0.1X mg to 2X mg, from 0.2X mg to 2X mg, from 0.3X mg to 2X mg, from 0.4X mg to 2X mg, or from 0.5X mg to 2X mg. In another example, in a plurality of tirzepatide-carrying biodegradable microspheres having an average release per week of X mg (after the optional burst release), the tirzepatide released per week (after the optional burst release) is, e.g., from 0.1X mg to 10X mg, from 0.2X mg to 10X mg, from 0.3X mg to 10X mg, from 0.4X mg to 10X mg, from 0.5X mg to 10X mg, from 0.1X mg to 9X mg, from 0.2X mg to 9X mg, from 0.3X mg to 9X mg, from 0.4X mg to 9X mg, from 0.5X mg to 9X mg, from 0.1X mg to 8X mg, from 0.2X mg to 8X mg, from 0.3X mg to 8X mg, from 0.4X mg to 8X mg, from 0.5X mg to 8X mg, from 0.1X mg to 7X mg, from 0.2X mg to 7X mg, from 0.3X mg to 7X mg, from 0.4X mg to 7X mg, from 0.5X mg to 7X mg, from 0.1X mg to 6X mg, from 0.2X mg to 6X mg, from 0.3X mg to 6X mg, from 0.4X mg to 6X mg, from 0.5X mg to 6X mg; from 0.1X mg to 5X mg, from 0.2X mg to 5X mg, from 0.3X mg to 5X mg, from 0.4X mg to 5X mg, from 0.5X mg to 5X mg, from 0.1X mg to 4X mg, from 0.2X mg to 4X mg, from 0.3X mg to 4X mg, from 0.4X mg to 4X mg, from 0.5X mg to 4X mg, from 0.1X mg to 3X mg, from 0.2X mg to 3X mg, from 0.3X mg to 3X mg, from 0.4X mg to 3X mg, from 0.5X mg to 3X mg, from 0.1X mg to 2X mg, from 0.2X mg to 2X mg, from 0.3X mg to 2X mg, from 0.4X mg to 2X mg, or from 0.5X mg to 2X mg. For example, the amount of tirzepatide released per day or per week can be indicated by the plasma concentration of tirzepatide in an animal (such as a mouse, a rat, a rabbit, or a dog) or a human after the tirzepatide-containing microspheres are injected into the animal or human. In another example, the amount of tirzepatide released per day or per week can be indicated by the concentration of tirzepatide in an appropriate medium that dissolves the released tirzepatide after the tirzepatide-containing microspheres are introduced into the medium. In yet another example, the amount of tirzepatide released per day or per week can be calculated as the difference in the amount of tirzepatide present in the microspheres before and after a period of time when the microspheres are present in an appropriate medium. The duration of continuous tirzepatide release characterized by an average release per day or per week within an abovementioned quantitative range is at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.

[0069] As used herein, the term “tirzepatide” encompasses the structure below (with CAS number 2023788-19-2) as well as its free base form and acetate salt. Tirzepatide is an agonist for both GLP-1 receptor and GIP receptor. It is commercially known and is sold by Eli Lilly and Company under the trade name Mounjaro® and Zepbound®.

[0070] As used herein, the term “subject” includes, without limitation, a mammal such as a human, a non-human primate, a dog, a cat, a horse, a sheep, a goat, a cow, a rabbit, a pig, a rat, and a mouse. In a preferred embodiment, the subject is a cat, a dog, or a horse. In another preferred embodiment, the subject is human (e.g., male or female, and adult or child). In one embodiment, the human subject has a body mass index (BMI) of 25 or higher, 27 or higher, 30 or higher, 35 or higher, 40 or higher, 45 or higher, 50 or higher, 55 or higher, 60 or higher, 65 or higher, or 70 or higher. In another embodiment, the human subject has a BMI from 25 to 27, from 25 to 30, from 27 to 30, from 30 to 35, from 30 to 40, from 30 to 45, from 30 to 50, from 30 to 55, from 30 to 60, from 30 to 65, from 30 to 70, from 40 to 45, from 40 to 50, from 40 to 55, from 40 to 60, from 40 to 65, from 40 to 70, from 45 to 50, from 45 to 55, from 45 to 60, from 45 to 65, from 45 to 70, from 50 to 55, from 50 to 60, from 50 to 65, from 50 to 70, from 55 to 60, from 55 to 65, from 55 to 70, from 60 to 65, from 60 to 70, or from 65 to 70.

[0071] As used herein, the term “therapeutically effective amount”, with respect to pharmaceutical tirzepatide carried in biodegradable microspheres, refers to the amount of pharmaceutical tirzepatide collectively carried by the total dose of biodegradable microspheres administered subcutaneously or intramuscularly. In one embodiment, the effective amount is 1 pg, 5 pg, 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850 pg, 900 pg, 950 pg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1 ,000 mg, 1 ,050 mg, 1 ,100 mg, 1 ,150 mg, 1 ,200 mg, 1 ,250 mg, 1 ,300 mg, 1 ,350 mg, 1 ,400 mg, 1 ,450 mg, 1 ,500 mg, 1 ,550 mg, 1 ,600 mg, 1 ,650 mg, 1 ,700 mg, 1 ,750 mg, 1 ,800 mg, 1 ,850 mg, 1 ,900 mg, 1 ,950 mg, or 2,000 mg. In another embodiment, the effective amount is from 1 pg to 10 pg, from 10 pg to 50 pg, from 50 pg to 100 pg, from 100 pg to 150 pg, from 150 pg to 200 pg, from 200 pg to 250 pg, from 250 pg to 300 pg, from 300 pg to 350 pg, from 350 pg to 400 pg, from 400 pg to 450 pg, from 450 pg to 500 pg, from 500 pg to 550 pg, from 550 pg to 600 pg, from 600 pg to 650 pg, from 650 pg to 700 pg, from 700 pg to 750 pg, from 750 pg to 800 pg, from 800 pg to 850 pg, from 850 pg to 900 pg, from 900 pg to 950 pg, from 950 pg to 1 mg, 1 mg to 10 mg, from 10 mg to 50 mg, from 50 mg to 100 mg, from 100 mg to 150 mg, from 150 mg to 200 mg, from 200 mg to 250 mg, from 250 mg to 300 mg, from 300 mg to 350 mg, from 350 mg to 400 mg, from 400 mg to 450 mg, from 450 mg to 500 mg, from 500 mg to 550 mg, from 550 mg to 600 mg, from 600 mg to 650 mg, from 650 mg to 700 mg, from 700 mg to 750 mg, from 750 mg to 800 mg, from 800 mg to 850 mg, from 850 mg to 900 mg, from 900 mg to 950 mg, from 950 mg to 1 ,000 mg, from 1 ,000 mg to 1 ,050 mg, from 1 ,050 mg to 1 ,100 mg, from 1 ,100 mg to 1 ,150 mg, from 1 ,150 mg to 1,200 mg, from

[0072] 1 ,200 mg to 1 ,250 mg, from 1 ,250 mg to 1 ,300 mg, from 1 ,300 mg to 1 ,350 mg, from

[0073] 1 ,350 mg to 1 ,400 mg, from 1 ,400 mg to 1 ,450 mg, from 1 ,450 mg to 1 ,500 mg, from

[0074] 1 ,500 mg to 1 ,550 mg, from 1 ,550 mg to 1 ,600 mg, from 1 ,600 mg to 1 ,650 mg, from

[0075] 1 ,650 mg to 1 ,700 mg, from 1 ,700 mg to 1 ,750 mg, from 1 ,750 mg to 1 ,800 mg, from

[0076] 1 ,800 mg to 1 ,850 mg, from 1 ,850 mg to 1 ,900 mg, from 1 ,900 mg to 1 ,950 mg, or from

[0077] 1 ,950 mg to 2,000 mg. In a further embodiment, the effective amount is from 1 pg to 250 pg, from 250 pg to 500 pg, from 500 pg to 750 pg, from 750 pg to 1 mg, 1 mg to 250 mg, from 250 mg to 500 mg, from 500 mg to 750 mg, from 750 mg to 1 ,000 mg, from 1 ,000 mg to 1 ,250 mg, from 1 ,250 mg to 1 ,500 mg, from 1 ,500 mg to 1 ,750 mg, or from 1 ,750 mg to 2,000 mg. In yet a further embodiment, the effective amount is from 1 pg to 500 pg, from 500 pg to 1 mg, 1 mg to 500 mg, from 10 mg to 500 mg, from 500 mg to 1 ,000 mg, from 1 ,000 mg to 1 ,500 mg, or from 1 ,500 mg to 2,000 mg.

[0078] As used herein, “treating” a subject afflicted with a disorder shall include, without limitation, (i) slowing, stopping or reversing the disorder's progression, (ii) slowing, stopping or reversing the progression of the disorder's symptoms, (iii) reducing the likelihood of the disorder’s recurrence, and / or (iv) reducing the likelihood that the disorder’s symptoms will recur. In the preferred embodiment, treating a subject afflicted with a disorder means (i) reversing the disorder's progression, ideally to the point of eliminating the disorder, and / or (ii) reversing the progression of the disorder’s symptoms, ideally to the point of eliminating the symptoms.

[0079] Embodiments of the Invention

[0080] This invention solves an unmet need in the art by providing an unexpectedly superior way to treat GLP-1 R-related disorders using tirzepatide. The invention does this via tirzepatide-carrying microspheres that can be administered to a subject subcutaneously or intramuscularly and release tirzepatide over time.

[0081] Specifically, this invention provides a biodegradable microsphere, wherein the microsphere (i) has a diameter of from 1 μm to 500 μm (e.g., from 5 μm to 100 pm); (ii) comprises a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carries pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, releases tirzepatide for at least four weeks. In one embodiment of the present biodegradable microsphere, the microsphere has a lactic acid to glycolic acid molar ratio of from 100:0 to 50:50. In another embodiment, the microsphere (i) has a diameter of from 5 μm to 100 pm; and (ii) has a lactic acid to glycolic acid molar ratio of 50:50, 65:35, 75:25, from 47:53 to 53:47, from 63:37 to 67:33, from 73:27 to 77:23, from 50:50 to 75:25, from 50:50 to 65:35, from 65:35 to 75:25, from 47:53 to 77:23, from 47:53 to 67:33, or from 63:37 to 77:23. In another embodiment, the microsphere further comprises polyethylene glycol (PEG). In another embodiment, the microsphere, when present subcutaneously, releases tirzepatide for longer than four weeks. In another embodiment, the microsphere, when present intramuscularly, releases tirzepatide for longer than four weeks. Preferably, the microsphere, when present subcutaneously, releases tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months. Also preferably, the microsphere, when present intramuscularly, releases tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.

[0082] In a further preferred embodiment of the present biodegradable microsphere, the microsphere has a pharmaceutical tirzepatide loading ratio of at least 10%. Preferably, the microsphere has a pharmaceutical tirzepatide loading ratio of from 10% to 12%, from 12% to 14%, from 14% to 16%, from 16% to 18%, from 18% to 20%, from 20% to 22%, from 22% to 24%, from 24% to 26%, from 26% to 28%, from 28% to 30%, from 30% to 32%, from 32% to 34%, from 34% to 36%, from 36% to 38%, from 38% to 40%, from 40% to 42%, from 42% to 44%, from 44% to 46%, from 46% to 48%, from 48% to 50%, from 50% to 52%, from 52% to 54%, from 54% to 56%, from 56% to 58%, from 58% to 60%, from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, 30% to 35%, from 35% to 40%, from 40% to 45%, from 45% to 50%, from 55% to 60%, from 15% to 25%, from 15% to 30%, from 15% to 35%, from 15% to 40%, from 15% to 45%, from 15% to 50%, from 15% to 55%, from 15% to 60%, from 20% to 30%, from 20% to 35%, from 20% to 40%, from 20% to 45%, from 20% to 50%, from 20% to 55%, from 20% to 60%, from 25% to 35%, from 25% to 40%, from 25% to 45%, from 25% to 50%, from 25% to 55%, from 25% to 60%, from 30% to 40%, from 30% to 45%, from 30% to 50%, from 30% to 55%, from 30% to 60%, from 35% to 45%, from 35% to 50%, from 35% to 55%, from 35% to 60%, from 40% to 50%, from 40% to 55%, from 40% to 60%, or from 50% to 60%. In yet a further preferred embodiment, the microsphere has a pharmaceutical tirzepatide loading ratio of from 15% to 60%.

[0083] This invention also provides a plurality of biodegradable microspheres, wherein the microspheres (i) have a dio value of at least 1 μm and a dgo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

[0084] In an embodiment of the present plurality of biodegradable microspheres, the microspheres further comprise polyethylene glycol (PEG). The PEG can be any type suitable for use in forming biodegradable microspheres (e.g., PEG1450 (Polysciences, Inc., Warrington, PA)). Moreover, the ratio of PEG to PLGA can be any ratio suitable for use in forming biodegradable microspheres (e.g., 25:100, 50:100, 75:100 or 100:100).

[0085] In another embodiment of the present plurality of biodegradable microspheres, the microspheres, when present subcutaneously, release tirzepatide for longer than four weeks. In a further embodiment of the present plurality of biodegradable microspheres, the microspheres, when present intramuscularly, release tirzepatide for longer than four weeks. Preferably, the microspheres, when present subcutaneously, release tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months. Also preferably, the microspheres, when present intramuscularly, release tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months. In a further embodiment of the present plurality of biodegradable microspheres, the microspheres (i) have a dw value of at least 5 μm and a dgo value of 100 μm or less; (ii) have a lactic acid to glycolic acid molar ratio of from 50:50 to 75:25: and (Hi) carry from 1 pg to 2,000 mg of pharmaceutical tirzepatide.

[0086] In a further preferred embodiment of the present plurality of biodegradable microspheres, the microspheres have a pharmaceutical tirzepatide loading ratio of at least 10%. Preferably, the microspheres have a pharmaceutical tirzepatide loading ratio of from 10% to 12%, from 12% to 14%, from 14% to 16%, from 16% to 18%, from 18% to 20%, from 20% to 22%, from 22% to 24%, from 24% to 26%, from 26% to 28%, from 28% to 30%, from 30% to 32%, from 32% to 34%, from 34% to 36%, from 36% to 38%, from 38% to 40%, from 40% to 42%, from 42% to 44%, from 44% to 46%, from 46% to 48%, from 48% to 50%, from 50% to 52%, from 52% to 54%, from 54% to 56%, from 56% to 58%, from 58% to 60%, from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%, from 35% to 40%, from 40% to 45%, from 45% to 50%, from 55% to 60%, from 15% to 25%, from 15% to 30%, from 15% to 35%, from 15% to 40%, from 15% to 45%, from 15% to 50%, from 15% to 55%, from 15% to 60%, from 20% to 30%, from 20% to 35%, from 20% to 40%, from 20% to 45%, from 20% to 50%, from 20% to 55%, from 20% to 60%, from 25% to 35%, from 25% to 40%, from 25% to 45%, from 25% to 50%, from 25% to 55%, from 25% to 60%, from 30% to 40%, from 30% to 45%, from 30% to 50%, from 30% to 55%, from 30% to 60%, from 35% to 45%, from 35% to 50%, from 35% to 55%, from 35% to 60%, from 40% to 50%, from 40% to 55%, from 40% to 60%, or from 50% to 60%. In yet a further preferred embodiment, the microspheres have a pharmaceutical tirzepatide loading ratio of from 15% to 60%.

[0087] This invention further provides an injectable formulation comprising (a) a pharmaceutically acceptable carrier and (b) a plurality of biodegradable microspheres wherein the microspheres (I) have a dw value of at least 1 μm and a dgo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

[0088] In an embodiment of the present injectable formulation, the microspheres further comprise polyethylene glycol (PEG). In another embodiment of the present injectable formulation, the microspheres, when present subcutaneously, release tirzepatide for longer than four weeks. In a further embodiment of the present injectable formulation, the microspheres, when present intramuscularly, release tirzepatide for longer than four weeks. Preferably, the microspheres, when present subcutaneously, release tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months. Also preferably, the microspheres, when present intramuscularly, release tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.

[0089] In a further preferred embodiment of the present injectable formulation, the microspheres have a pharmaceutical tirzepatide loading ratio of at least 10%. Preferably, the microspheres have a pharmaceutical tirzepatide loading ratio of from 10% to 12%, from 12% to 14%, from 14% to 16%, from 16% to 18%, from 18% to 20%, from 20% to 22%, from 22% to 24%, from 24% to 26%, from 26% to 28%, from 28% to 30%, from 30% to 32%, from 32% to 34%, from 34% to 36%, from 36% to 38%, from 38% to 40%, from 40% to 42%, from 42% to 44%, from 44% to 46%, from 46% to 48%, from 48% to 50%, from 50% to 52%, from 52% to 54%, from 54% to 56%, from 56% to 58%, from 58% to 60%, from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%, from 35% to 40%, from 40% to 45%, from 45% to 50%, from 50% to 55%, from 55% to 60%, from 15% to 25%, from 15% to 30%, from 15% to 35%, from 15% to 40%, from 15% to 45%, from 15% to 50%, from 15% to 55%, from 15% to 60%, from 20% to 30%, from 20% to 35%, from 20% to 40%, from 20% to 45%, from 20% to 50%, from 20% to 55%, from 20% to 60%, from 25% to 35%, from 25% to 40%, from 25% to 45%, from 25% to 50%, from 25% to 55%, from 25% to 60%, from 30% to 40%, from 30% to 45%, from 30% to 50%, from 30% to 55%, from 30% to 60%, from 35% to 45%, from 35% to 50%, from 35% to 55%, from 35% to 60%, from 40% to 50%, from 40% to 55%, from 40% to 60%, or from 50% to 60%. In yet a further preferred embodiment, the microspheres have a pharmaceutical tirzepatide loading ratio of from 15% to 60%. This invention still further provides a method for treating a subject afflicted with a GLP- 1 R~related disorder comprising administering biodegradable microspheres to the subject, wherein the microspheres (i) have a dw value of at least 1 μm and a dgo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

[0090] In an embodiment of the present therapeutic method, the microspheres further comprise polyethylene glycol (PEG).

[0091] In the preferred embodiment of the present therapeutic method, the subject is human (e.g., male orfemale).

[0092] In another embodiment of the present therapeutic method, the microspheres (i) have a d10value of at least 5 μm and a dgo value of 100 μm or less; (ii) have a lactic acid to glycolic acid molar ratio of from 50:50 to 75:25; and (iii) carry from 1 pg to 2,000 mg of pharmaceutical tirzepatide. Preferably, the microspheres have an average lactic acid to glycolic acid molar ratio of 50:50, 65:35, 75:25, from 47:53 to 53:47, from 63:37 to 67:33, from 73:27 to 77:23, from 47:53 to 77:23, from 47:53 to 67:33, from 63:37 to 77:23, or from 50:50 to 75:25.

[0093] In another embodiment of the present therapeutic method, the microspheres, when present subcutaneously, release tirzepatide for longer than four weeks. In a further embodiment of the present therapeutic method, the microspheres, when present intramuscularly, release tirzepatide for longer than four weeks. Preferably, the microspheres, when present subcutaneously, release tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months. Also preferably, the microspheres, when present intramuscularly, release tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months. In a further preferred embodiment of the present method, the microspheres have a pharmaceutical tirzepatide loading ratio of at least 10%. Preferably, the microspheres have a pharmaceutical tirzepatide loading ratio of from 10% to 12%, from 12% to 14%, from 14% to 16%, from 16% to 18%, from 18% to 20%, from 20% to 22%, from 22% to 24%, from 24% to 26%, from 26% to 28%, from 28% to 30%, from 30% to 32%, from 32% to 34%, from 34% to 36%, from 36% to 38%, from 38% to 40%, from 40% to 42%, from 42% to 44%, from 44% to 46%, from 46% to 48%, from 48% to 50%, from 50% to 52%, from 52% to 54%, from 54% to 56%, from 56% to 58%, from 58% to 60%, from 10% to 15%, from 15% to 20%, from 20% to 25%, from 25% to 30%, from 30% to 35%, from 35% to 40%, from 40% to 45%, from 45% to 50%, from 50% to 55%, from 55% to 60%, from 15% to 25%, from 15% to 30%, from 15% to 35%, from 15% to 40%, from 15% to 45%, from 15% to 50%, from 15% to 55%, from 15% to 60%, from 20% to 30%, from 20% to 35%, from 20% to 40%, from 20% to 45%, from 20% to 50%, from 20% to 55%, from 20% to 60%, from 25% to 35%, from 25% to 40%, from 25% to 45%, from 25% to 50%, from 25% to 55%, from 25% to 60%, from 30% to 40%, from 30% to 45%, from 30% to 50%, from 30% to 55%, from 30% to 60%, from 35% to 45%, from 35% to 50%, from 35% to 55%, from 35% to 60%, from 40% to 50%, from 40% to 55%, from 40% to 60%, or from 50% to 60%. In yet a further preferred embodiment, the microspheres have a pharmaceutical tirzepatide loading ratio of from 15% to 60%.

[0094] In this invention, the biodegradable microspheres can be administered to the subject using any known method (e.g., injection) appropriate for administering agents subcutaneously or intramuscularly. In one embodiment, the present method is performed only once. In another embodiment, the present method is performed a plurality of times (e.g., two times, three times, four times, five times, or more). In that embodiment, each subsequent time the method is performed, it is performed after a suitable period has lapsed since the preceding time the method was performed. This suitable time can be, for example, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer. Microsphere-based drug products and methods of delivering them are known, at least generally (e.g., Lupron® (leuprolide acetate microspheres for depot suspension (Abbvie)); and Sandostatin LAR® Depot (octreotide acetate for injectable suspension) (Novartis)). In the present method for treating a subject afflicted with a GLP-1 R-related disorder, the GLP-1 R-related disorder can be, for example, obesity, diabetes (e.g., type 1 diabetes and type 2 diabetes), poor glycemic control (e.g., in type 2 diabetes), cardiovascular disease, heart failure, metabolic associated steatohepatitis, obstructive sleep apnea, osteoarthritis, chronic kidney disease, alcoholic fatty liver disease, a neurodegenerative disease (e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Lou Gehrig’s disease, Creutzfeldt-Jakob disease, stroke, or multiple sclerosis) and an addictive disorder (e.g., alcohol use disorder, substance use disorder). As such, the following are exemplary embodiments of the present therapeutic method.

[0095] In a first embodiment, this invention provides a method for treating a human subject afflicted with obesity (i.e. , a method for causing weight loss in the subject) comprising administering the present biodegradable microspheres to the subject. The human subject treated in the present method can have any BMI above normal, such as a BMI of 25 or higher, 27 or higher, 30 or higher, 40 or higher, or 50 or higher. Preferably, the subject has a BMI of 27 or higher.

[0096] In a second embodiment, this invention provides a method for treating a human subject (e.g., an adult human subject) afflicted with diabetes (e.g., type 1 diabetes or type 2 diabetes) comprising administering the present biodegradable microspheres to the subject. Preferably, this invention provides a method for treating a human subject afflicted with type 2 diabetes comprising administering the present biodegradable microspheres to the subject.

[0097] In a third embodiment, this invention provides a method for treating a human subject (e.g., an adult human subject) afflicted with poor glycemic control (e.g., in type 2 diabetes) comprising administering the present biodegradable microspheres to the subject. That is, in this embodiment, the method improves glycemic control in the subject (e.g., lowering fasting and post-prandial glucagon levels in subjects afflicted with type 2 diabetes).

[0098] In a fourth embodiment, this invention provides a method for treating a human subject afflicted with cardiovascular disease comprising administering the present biodegradable microspheres to the subject. In a preferred embodiment, the cardiovascular disease is stroke, myocardial infarction, cardiovascular death, angina, or heart failure.

[0099] In a fifth embodiment, this invention provides a method for treating a human subject afflicted with metabolic associated steatohepatitis comprising administering the present biodegradable microspheres to the subject.

[0100] In a sixth embodiment, this invention provides a method for treating a human subject afflicted with obstructive sleep apnea comprising administering the present biodegradable microspheres to the subject.

[0101] In a seventh embodiment, this invention provides a method for treating a human subject afflicted with osteoarthritis comprising administering the present biodegradable microspheres to the subject.

[0102] In an eighth embodiment, this invention provides a method for treating a human subject afflicted with chronic kidney disease comprising administering the present biodegradable microspheres to the subject.

[0103] In a ninth embodiment, this invention provides a method for treating a human subject afflicted with alcoholic fatty liver disease comprising administering the present biodegradable microspheres to the subject.

[0104] In a tenth embodiment, this invention provides a method for treating a human subject afflicted with a neurodegenerative disease comprising administering the present biodegradable microspheres to the subject. In a preferred embodiment, the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Lou Gehrig’s disease, Creutzfeldt-Jakob disease, stroke, or multiple sclerosis.

[0105] Finally, in an eleventh embodiment, this invention provides a method for treating a human subject afflicted with an addictive disorder comprising administering the present biodegradable microspheres to the subject. In a preferred embodiment, the addictive disorder is alcohol use disorder, or substance use disorder. This invention also provides an article of manufacture (kit) comprising, in separate compartments, (a) (i) a diluent and (ii) optionally, a label instructing the user to subcutaneously or intramuscularly administer the present biodegradable microspheres to a subject, and (b) plurality of biodegradable microspheres, wherein the microspheres (i) have a dw value of at least 1 μm and a dgo value of 500 μm or less (e.g., a dw value of least 5 μm and a dgo value of 100 μm or less); (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix (preferably having a lactic acid to glycolic acid molar ratio of from 100:0 to 50:50 (e.g., 50:50, 65:35, 75:25, from 47:53 to 53:47, from 63:37 to 67:33, from 73:27 to 77:23, from 47:53 to 77:23, from 47:53 to 67:33, 63:37 to 77:23, or from 50:50 to 75:25)); (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide (e.g., from 1 pg to 2,000 mg of pharmaceutical tirzepatide); and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks (and optionally release tirzepatide for at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months). In one embodiment of the present kit, the microspheres further comprise polyethylene glycol (PEG). Where applicable, the embodiments described above for the present method are also envisioned for this article of manufacture.

[0106] In a preferred embodiment, the present kit is supplied as a single-dose kit and contains (i) a single dose vial of tirzepatide-carrying biodegradable microspheres, and (ii) a single dose vial of diluent (e.g., sterile, clear liquid solution of 0.9% w / w sodium chloride, 0.5% - 1 % w / w sodium carboxymethylcellulose, and 0.1 % w / w polysorbate-80, or sterile liquid of medium-chain fatty acids).

[0107] This invention further provides an article of manufacture comprising a syringe having therein the present injectable formulation. Ideally, this article of manufacture is ready for use without further manipulation.

[0108] This invention still further provides an article of manufacture comprising a dual chamber syringe having therein, in separate compartments, (a) a diluent, and (b) plurality of biodegradable microspheres, wherein the microspheres (i) have a dw value of at least 1 μm and a dgo value of 500 μm or less (e.g., a dw value of least 5 μm and a dgo value of 100 μm or less); (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks, wherein the diluent and microspheres can be admixed within the syringe to form an injectable formulation immediately prior to use (e.g., within 30 minutes, 20 minutes, 10 minutes, five minutes, or one minute of use).

[0109] Where applicable, the embodiments described above for the present method and present kit are also envisioned for the above two syringe-based articles of manufacture.

[0110] This invention will be better understood by reference to the examples which follow, but those skilled in the art will readily appreciate that the specific examples detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

[0111] Examples

[0112] Example 1 . Screening for conditions to prepare tirzepatide microspheres in a beaker

[0113] PLGA refers to poly-lactic-co-glycolic acid; PDLA refers to poly-D-lactic acid, which is one kind of poly-lactic acid (PLA); DMSO refers to dimethyl sulfoxide; PVA refers to polyvinyl alcohol; PBS refers to phosphate buffered saline, pH 7.4; and MW refers to molecular weight.

[0114] (i) PLGA50:50, 0.2 dl / g, acid-terminated: LG ratio = 47:53 to 53:47, Inherent viscosity = 0.16-0.24 dl / g. MW:7, 000-17,000; (ii) PLGA50:50, LG ratio = 47:53 to 53:47, 0.2 dl / g, ester-term inated: Inherent viscosity = 0.16-0.24 dl / g. MW:7, 000-17,000; (ill) PLGA50:50, 0.4 dl / g, acid-terminated: LG ratio = 47:53 to 53:47, Inherent viscosity = 0.32-0.44 dl / g. MW:24, 000-38, 000; (iv) PLGA50:50, 0.4 dl / g, ester-term inated: LG ratio = 47:53 to 53:47, Inherent viscosity = 0.32-0.44 dl / g. MW:24, 000-38, 000; (v) PLGA50:50, 0.5 dl / g, acid-terminated: LG ratio = 47:53 to 53:47, Inherent viscosity = 0.45-0.6 dl / g. MW:38, 000-54, 000; (vi) PLGA50:50, 0.5 dl / g, ester-terminated: LG ratio = 47:53 to 53:47, Inherent viscosity = 0.45-0.6 dl / g. MW: 38, 000-54, 000; (vii) PLGA50:50, 0.6 dl / g, ester-terminated: LG ratio = 47:53 to 53:47, Inherent viscosity = 0.50-0.65 dl / g; (viii) PLGA50:50, 0.7 dl / g, ester-terminated: LG ratio = 47:53 to 53:47, Inherent viscosity = 0.61-0.74 dl / g. MW: 54, 000-69, 000; (ix) PLGA65:35, 0.4 dl / g, acid- terminated: LG ratio = 63:37 to 67:33, Inherent viscosity = 0.32-0.44 dl / g. MW:24,000- 38,000; (x) PLGA75:25, 0.2 dl / g, acid-terminated: L:G ratio = 73:27 to 77:23, Inherent viscosity = 0.14-0.22 dl / g. MW: 4,000-15,000; (xi) PLGA75:25, 0.2 dl / g, ester- terminated: LG ratio = 73:27 to 77:23, Inherent viscosity = 0.16-0.24 dl / g. MW: 4,000- 15,000; (xii) PLGA75:25, 0.4 dl / g, acid-terminated: LG ratio = 73:27 to 77:23, Inherent viscosity = 0.32-0.44 dl / g. MW: 15,000-35,000; (xiii) PLGA75:25, 0.4 dl / g, ester- terminated: LG ratio = 73:27 to 77:23, Inherent viscosity = 0.32-0.44 dl / g. MW: 15,000- 35,000; (xiv) PLGA75:25, 0.6 dl / g, ester-term inated: LG ratio = 73:27 to 77:23, Inherent viscosity = 0.5-0.7 dl / g. MW: 35,000-76,000 with average about 61 ,100; (xv) PLGA75:25, 0.65 dl / g, ester-term inated: LG ratio = 73:27 to 77:23, Inherent viscosity = 0.55-0.75 dl / g. MW: about 97,000; (xvi) PLGA75:25, 0.9 dl / g, ester-term inated: LG ratio = 73:27 to 77:23, Inherent viscosity = 0.71-1.0 dl / g. MW: 76, 000-115,000; (xvii) PLGA75:25, 1.1 dl / g, ester-term inated: LG ratio = 73:27 to 77:23, Inherent viscosity = 0.9-1.3 dl / g. MW: 115,000-190,000; (xviii) PLGA85:15, 1.5 dl / g, ester-term inated: LG ratio = 83:17 to 87:13, Inherent viscosity = 1.3-1.7 dl / g. MW: 190,000-240,000.

[0115] In experiments described herein, one type of PVA (i.e. , of one molecular weight and degree of hydrolysis) at one concentration (i.e., 1 %) is used to produce microspheres. However, in this invention, other types of PVA and ether PVA concentrations are also envisioned to yield the same microspheres. For example, where 0.11 % PVA4-88, 200 pl dichloromethane and 1 ,400 r μm stirring is used to produce a certain population of microspheres, 0.05% PVA4-88, 300 pl dichloromethane and 1 ,000 r μm stirring may also be used to produce essentially the same population of microspheres. Surfactants other than PVA can also be used in this invention to produce microspheres. These other surfactants include, for example, the commonly known surfactants vitamin E, Tween-20, Tween-80, poloxamers, poloxamines, pluronic polymers (such as F68 and F127), and sodium cholate. Similarly, in experiments described herein, dichloromethane, in combination with DMSO or acetic acid, is used to produce microspheres. However, in this invention, other types of organic solvents (e.g., ethyl acetate, chloroform, acetone, propylene carbonate, and tetrahydrofuran) may be used instead of dichloromethane to yield essentially the same microspheres, assuming other experimental parameters are adjusted accordingly. Moreover, in this invention, any of a plurality of physical methods for preparing PLGA microspheres (e.g., spinning disk, spray drying, and microfluidics) may be used to yield the subject microspheres. Tirzepatide free base (Thinheal Pharmaceutical China) or tirzepatide acetate (Xingcan Pharmaceutical China) was dissolved in co-solvent #1 with sonication and shaking, and mixed with co-solvent #2, into which PLGA was added and dissolved to form the oil phase. The oil phase was injected into different volumes of PVA4-88 solution in a 50 ml beaker and emulsified with a stir bar for at least 30 minutes to form microspheres in an oil-in-water single emulsion. The microspheres were observed under light microscopy for morphology.

[0116] In general, tirzepatide free base and tirzepatide acetate were soluble in acetic acid or DMSO. Additional DCM could be mixed in without causing precipitation of tirzepatide, up to a limit. From the emulsification process, there were two types of outcomes, including good microsphere formation or poor microsphere formation (Table 1 ). The result from the emulsification process depended on the amount of tirzepatide, amount of PLGA, type of PLGA, amounts of co-solvents, among others. The outcome was sensitive to the reaction condition and unpredictable.

[0117] Table 1 . Screening for conditions to prepare tirzepatide microspheres

[0118] Example 2. Preparing tirzepatide acetate microspheres

[0119] 3 mg of tirzepatide acetate was dissolved in 100 pl DMSO with sonication and mixed with 400 pl DCM and 8 mg PLGA (Table 2). The oil phase was injected into 50 ml 1 % PVA4-88 with a stir bar at 1 ,400 r μm for 30 minutes in an oil-in-water single emulsion. The microspheres were collected by centrifugation at 500 g for 1 minute, washed with PBS, filtered with a 100 μm strainer, washed with water, and lyophilized.

[0120] To quantify tirzepatide acetate loading in microspheres, about 4 mg microspheres were dissolved in 0.5 ml acetonitrile (ACN), precipitated in 4.5 ml 10mM NaOH, and centrifuged at 4,000 g for 3 minutes to collect supernatant, which contained the extracted tirzepatide. Tirzepatide concentrations were analyzed with an Agilent HPLC system (mobile phase: 0.1 % trifluoroacetic acid (TFA) in ACN:0.1 % TEA in water=55:45; Zorbax SB~phenyl L11 column; flow rate: 1.5 ml / min; temperature: 30 °C; injection: 30 pl; detection: 230 nm).

[0121] To quantify the initial burst release of tirzepatide acetate from microspheres, about 4 mg of microspheres were mixed with 1 ml PBS in a centrifuge tube and shaken at 60 rpm at 37 °C. After two days, the supernatant was collected and analyzed with the HPLC method above. The percentage of burst release was calculated as the amount of tirzepatide acetate in the supernatant divided by total drug loading in the microsphere.

[0122] Table 2. Drug loading of tirzepatide acetate in microspheres and burst release (PLGA50:50 has L:G ratio from 47:53 to 53:47; PLGA65:35 has LG ratio from 63:37 to 67:33; PLGA75:25 has L:G ratio from 73:27 to 77:23; Viscosity of 0.2 dl / g indicates a range of 0.16-0.24 dl / g; Viscosity of 0.4 dl / g indicates a range of 0.32-0.44 dl / g;

[0123] Viscosity of 0.5 dl / g indicates a range of 0.45-0.6 dl / g) Example 3. Increasing tirzepatide acetate loading in microspheres

[0124] To increase tirzepatide acetate loading in microspheres, 3-6 mg of tirzepatide acetate was dissolved in 100 pl DMSO with sonication and mixed with 400 pl DCM and 6 mg PLGA65:35 (with LG ratio from 63:37 to 67:33), 0.4 dl / g (with viscosity range from 0.32-0.44 dl / g), acid-terminated (Table 3). The oil phase was injected into 50 ml 1 % PVA4-88 with a stir bar at 1 ,400 r μm for 30 minutes in an oil-in-water single emulsion. The microspheres were collected by centrifugation at 500 g for 1 minute, washed with PBS, filtered with a 100 μm strainer, washed with water, and lyophilized.

[0125] Tirzepatide acetate loading was analyzed with the same HPLC method in Example 2 and shown in Table 3. The highest drug loading reached 35.7%, which is very high relative to existing FDA-approved peptide-loaded PLGA microspheres. The particle sizes of microspheres were analyzed with a laser-diffraction particle size analyzer (LS13320, Beckman Coulter, US).

[0126] Table 3. Particle sizes of microspheres with increased tirzepatide acetate loading acetate microspheres in rats

[0127] 4 mg of tirzepatide acetate was dissolved in 100 pl DMSO with sonication and mixed with 400 pl DCM and 6 mg PLGA (Table 4). The oil phase was injected into 50 ml 1 % PVA4-88 with a stir bar at 1 ,400 r μm for 30 minutes in an oil-in-water single emulsion. The microspheres were collected by centrifugation at 500 g for 1 minute, washed with PBS, filtered with a 100 μm strainer, washed with water, and lyophilized. These reactions were conducted in multiple beakers in parallel and the microspheres from the same group were pooled into a higher dose. Tirzepatide acetate loading was analyzed with the same HPLC method in Example 2 and shown in Table 4. The particle sizes of microspheres were analyzed with a laserdiffraction particle size analyzer (LS13320, Beckman Coulter, US).

[0128] Table 4. Characteristics of tirzepatide acetate microspheres injected to rats (PLGA50:50 has LG ratio from 47:53 to 53:47; PLGA65:35 has LG ratio from 63:37 to 67:33;

[0129] PLGA75:25 has LG ratio from 73:27 to 77:23; Viscosity of 0.2 dl / g indicates a range of 0.16-0.24 dl / g; Viscosity of 0.4 dl / g indicates a range of 0.32-0.44 dl / g; Viscosity of 0.5 dl / g indicates a range of 0.45-0.6 dl / g)

[0130] 40 mg of microspheres from each group were resuspended in 1 ml saline and injected subcutaneously to male Sprague Dawley rats. Plasma samples were taken periodically to analyze tirzepatide concentrations (SCIEX Exion LC-MS / MS System with Triple Quad 6500+ and Analyst 1 .7.1 AB Sciex). In Table 5, all groups showed continuous tirzepatide release over at least 28 days (=672 hours). Notably, formulations 4D and 4F showed continuous tirzepatide release over 56 days (=1 ,344 hours). Table 5. Plasma tlrzepatide concentration in rats at different sampling timepoints (hour)

[0131] 4-8 mg of tlrzepatide acetate was dissolved in 200 pl DMSO with sonication and mixed with 800 pl DCM and 8-12 mg PLGA (Table 6). The oil phase was injected into 50 ml 1 % or 5% PVA4-88 with a stir bar at 1 ,500 r μm for 30 minutes in an oil-in-water single emulsion. The microspheres were collected by centrifugation at 500 g for 1 minute, washed with PBS, filtered with an 85 μm strainer, washed with water, and lyophilized. These reactions were conducted in multiple beakers in parallel and the microspheres from the same group were pooled into a higher dose.

[0132] Tlrzepatide acetate loading was analyzed with the same HPLC method in Example 2 and shown in Table 6. The particle sizes of microspheres were analyzed with a laserdiffraction particle size analyzer (LS13320, Beckman Coulter, US).

[0133] 20 mg of microspheres from each group were resuspended in 1 ml saline and injected subcutaneously to male Sprague Dawley rats. Plasma samples were taken periodically to analyze tirzepatide concentrations (SCIEX Exion LC-MS / MS System with Triple Quad 6500+ and Analyst 1 .7.1 AB Sciex). In Table 7, formulations 5A, 5B, 50, 5D, 5I, 5J, and 5K showed continuous tirzepatide release over 31 days, and formulations 5E, 5F, 5G, 5M, 5N, and 50 showed continuous tirzepatide release over 56 days.

[0134] Table 6. Characteristics of tirzepatide acetate microspheres injected to rats (PLGA50:50 has L:G ratio from 47:53 to 53:47; PLGA75:25 has L:G ratio from 73:27 to 77:23;

[0135] Viscosity of 0.4 dl / g indicates a range of 0.32-0.44 dl / g; Viscosity of 0.5 dl / g indicates a range of 0.45-0.6 dl / g)

[0136]

[0137] Table 7. Plasma tirzepatide concentration in rats Example 6. In vivo release of tirzepatide acetate microspheres in rats and dogs

[0138] 6-8 mg of tirzepatide acetate was dissolved in 200 pl DMSO with sonication and mixed with 800 pl DCM and 12 mg PLGA50:50 (with L:G ratio from 47:53 to 53:47), 0.5 dl / g (with viscosity range from 0.45-0.6 dl / g), acid-terminated (Table 8). The oil phase was injected into 50 ml 1 % or 5% PVA4-88 with a stir bar at 1 ,200 r μm or 1 ,400 r μm for 30 minutes in an oil-in-water single emulsion. The microspheres were collected by centrifugation at 500 g for 1 minute, washed with PBS, filtered with an 85 μm strainer, washed with water, and lyophilized. These reactions were conducted in multiple beakers in parallel and the microspheres from the same group were pooled into a higher dose.

[0139] Tirzepatide acetate loading was analyzed with the same HPLC method in Example 2 and shown in Table 8. The particle sizes of microspheres were analyzed with a laserdiffraction particle size analyzer (LS13320, Beckman Coulter, US).

[0140] 10 mg or 100 mg of microspheres from each group were resuspended in 1 ml saline and injected subcutaneously to male Sprague Dawley rats or male Beagle dogs, respectively. Plasma samples were taken periodically to analyze tirzepatide concentrations {SCIEX Exion LC-MS / MS System with Triple Quad 6500+ and Analyst 1 .7.1 AB Sciex). In Table 9, all formulations showed 63 days of continuous tirzepatide release in dogs. Table 8. Characteristics of tirzepatide acetate microspheres injected to rats and dogs Table 9. Plasma tirzepatide concentration in rats and dogs Example 7. Preparing tirzepatide acetate microsphere in a Silverson inline Mixer with acetic acid as co-solvent

[0141] Figure 1 shows an inline mixing chamber (Silverson Machines, Massachusetts, USA) where the oil phase (organic solution with dissolved PLGA and tirzepatide acetate) and the water phase (PVA4-88 solution) can be mixed to prepare microspheres. To prepare the oil phase, acetic acid was optionally used as a co-solvent to increase solubility of tirzepatide acetate, and its effect on drug release in vivo was studied. Specifically, 30-60 mg tirzepatide acetate was dissolved in 100 pl acetic acid in a sonicator bath, and 1 ml DMSO was added and mixed. 4 ml DCM was injected into this DMSO-acetic acid solution and mixed by vortex. Then, 60 mg PLGA50:50 (with L:G ratio from 47:53 to 53:47), 0.5 dl / g (with viscosity range from 0.45-0.6 dl / g), acid- terminated was dissolved into the solution. Five batches of this DCM-DMSO-acetic acid solution were combined and pumped into the inline mixing chamber at 8 ml / min, and the water phase (0.11 % PVA488) was pumped into the inline mixing chamber at 800 ml / min. The mixer was set at 700 r μm to prepare an oil-in-water single emulsion, which was solidified in a beaker for 5 minutes under stirring and collected on a 10 pm sieve. The microspheres were filtered through a 90 μm strainer, washed in PBS (pH=7.4) twice, then washed in water once, and lyophilized.

[0142] For microspheres prepared without acetic acid co-solvent, 30-60 mg tirzepatide acetate was dissolved in 1 ml DMSO in a sonicator bath and with a sonicator probe, and 4m I DCM was injected into this DMSO solution and mixed by vortex. Then, 60 mg PLGA50:50 (with L:G ratio from 47:53 to 53:47), 0.5 dl / g (with viscosity range from 0.45-0.6 dl / g), acid-terminated was dissolved into the solution. Five batches of this DCM-DMSO solution were combined and pumped into the inline mixing chamber at 8 ml / min, and the water phase (0.11 % PVA488) was pumped into the inline mixing chamber at 800 ml / min. The mixer was set at 1 ,000 r μm to prepare an oil-in-water single emulsion, which was solidified in a beaker for 5 minutes under stirring and collected on a 10 μm sieve. The microspheres were filtered through a 90 μm strainer, washed in PBS (pH=7.4) twice, then washed in water once, and lyophilized.

[0143] Tirzepatide acetate loading was analyzed with the same HPLC method in Example 2 and shown in Table 10. The particle sizes of microspheres were analyzed with a laserdiffraction particle size analyzer (LS13320, Beckman Coulter, US).

[0144] 100 mg of microspheres from each group were resuspended in 1 ml saline and injected subcutaneously to male Beagle dogs. Plasma samples were taken periodically to analyze tirzepatide concentrations (SCIEX Exion LC-MS / MS System with Triple Quad 6500+ and Analyst 1.7.1 AB Sciex). In Table 11 , formulations 7A, 7B, 7C, 7D, 7F, 7G, and 7H showed continuous tirzepatide release over 56 days. Table 10. Characteristics of tirzepatide acetate microspheres injected to dogs Table 11 . Plasma tirzepatide concentration In dogs Example 8. Preparing tirzepatide acetate microsphere in a Silverson inline Mixer with acetic acid as co-solvent and PLGA75:25

[0145] 20-60 mg tirzepatide acetate was dissolved in 100 pl acetic acid in a sonicator bath, and 1 ml DMSO was added and mixed. 4 ml DCM was injected into this DMSO-acetic acid solution and mixed by vortex. Then, 60 mg PLGA75:25 (with LG ratio from 73:27 to 77:23), 0.4 dl / g (with viscosity range of 0.32-0.44 dl / g), acid-terminated was dissolved into the solution. This DCM-DMSO-acetic acid solution was pumped into the Silverson inline mixing chamber at 8 ml / min, and the water phase (0.11 % PVA488) was pumped into the inline mixing chamber at 800 ml / min. The mixer was set at 800-1800 r μm to prepare an oil-in-water single emulsion, which was solidified in a beaker for 5 minutes under stirring and collected on a 10 μm sieve. The microspheres were filtered through a 75 μm or 90 μm strainer, washed in PBS (pH=7.4) twice, then washed in water once, and lyophilized.

[0146] Tirzepatide acetate loading was analyzed with the same HPLC method in Example 2 and shown in Table 12. The particle sizes of microspheres were analyzed with a laserdiffraction particle size analyzer (LS13320, Beckman Coulter, US).

[0147] Table 12. Characteristics of tirzepatide acetate microspheres made from acetic acid cosolvent and PLGA75:25 (with LG ratio from 73:27 to 77:23), 0.4 dl / g (with viscosity range of 0.32-0.44 dl / g), acid-terminated References

[0148] FDA label for Bydureon BCise.

[0149] FDA label for Trelstar.

[0150] FDA label for Tirzepatide.

[0151] Han et al. (2016) Bioerodable PLGA-based microparticles for producing sustained- release drug formulations and strategies for improving drug loading. Frontiers in Pharmacology. 7:185.

[0152] Park et al. (2019) Injectable, long-acting RIGA formulation: Analyzing PLGA and understanding microparticle formation. Journal of Controlled Release. 304:125-134.

[0153] Song et al. (2022) IVIVC of octreotide in PLGA-glucose microsphere formulation, Sandostatin LAR. AAPS PharmSciTech. 23:258.

[0154] Zhou et al. (2018) Reverse engineering the 1 -month Lupron Depot. The AAPS Journal. 20:105.

Claims

What is claimed is:1 . A biodegradable microsphere, wherein the microsphere (i) has a diameter of from 1 μm to 500 pm; (ii) comprises a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carries pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, releases tirzepatide for at least four weeks.

2. The biodegradable microsphere of claim 1 , wherein the microsphere has a lactic acid to glycolic acid molar ratio of from 100:0 to 50:50.

3. The biodegradable microsphere of claim 1 or 2, wherein the microsphere (i) has a diameter of from 5 μm to 100 pm; and (ii) has a lactic acid to glycolic acid molar ratio of 50:50, 65:35, 75:25, from 47:53 to 53:47, from 63:37 to 67:33, from 73:27 to 77:23, from 47:53 to 77:23, from 47:53 to 67:33, from 63:37 to 77:23, or from 50:50 to 75:25.

4. The biodegradable microsphere of any of claims 1-3, wherein the microsphere further comprises polyethylene glycol (PEG).

5. The biodegradable microsphere of any of claims 1-4, wherein the microsphere, when present subcutaneously or intramuscularly, releases tirzepatide for longer than four weeks.

6. The biodegradable microsphere of any of claims 1-5, wherein the microsphere has a pharmaceutical tirzepatide loading ratio of at least 10%.

7. A plurality of biodegradable microspheres, wherein the microspheres (i) have a dw value of at least 1 μm and a doo value of 500 μm or less; (ii) comprise a polylactic- co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

8. The plurality of biodegradable microspheres of claim 7, wherein the microspheres further comprise polyethylene glycol (PEG).

9. The plurality of biodegradable microspheres of claim 7 or 8, wherein the microspheres, when present subcutaneously or intramuscularly, release tirzepatide for at least eight weeks.

10. The plurality of biodegradable microspheres of any of claims 7-9, wherein the microspheres (i) have a dio value of at least 5 μm and a doo value of 100 μm or less; (ii) have a lactic acid to glycolic acid molar ratio of from 50:50 to 75:25; and (iii) carry from1 pg to 2,000 mg of pharmaceutical tirzepatide.11 . The plurality of biodegradable microspheres of any of claims 7-10, wherein the microspheres have a pharmaceutical tirzepatide loading ratio of at least 10%.

12. An injectable formulation comprising (a) a pharmaceutically acceptable carrier and (b) a plurality of biodegradable microspheres wherein the microspheres (i) have a dw value of at least 1 μm and a doo value of 500 μm or less; (ii) comprise a polylactic- co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

13. The formulation of claim 12, wherein the microspheres further comprise polyethylene glycol (PEG).

14. The formulation of claim 12 or 13, wherein the microspheres, when present subcutaneously or intramuscularly, release tirzepatide for at least eight weeks.

15. A method for treating a subject afflicted with a GLP-1 R-related disorder comprising administering biodegradable microspheres to the subject, wherein the microspheres (i) have a dio value of at least 1 μm and a doo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

16. The method of claim 15, wherein the microspheres further comprise polyethylene glycol (PEG).

17. The method of claim 15 or 16, wherein the subject is human.

18. The method of any of claims 15-17, wherein the microspheres (i) have a dio value of at least 5 μm and a doo value of 100 μm or less; (ii) have a lactic acid to glycolic acid molar ratio of from 50:50 to 75:25; and (iii) carry from 1 pg to 2,000 mg of pharmaceutical tirzepatide.

19. The method of any of claims 15-18, wherein the microspheres have an average lactic acid to glycolic acid molar ratio of 50:50, 65:35, 75:25, from 47:53 to 53:47, from 63:37 to 67:33, from 73:27 to 77:23, from 47:53 to 77:23, from 47:53 to 67:33, from 63:37 to 77:23, or from 50:50 to 75:25.

20. The method of any of claims 15-19, wherein the microspheres release tirzepatide for at least eight weeks.21 . The method of any of claims 15-20, wherein the microspheres release tirzepatide for at least three months.

22. The method of any of claims 15-21 , wherein the microspheres release tirzepatide for at least six months.

23. The method of any of claims 15-22, wherein the microspheres have a pharmaceutical tirzepatide loading ratio of at least 10%.

24. A kit comprising, in separate compartments, (a) a diluent, and (b) plurality of biodegradable microspheres, wherein the microspheres (i) have a dw value of at least 1 μm and a doo value of 500 μm or less; (ii) comprise a polylactic-co-glycolic acid copolymer (PLGA) matrix; (iii) carry a therapeutically effective amount of pharmaceutical tirzepatide; and (iv) when present subcutaneously or intramuscularly, release tirzepatide for at least four weeks.

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