WO2024151625A1

WO2024151625A1 - Non-toxic plga compositions and methods of making and using same

Info

Publication number: WO2024151625A1
Application number: PCT/US2024/010868
Authority: WO
Inventors: Barath RAMASUBRAMANIAN; Doug SOBEL
Original assignee: Georgetown University
Priority date: 2023-01-10
Filing date: 2024-01-09
Publication date: 2024-07-18

Abstract

Provided are poly(D,L-lactide-co-glycolide) copolymer (PLGA) constructs and compositions prepared using non-toxic solvent glycofurol. The PLGA constructs and compositions include very low residual amounts of glycofurol and exhibit unexpectedly prolonged in vitro and in vivo release of drugs incorporated therein. Also provided are methods for making and using the PLGA constructs and compositions and pharmaceutical compositions comprising same.

Description

NON-TOXIC PLGA COMPOSITIONS AND METHODS OF MAKING

AND USING SAME

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/438,074, filed January 10, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Issues surrounding the clinical treatment of systemic and local inflammation and other diseases, particularly in situations where repeat parenteral injections are required, poor medication compliance is suspected, or wide swings in drug levels are hazardous, has led to the development of methodologies to administer drug continuously and automatically. Such drug delivery methodologies have included pump administration of drugs and parenteral administration of drug-laden microcrystals, lipospheres, and polymers.

Poly(D,L-lactide-co-glycohde) copolymer (PLGA), a composition biodegradable compound made up of lactic acid and glycolic acid, is of great interest as a drug releasing system. The polymer can release many kinds of substances when placed in the body and is FDA-approved for human use. Such FDA-approved PLGA products include medications, growth factors, and hormones.

There have been some drawbacks using PLGA. The kinetics of PLGA drug release is mostly triphasic, so from the time of placement, there is no overall drug delivery with zeroorder (constant) kinetics. To try' to solve this problem, drug-laden PLGA has been added to gels or has been pre-incubated for days or weeks prior to use. Further, drugs within PLGA may become denatured, particularly proteins, due to the acidic environment which is generated as PLGA is hydrolyzed. Moreover, PLGA spheres are constructed with organic (volatile) solvents, most commonly chloroform and dichloromethane (DCM), which are deemed Class 2 (‘‘Solvents to Be Limited”) by the United States Pharmacopeia (USP) and are recommended to be limited to 60 and 600 ppm, respectively, by the International Conference of Harmonization (ICH) because of their inherent toxicity.

Although approved for clinical use, adverse side effects of currently used drug-laden PLGA spheres (e.g., microspheres) include sterile abscess, pain and swelling which develop in response to residual organic solvents used to dissolve the PLGA when constructing PLGA spheres. These problems still occur even after extracting the solvent from the spheres. To avert these problems, somewhat less toxic solvents have been tried with limited success.

One of the least toxic solvents that has been explored is glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether; also known as tetraglycol), a semi-polar solvent. Glycofurol is a good candidate as a PLGA solvent because it is a non-toxic solvent, biocompatible, and approved for human use. However, preparing these spheres has met very limited success. One drawback is that the duration of drug release has been short. Another drawback has been extreme variability of drug releasing properties, even among drugs of similar lipophilicity. Allhenn et al., Pharm. Res. (2011) 28:563-571.

Thus, there still exists a need for active pharmaceutical ingredients formulated for extended delivery with reduced toxicity.

SUMMARY OF THE INVENTION

As disclosed herein, improved methods for preparing PLGA-based compositions comprising any of a variety of active pharmaceutical ingredients have been developed. Advantageously, the PLGA compositions prepared according to the methods of the instant disclosure are formed with non-toxic glycofurol and without the need for toxic solvents such as chloroform and dichloromethane. As used herein, the present invention also provides compositions of various shapes and sizes comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA) in glycofurol, and at least one active pharmaceutical ingredient (API), wherein the compositions are at least substantially free of toxic volatile solvents like chloroform, dichloromethane, and dioxane. Furthermore, the PLGA compositions prepared in accordance with the methods have very reduced residual amounts of solvent, i.e., glycofurol, compared to existing methods, further reducing essentially to zero any potential toxicity attributable to the solvent used in their preparation. In addition. PLGA compositions prepared in accordance with the methods described herein show extended release of drug as compared to existing methods.

An aspect of the instant disclosure is a method of making a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API), comprising: suspending PLGA in glycofuroL thereby providing a polymer solution; combining at least one API with the polymer solution, thereby providing an API- polymer solution; and contacting the API-polymer solution with an aqueous phase or solution, thereby forming the composition.

An aspect of the instant disclosure is a method of making a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API) comprising: suspending PLGA in glycofurol, thereby providing a polymer solution; combining at least one API with the polymer solution, thereby providing an API- polymer solution; and combining the API-polymer solution with an aqueous solution, thereby forming the composition.

In certain embodiments, the API-polymer is sprayed into the aqueous solution.

An aspect of the instant disclosure is a method of making a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API): suspending PLGA in glycofurol, thereby providing a polymer solution; combining at least one API with the polymer solution, thereby providing an API- polymer solution; and pumping the API-polymer solution through a droplet generator into aqueous solution, wherein the droplet generator comprises a fine needle surrounded by an air jacket, thereby forming the composition.

In an embodiment, the fine needle consists of a 14s - 30s gauge needle.

An aspect of the instant disclosure is a method of making a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API) comprising: suspending PLGA in glycofurol, thereby providing a polymer solution; combining at least one API with the polymer solution, thereby providing an API- polymer solution; placing the API-polymer solution into a mold having a desired shape and size; and placing the mold containing the API-polymer solution into an aqueous phase, thereby forming a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), gly cofurol, and at least one API, wherein the composition has the desired shape and size of the mold.

In accordance with each of the foregoing aspects and embodiments of the instant disclosure, in an embodiment, the composition is substantially free of dichloromethane.

In accordance with each of the foregoing aspects and embodiments of the instant disclosure, in an embodiment the PLGA accounts for about 0.1% weight to about 50% weight of the polymer solution.

In accordance with each of the foregoing aspects and embodiments of the instant disclosure, in an embodiment the API accounts for about 1 % weight to about 50% weight of the polymer solution.

An aspect of the instant disclosure is a composition comprising poly(D,L-lactide-co- glycolide copolymer (PLGA) made with a PLGA concentration of I%-50% in the polymer solution and at least one active pharmaceutical ingredient (API), wherein the composition is at least substantially free of toxic volatile solvents like chloroform, dichloromethane, and dioxane.

In certain embodiments, the composition is at least substantially free of dichloromethane.

An aspect of the instant disclosure is a composition comprising poly(D,L-lactide-co- glycolide) copolymer (PLGA) and at least one active pharmaceutical ingredient (API), wherein the composition is at least substantially free of dichloromethane.

In certain embodiments, the composition comprises substantially spherical components, i.e.. takes the form of or is a sphere.

In certain embodiments, the composition takes the form of or is a microsphere. In certain embodiments, the microsphere has a diameter of about 1 micrometer to about 1000 micrometers (pm).

In certain embodiments, the PLGA comprises alactide-to-glycolide ratio of about 100:0 to about 40:60. In certain embodiments, the PLGA comprises a lactide-to-glycolide ratio of about 40:60 to about 85: 15.

In certain embodiments, the PLGA comprises a lactide-to-glycolide ratio of about

100:0. In certain embodiments, the PLGA comprises a lactide-to-glycolide ratio of about

85: 15.

In certain embodiments, the PLGA comprises a lactide-to-glycolide ratio of about 40:60.

In certain embodiments, the PLGA has a molecular weight of about 2 kDa to about 2,000 kDa.

In certain embodiments, the PLGA accounts for about 0.1% weight to about 50% weight of the polymer solution.

In certain embodiments, the concentration of API in the polymer solution is about 0.1% to about 50%. In certain embodiments, the concentration of API in the polymer solution is about 0.1% to about 40% weight of the polymer solution. In certain embodiments, the API accounts for about 1% weight to about 50% weight of the polymer solution.

A further aspect of the instant disclosure is a pharmaceutical composition comprising a composition of the disclosure and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formed by contacting a composition of the disclosure with a suitable pharmaceutically acceptable carrier.

Yet a further aspect of the instant disclosure is a method of treating or preventing a disease, disorder, or condition, comprising administering to a subject in need thereof an effective amount of a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA) glycofurol, and at least one active pharmaceutical ingredient (API), wherein the composition is substantially free of dichloromethane, and wherein the API is therapeutically effective for treating or preventing the disease, disorder, or condition.

In certain embodiments, the disease, disorder, or condition is selected from the group comprising of cancer, autoimmune disease, transplant rejection, allergy, asthma, anemia, viral infection, bacterial infection, fungal infection, genetic disorder, infertility, pregnancy, and any combination thereof.

In certain embodiments, the subject is an animal.

In certain embodiments, the subject is a human.

In certain embodiments, the subject is a plant and the disease, disorder, or condition is selected from the group consisting of tumor, viral infection, bacterial infection, fungal infection, genetic disorder, and any combination thereof. In accordance with each of the foregoing aspects and embodiments, in certain embodiments the API is any substance or combination of substances, including any organic or inorganic substances, which are capable of exerting pharmacological activity or otherwise have effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or condition in a subj ect.

In accordance with each of the foregoing aspects and embodiments, in certain embodiments the API is selected from the group comprising of anti-inflammatory agents, anti- angiogenic agents, anti-cancer agents, anti-viral agents, anti-bacterial agents, anti-fungal agents, anti-hypertensive agents, hormones, insulin, clotting factors, cytokines, growth factors, enzymes, other polypeptides, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a PLGA droplet generator appartus used in Example 1. Shown are Syringe S, Syringe Pump D, Flow Regulator F, and Pump P, where dl is the distance between the needle tip and the air flow tube and d2 is the distance between the needle tip and the surface of the double-distilled water (ddELO).

FIG. 2A is a photomicrographic image of glycofurol formulated spheres prepared with 20% PLGA and 16.089% (w/w) dexamethasone load.

FIG. 2B is a graph depicting size distribution of glycofurol formulated compositions determined by software image measurements (ImageJ).

FIG. 3 A - FIG. 3D are HPLC chromatograms of the following preparations: FIG. 3A, PLGA without dexamethasone (DEX). FIG 3B, dexamethasone (DEX) alone at a concentration of 2 pg mL'¹. FIG. 3C, dexamethasone-loaded PLGA spheres at one week. FIG. 3D, dexamethasone-loaded PLGA spheres at 5 weeks.

FIG. 4A - FIG. 4B are graphs depicting results of an Alamar Blue assay to determine cytotoxicity of PLGA spheres made from dichlormethane (DCM) and glycofurol (Glyc) on splenocytes (FIG. 4A) and fibroblasts (FIG. 4B). * indicates p<0.05, ** indicates p<0.01 compared to control (cells with no PLGA spheres).

FIG. 5 is a graph depicting cumulative drug release (percent) over time from PLGA compositions constructed with either 20% or 5% PLGA and 20% dexamethasone in glycofurol. * indicates p<0.05. ** indicates pO.OOOl. FIG. 6 is a graph depcting accumulated drug release (percent) over time from PLGA compositions made from a 20% PLGA solution constructed with a drug load with 20% and 1% dexamethasone in glycofurol.

FIG. 7 is a graph depicting accumulated drug release (percent) over time from 20% drug (DEX) in 20% PLGA spheres made from glycofurol and di chlormethane (DCM). * indicates p<0.05, ** indicates p<0.001.

FIG. 8 is a graph depicting percent water diffusion into PLGA spheres constructed with either glycofurol or dichlormethane (DCM) as a solvent with a PLGA concentration of 20% and drug (dexamethasone) of 20%. ** indicates p <0.005.

FIG. 9 is a graph depicting accumulated release (percent) over time of lipophilic dexamethsone (DEX) and hydrophilic dexamethasone salt (DSP) from PLGA compositions.

FIG. 10 is a graph depicting accumulated release (percent) over time of Enbrel from 20% and 30% PLGA glycofurol formulated compositions.

FIG. 11 is a graph depicting accumulated release (percent) over time of tacrolimus and rapamycin from 30% PLGA glycofurol formulated compositions.

FIG. 12 is a graph depicting accumulated release (percent) over time of lenalidomide and hydrocortisone (HC) from 20% PLGA glycofurol formulated compositions.

FIG. 13 is a graph depicting accumulated release (percent) over time of heparin from 20% PLGA (75:25) glycofurol formulated compositions.

FIG. 14 is a graph depicting accumulated release (percent) over time of tafacinib from PLGA glycofurol formulated compositions.

FIG. 15 is a graph depicting average blood glucose in streptozoticin-induced diabetic mice treated with mouse islet cell line (Min6 cells) mixed with matrigel cell biomatrix either with (Test) or without (Control) PLGA compositions. N = 5 in each group.

FIG. 16A is a graph depicting accumulated release (percent) over time of 20% rabbit IgG (RIgG) from 20%, 30%, and 40% PLGA glycofurol formulated compositions, with and without titanium dioxide (TiCh). X-axis, days; Y-axis, accumulated release (percent).

FIG. 16B is a graph depicting accumulated release (percent) over time of 20% dexamethasone from 20%, 30%, and 40% PLGA glycofurol formulated compositions, with and without titanium dioxide (TiCh). X-axis, days; Y-axis, accumulated release (percent). FIG. 17 is a graph depicting accumulated release (percent) over time of 1%, 5%, and 20% rapamycin from 20% PLGA (75:25) glycofurol formulated compositions.

FIG. 18 is a graph depicting the effect of lactide:glycolide ratio of PLGA on drug release characteristics for 20% lipophilic dexamethasone (DEX), 20% PLGA in glycofurol formulated compositions.

FIG. 19A is a graph depicting the effect of molecular weight of PLGA on drug release characteristics for 20% lipophilic dexamethasone (DEX), 20% 50:50 PLGA in glycofurol formulated compositions.

FIG. 19B is a graph depicting the effect of molecular weight of PLGA on drug release characteristics for 20% lipophilic dexamethasone (DEX), 20% 50:50 PLGA in glycofurol formulated compositions.

FIG. 20A is a graph depicting the effect of polymer concentration on release of rapamycin from PLGA (75:25) glycofurol formulated compositions.

FIG. 20B is a graph depicting the effect of polymer concentration on release of dexamethasone (DEX) from PLGA (75:25) glycofurol formulated compositions.

FIG. 21 is a graph depicting the effect of different concentrations of drug in polymer solution on release rate of non-peptide drugs dexamethasone (Dex), hydrocortisone (HC), rapamycin, and lenalidomide from PLGA 20% glycofurol formulated compositions. X-axis, days; Y-axis, accumulated release percent.

FIG. 22 is a graph depicting the effect of different concentrations of drug in polymer solution on release rate of non-peptide drugs rapamycin and tacrolimus from 30% PLGA glycofurol formulated compositions.

FIG. 23 is a graph depicting the effect of different concentrations of drug in polymer solution (5% vs. 20%) on release of non-peptide drug tofacitinib from 20% PLGA glycofurol formulated compositions.

FIG. 24 is a graph depicting the effect of different concentrations of drug in polymer solution (1% vs. 5% vs. 20%) on release of peptide drug Enbrel from 20% PLGA glycofurol formulated compositions.

FIG. 25 is a graph depicting the effect of different concentrations of drug in polymer solution (1% vs. 5% vs. 20%) and polymer concentration (20% vs. 30%) on release of peptide drug Enbrel from PLGA glycofurol formulated compositions. FIG. 26A is a graph depicting the effect of different concentrations of drug in polymer solution (5% vs. 20%) on release of peptide drug heparin from 20% PLGA (75:25) glycofurol formulated compositions. These data are important since they demonstrate that the delivery PLGA composition described herein can uniquely also be used to release hydrophobic drugs.

FIG. 26B is a graph depicting the effect of different concentrations of drug in polymer solution (1% vs. 5% vs. 20%) and polymer concentration (20% vs. 30%) on release of peptide drug heparin from PLGA (75:25) glycofurol formulated compositions. X-axis, days; Y-axis, accumulated release percent.

FIG. 27 is a graph depicting the effect of different concentrations of drug in polymer solution (5% vs. 20%) on release of the peptide drug Abatacept from 20% PLGA glycofurol formulated compositions.

FIG. 28 is a graph depicting the effect of different concentrations of drug in polymer solution (5% vs. 20%) on release of rabbit IgG from 20% PLGA glycofurol formulated compositions. Data for 5% dexamethasone (Dex) is shown for comparison. X-axis, days; Y- axis, accumulated release percent.

FIG. 29 is a graph depicting peptide and non-peptide drug release from PLGA formulations with gycofurol. All drugs at 20% load; all PLGA 20% glycofurol formulated compositions. X-axis, days; Y-axis, accumulated release percent.

FIG. 30 is a graph depicting the effect of 1% T1O2 on release of dexamethasone from glycofurol formulated compositions X-axis, days; Y-axis, accumulated release percent.

FIG. 31 is a graph depicting the effects of PLGA polymer concentration (20% vs. 30% vs. 40%) and T1O2 (1% or none) on release of 5% dexamethasone (Dex). X-axis, days; Y-axis, accumulated release percent.

FIG. 32 is a graph depicting the effects of PLGA polymer concentration (20% vs. 30% vs. 40%) and TiCh (1% or none) on release of 20% rabbit IgG (RIgG) from glycofurol formulated compositions. X-axis, days; Y-axis, accumulated release percent.

FIG. 33 is a graph depicting the effects of PLGA polymer concentration (20% vs 30% vs 40%) and T1O2 (1% or none) on release of 5% rabbit IgG (RIgG) from glycofurol formulated compositions. X-axis, days; Y-axis, accumulated release percent. FIG. 34 is a graph depicting accumulated release of dexamethasone (DEX) (1% vs 20%), rapamycin (RAP) (1% vs. 20%), and Enbrel (1% vs. 20%) from 20% PLGA compositions.

FIG. 35A is a graph depicting the effect on proliferation of ConA- or lipopolysaccharide (LPS)-stimulated splenocytes of dexamethasone (DEX) (1 % vs. 20%), rapamycin (RAP) (1% vs. 20%), and Enbrel (1% vs. 20%) released from 20% PLGA glycofurol formulated compositions.

FIG. 35B is a graph depicting the effect on proliferation of Con-A- or lipopolysaccharide (LPS)-stimulated splenocytes of dexamethasone (DEX) (1% vs. 20%), rapamycin (RAP) (1% vs. 20%), and Enbrel (1% vs. 20%) released from 20% PLGA glycofurol formulated compositions Data is for days 14-16. Y-axis, net absorbance.

FIG. 35C is a graph depicting the effect on proliferation of Con-A- or lipopolysaccharide (LPS)-stimulated splenocytes of dexamethasone (DEX) (1% vs. 20%), rapamycin (RAP) (1% vs. 20%), and Enbrel (1% vs. 20%) released from 20% PLGA glycofurol formulated compositions. Data is for days 21-23. Y-axis, net absorbance.

FIG. 35D is a graph depicting the effect on proliferation of Con-A- or lipopolysaccharide (LPS)-stimulated splenocytes of dexamethasone (DEX) (1% vs. 20%), rapamycin (RAP) (1% vs. 20%). and Enbrel (1% vs. 20%) released from 20% PLGA glycofurol formulated compositions. Data is for days 42-45. Y-axis, net absorbance.

FIG. 36A is a graph depicting accumulated release of immunoglobulin from PLGA glycofurol formulated PLGA composition shaped as rods.

FIG. 36B is a graph depicting accumulated release of rapamycin from PLGA- gly cofurol formulated PLGA composition shaped as rods.

FIG. 37 is a graph depicting accumulated release of dexamethasone from PLGA- glycofurol formulated PLGA microspheres produced by an electrospray technique.

FIG. 38 is a graph depicting accumulated release of sodium citrate, an inorganic molecule, from PLGA microspheres using a glycofurol technique.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions Glycofurol is represented by the structure

where n is an integer > 1 . The molecular weight of glycofurol will vary in accordance with the value(s) of n. Glycofurol is readily available from any of a variety of commercial suppliers.

Biocompatible, biodegradable polymers suitable for controlled drug delivery are known in the art and include polylactic acid (PLA), polygly colic acid (PGA), poly(lactic-co-glycolic acid) (PLGA). polycaprolactone (PCL), polyhydroxyalkanoate (PHA), poly(lactic acid)- poly(ethylene oxide) (PLA-PEG), polyanhydrides, poly(ester anhydrides), polymethylmethacrylate (PMMA), poly(2-hydroxyethyl methacrylate) (pHEMA), poly caprolactone (PCL), cellulose acetate, chitosan, and copolymers and blends thereof. While the instant disclosure contemplates using any of the foregoing polymers, a particularly exemplary polymer for controlled delivery’ of drugs is PLGA. PLGA is well-described in the art and is readily available from any of a variety of commercial suppliers. Depending on the ratio of lactide to glycolide used for the polymerization, different forms of PLGA can be obtained; these are usually identified in regard to the molar ratio of the monomers used (e.g., PLGA 75:25 identifies a copolymer whose composition is 75% lactic acid and 25% glycolic acid).

As used herein, a ‘"composition” or “composite” refers to generally a solid structure of various shapes and sizes, including but not limited to cube, cuboid, triangular, or cylindrical, configurations. As used herein, a “microsphere” refers to a generally spherical solid structure having a diameter of about 1 pm to about 999 pm. For ease of reference, in certain embodiments, the diameter of a microsphere may be somewhat less than about 1 pm, e.g., about 0.01 pm (10 nm) to about 0.99 pm. For ease of reference, in certain embodiments, the diameter of a microsphere may be somewhat greater than about 999 pm, e.g., about 1000 pm (1 mm) to about 10,000 pm.

As used herein, “anti-inflammatory agent” refers to any pharmaceutically acceptable agent capable of reducing an inflammatory response in a mammal. Anti-inflammatory agents include corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs), anti-leukotrienes, and immune-selective anti-inflammatory derivatives (ImSAIDs). Corticosteroids include, without limitation, prednisone, prednisolone, methylprednisolone, cortisol (hydrocortisone), cortisone, beclometasone, betamethasone, and dexamethasone. NSAIDs include, without limitation, aspirin, ibuprofen, naproxen, fenoprofen, ketoprofen, indomethacin, tolmetin, sulindac, diclofenac, etodolac, ketorolac, piroxicam, meloxicam, tenoxicam, droxicam, and lomoxicam. Anti-leukotrienes include, without limitation, montelukast, zafirlukast, pranlukast, and 5 -lipoxygenase inhibitors, such as zileuton.

As used herein, the phrase “substantially free"’ means at least 80% free. In some embodiments, the phrase “substantially free” means at least 81% free. In some embodiments, the phrase “substantially free” means at least 82% free. In some embodiments, the phrase “substantially free” means at least 83% free. In some embodiments, the phrase “substantially free” means at least 84% free. In some embodiments, the phrase “substantially free” means at least 85% free. In some embodiments, the phrase “substantially free” means at least 86% free. In some embodiments, the phrase “substantially free” means at least 87% free. In some embodiments, the phrase “substantially free” means at least 88% free. In some embodiments, the phrase “substantially free” means at least 89% free. In some embodiments, the phrase “substantially free” means at least 90% free. In some embodiments, the phrase “substantially free” means at least 91% free. In some embodiments, the phrase “substantially free” means at least 92% free. In some embodiments, the phrase “substantially free” means at least 93% free. In some embodiments, the phrase “substantially free” means at least 94% free. In some embodiments, the phrase “substantially free” means at least 95% free. In some embodiments, the phrase “substantially free” means at least 96% free. In some embodiments, the phrase “substantially free” means at least 97% free. In some embodiments, the phrase “substantially free” means at least 98% free. In some embodiments, the phrase “substantially free” means at least 99% free. In some embodiments, the phrase “substantially free” means 100% free.

Examples of Class 2 solvents are show n in Table 1, where PDE refers to permitted daily exposure.

Table 1: Class 2 solvents

Solvent PDE Cone. Limit

(mg/ day) (ppm)

Acetonitrile 4.1 410

Chlorobenzene 3.6 360

Chloroform 0.6 60

Cyclohexane 38.8 3880

1,2-Dichloroethene 18.7 1870 1 ,2-Dimethoxy ethane 1.0 100 N,N-Dimethylacetamide 10.9 1090 N,N-Dimethylformamide 8.8 880 1,4-Dioxane 3.8 380 2-Ethoxy ethanol 1.6 160 Ethylene glycol 6.2 620 Formamide 2.2 220

Hexane 2.9 290

Methanol 30.0 3000

2-Methoxy ethanol 0.5 50 Methylbutylketone 0.5 50 Methyl cy cl ohexane 11.8 1180 Methylene chloride 6.0 600 N-Methylpyrrolidone 5.3 530 Nitromethane 0.5 50 Pyridine 2.0 200 Sulfolane 1.6 160 Tetrahydrofuran 7.2 720 Tetralin 1.0 100 Toluene 8.9 890 Trichloroethylene 0.8 80 Xylene 21.7 2170

II. Compositions

An aspect of the disclosure is a composition such as but not limited to a sphere or microsphere comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol and at least one active pharmaceutical ingredient (API), wherein the microsphere is substantially free of dichloromethane. Such compositions can be prepared, for example, using the methods disclosed herein.

In certain embodiments, the glycofurol content of the liquid portion of the polymer solution is approximately 60-100% although 80-100 % is best.

In certain embodiments, the microsphere has a diameter of about 1 to about 1000 micrometers (pm). In certain embodiments, the microsphere has a diameter of about 1 to about 500 pm. In certain embodiments, the microsphere has a diameter of about 1 to about 250 pm. In certain embodiments, the microsphere has a diameter of about 1 to about 100 pm. In certain embodiments, the microsphere has a diameter of about 1 to about 50 pm. In certain embodiments, the microsphere has a diameter of about 1 to about 10 pm. In certain embodiments, the microsphere has a diameter of about 1 pm.

In certain embodiments, the microsphere has a diameter of about 10 to about 1000 pm.

In certain embodiments, the microsphere has a diameter of about 10 to about 500 pm. In certain embodiments, the microsphere has a diameter of about 10 to about 250 pm. In certain embodiments, the microsphere has a diameter of about 10 to about 100 pm. In certain embodiments, the microsphere has a diameter of about 10 to about 50 pm. In certain embodiments, the microsphere has a diameter of about 10 to about 20 pm. In certain embodiments, the microsphere has a diameter of about 10 pm.

In certain embodiments, the microsphere has a diameter of about 25 to about 1000 pm. In certain embodiments, the microsphere has a diameter of about 25 to about 500 pm. In certain embodiments, the microsphere has a diameter of about 25 to about 250 pm. In certain embodiments, the microsphere has a diameter of about 25 to about 100 pm. In certain embodiments, the microsphere has a diameter of about 25 to about 50 pm. In certain embodiments, the microsphere has a diameter of about 25 pm.

In certain embodiments, the microsphere has a diameter of about 50 to about 1000 pm. In certain embodiments, the microsphere has a diameter of about 50 to about 500 pm. In certain embodiments, the microsphere has a diameter of about 50 to about 250 pm. In certain embodiments, the microsphere has a diameter of about 50 to about 200 pm. In certain embodiments, the microsphere has a diameter of about 50 to about 100 pm. In certain embodiments, the microsphere has a diameter of about 50 pm.

In certain embodiments, the microsphere has a diameter of about 100 to about 1000 pm. In certain embodiments, the microsphere has a diameter of about 100 to about 500 pm. In certain embodiments, the microsphere has a diameter of about 100 to about 250 pm. In certain embodiments, the microsphere has a diameter of about 100 to about 200 pm. In certain embodiments, the microsphere has a diameter of about 100 pm.

In certain embodiments, the microsphere has a diameter of about 200 to about 1000 pm. In certain embodiments, the microsphere has a diameter of about 200 to about 500 pm. In certain embodiments, the microsphere has a diameter of about 200 to about 250 pm. In certain embodiments, the microsphere has a diameter of about 200 pm.

In certain embodiments, the microsphere has a diameter of about 250 to about 1000 pm. In certain embodiments, the microsphere has a diameter of about 250 to about 500 pm. In certain embodiments, the microsphere has a diameter of about 250 pm. In certain embodiments, the microsphere has a diameter of about 500 to about 1000 m. In certain embodiments, the microsphere has a diameter of about 500 pm.

In certain embodiments, the microsphere has a diameter of about 1000 pm.

In certain embodiments, the PLGA compositions can be molded to different shapes and sizes.

In certain embodiments, the PLGA composition comprises a lactide:glycolide ratio of 100:0 to 30:60 with 40:60 to 85:15 as an exemplary ratio.

In certain embodiments, the PLGA composition comprises a lactide:glycolide ratio of 50:50. In certain embodiments, the PLGA composition comprises a lactide:glycolide ratio > 60:40. In certain embodiments, the PLGA comprises a lactide:glycolide ratio > 65:35. In certain embodiments, the PLGA composition comprises a lactide:glycolide ratio > 70:30. In certain embodiments, the PLGA composition comprises a lactide:glycolide ratio > 75:25. In certain embodiments, the PLGA composition comprises a lactide:glycolide ratio equal to about 75:25. In certain embodiments, the PLGA composition comprises a lactide:glycolide ratio equal to 75:25.

In certain embodiments, the PLGA has a molecular weight of about 2.0 kDa to about 2,000 kDa. In certain embodiments, the PLGA has a molecular weight of about 10-75 kDa. In certain embodiments, the PLGA has a molecular weight of about 20-75 kDa. In certain embodiments, the PLGA has a molecular weight of about 30-75 kDa. In certain embodiments, the PLGA has a molecular weight of about 40-75 kDa. In certain embodiments, the PLGA has a molecular weight of about 50-75 kDa. In certain embodiments, the PLGA has a molecular weight of about 60-75 kDa. In certain embodiments, the PLGA has a molecular weight of about 70-75 kDa. In certain embodiments, the PLGA has a molecular weight of about 75 kDa.

In certain embodiments, the PLGA has a molecular weight of about 10-65 kDa (1-300 kDa). In certain embodiments, the PLGA has a molecular weight of about 20-65 kDa. In certain embodiments, the PLGA has a molecular weight of about 30-65 kDa. In certain embodiments, the PLGA has a molecular weight of about 40-65 kDa. In certain embodiments, the PLGA has a molecular weight of about 50-65 kDa. In certain embodiments, the PLGA has a molecular weight of about 60-65 kDa. In certain embodiments, the PLGA has a molecular weight of about 65 kDa. In certain embodiments, the PLGA has a molecular weight of about 10-55 kDa. In certain embodiments, the PLGA has a molecular weight of about 20-55 kDa. In certain embodiments, the PLGA has a molecular weight of about 30-55 kDa. In certain embodiments, the PLGA has a molecular weight of about 40-55 kDa. In certain embodiments, the PLGA has a molecular weight of about 50-55 kDa. In certain embodiments, the PLGA has a molecular weight of about 55 kDa.

In certain embodiments, the PLGA has a molecular weight of about 10-45 kDa. In certain embodiments, the PLGA has a molecular weight of about 20-45 kDa. In certain embodiments, the PLGA has a molecular weight of about 30-45 kDa. In certain embodiments, the PLGA has a molecular w eight of about 40-45 kDa. In certain embodiments, the PLGA has a molecular w eight of about 45 kDa.

In certain embodiments, the PLGA has a molecular weight of about 10-35 kDa. In certain embodiments, the PLGA has a molecular weight of about 20-35 kDa. In certain embodiments, the PLGA has a molecular weight of about 30-35 kDa. In certain embodiments, the PLGA has a molecular w eight of about 35 kDa.

In certain embodiments, the PLGA has a molecular weight of about 10-25 kDa. In certain embodiments, the PLGA has a molecular weight of about 20-25 kDa. In certain embodiments, the PLGA has a molecular weight of about 25 kDa.

In certain embodiments, the PLGA concentration within the polymer solution is 1%- 50%. In certain embodiments, the PLGA concentration within the polymer solution is 15-40%.

In certain embodiments, the API accounts for 0. 1 to 50% of the polymer solution. In certain embodiments, the API accounts for 1 to 30% of the polymer solution.

In certain embodiments, the API is selected from the group consisting of antiinflammatory agents, anti-angiogenic agents, anti-cancer agents, anti-viral agents, antibacterial agents, anti-fungal agents, anti-hypertensive agents, hormones, insulin, clotting factors, cytokines, growth factors, enzymes, other polypeptides, and any combination thereof.

III. Pharmaceutical Compositions

Also provided is a pharmaceutical composition comprising a microsphere as disclosed herein and a pharmaceutically acceptable carrier. The pharmaceutical composition generally includes a therapeutically effective amount of the API or combination of APIs. The term “pharmaceutically acceptable carrier” relates to carriers or excipients, which are generally non-toxic. Examples of such excipients are, but are not limited to, saline. Ringer’s solution, dextrose solution, Hanks’ solution, and water for injection. Non-aqueous excipients such as fixed oils and ethyl oleate may also be used.

Pharmaceutical compositions ty pically are sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, suspension, dispersion, gel, or other ordered structure suitable to high drug concentration. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonicity agents, such as sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the compositions. Pharmaceutically-acceptable antioxidants may also be included, for example (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, bufy dated hydroxyanisole (BHA), butylated hydroxy toluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Sterile injectable solutions can be prepared by incorporating the compositions in the required amount in an appropriate solvent or other carrier with one or a combination of ingredients, e.g., as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients, e.g., from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The pharmaceutical composition can be prepared with carriers that will further protect the compositions against rapid release, such as a controlled release formulation, including implants, transdermal patches, and encapsulated (e.g., microencapsulated or macroencapsulated) deliver}' systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poly lactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g.. Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The pharmaceutical compositions can be administered with medical devices known in the art.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

In certain embodiments, the pharmaceutical composition can be conveniently formulated in single unit doses.

The pharmaceutical composition is generally administered parenterally, locally, or orally.

The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection or infusion, and include, without limitation, intravenous, intraperitoneal, subcutaneous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital (intraocular), intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracistemal, intratumoral, peritumoral, intracavitary', intrahepatic, intracranial, intralumenal, and intravesical injection and infusion.

The pharmaceutical composition can be formulated for administration to a subject by any suitable route of administration. Such routes of administration can include, for example, intravenous, subcutaneous, intraperitoneal, intraorbital (intraocular), intratumoral, peritumoral, intracavitary, intrahepatic, intracranial, intralumenal, and intravesical.

In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for subcutaneous administration. IV. Methods of Making

An aspect of the disclosure is a method of making compositions comprising poly(D,L- lactide-co-glycolide) copolymer (PLGA), glycofurol and at least one active pharmaceutical ingredient (API), wherein the microsphere is substantially free of dichloromethane. The method comprises: suspending PLGA in glycofurol, thereby providing a polymer solution; combining API to the polymer solution, thereby providing an API-polymer solution; and pumping the API-polymer solution through a droplet generator into water, wherein the droplet generator comprises a fine needle surrounded by an air jacket, thereby forming the composition.

In certain embodiments, the fine needle is a 22s gauge (0.00625inch inner diameter) or finer (smaller inner diameter) needle.

An exemplary method is disclosed in the Examples below. Notably, the method can be used to make PLGA spheres with the non-toxic solvent glycofurol, and these compositions are capable of releasing any of a wide variety of different kinds of drugs or substances for 6-12 months (or more). The PLGA polymer sy stem can be customized to release any of a number of different substances, both hydrophobic and hydrophilic, such as drugs (e.g., steroids, antibodies, medications for cancer, inflammatory disease, pain relief, tissue rejection, genetic materials such as DNA, etc.).

Alternatively or in addition, additives such as titanium dioxide can be incorporated into the compositions to slow further the release of drug or substance from the compositions.

Optionally, the drug-laden PLGA compositions can be coated or surrounded, completely or partially, with gels to extend duration of release and alter the solvent and aqueous phases.

V. Methods of Using

An aspect of the disclosure is a method of treating or preventing a disease, disorder, or condition, comprising administering to a subject in need thereof an effective amount of a PLGA composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA) in glycofurol, and at least one active pharmaceutical ingredient (API), wherein the composition is free of dichloromethane, and wherein the API is therapeutically effective for treating or preventing the disease, disorder, or condition. Any embodiment of the compositions disclosed herein can be used in practicing the method.

As used herein, an “effective amount” refers to an amount that is sufficient to achieve a desired outcome or result. As used herein, a “therapeutically effective amount” refers to an amount that is sufficient to achieve a desired biological or therapeutic outcome or result.

As used herein, “therapeutically effective” means capable of achieving a desired therapeutic outcome or result.

As used herein, “treat” or “treating” refers to reducing, ameliorating, or slowing or halting progression of at least one objective or subjective sign, symptom, or characteristic of a particular disease or condition of a subject. In certain embodiments, “treat” or “treating” refers to reducing, ameliorating, or slowing or halting progression of at least one objective sign, symptom, or characteristic of a particular disease or condition of a subject by a measurable degree or amount. In certain embodiments, “treat” or “treating” refers to reducing, ameliorating, or slowing or halting progression of at least one objective or subjective sign, symptom, or characteristic of a particular disease or condition of a subject or group of subjects by a statistically significant degree or amount.

As used herein, “preventing” refers to at least substantially blocking the development or occurrence of a disease, disorder, condition, or event. In certain embodiments, “preventing” refers to blocking the development or occurrence of a disease, disorder, condition, or event.

The pharmaceutical composition can be administered to a subject by any suitable route of administration. Such routes of administration can include, for example, intravenous, subcutaneous, intraperitoneal, intraorbital (intraocular), intratumoral, peritumoral, intracavitary, intrahepatic. intracranial, intralumenal, and intravesical.

In certain embodiments, the route of administration is intravenous.

In certain embodiments, the route of administration is subcutaneous.

In certain embodiments, the route of administration is intratumoral.

In certain embodiments, the pharmaceutical composition can be administered as a bolus injection. In certain embodiments, the pharmaceutical composition can be administered as an infusion. In certain embodiments, the pharmaceutical composition is administered only once. In certain other embodiments, the pharmaceutical composition is administered more than just once. In the case of repeat dosing, the dosing frequency can be determined by a health professional based on considerations such as the condition to be treated, the overall condition of the subject being treated (e.g., age, general health, sex, body weight), the route of administration or site of the pharmaceutical composition, and response of the condition to the treatment. Alternatively, in certain embodiments, in the case of repeat dosing, the dosing frequency can be determined by the subject being treated based on considerations such as the condition to be treated, the overall condition of the subject being treated (e.g., age, general health, sex, body weight), and response of the condition to the treatment.

The method can further include administration of one or more additional agents useful in the treatment of the disease, disorder, or condition. Such one or more additional agents can be administered to the subject before, simultaneously, or following administration of the compositions or pharmaceutical composition of the instant disclosure. Additionally, such one or more additional agents can be administered to the subject by the same or different route of administration of the compositions or pharmaceutical composition of the instant disclosure.

In certain embodiments, the disease, disorder, or condition is selected from the group comprising of cancer, autoimmune disease, diabetes mellitus (type 1 or type 2), transplant rejection, allergy, asthma, anemia, glaucoma, benign prostatic hypertrophy, addiction, viral infection, bacterial infection, fungal infection, genetic disorder, hypertension, infertility, pregnancy, and any combination thereof.

In certain embodiments, the disease, disorder, or condition is cancer.

In certain embodiments, the disease, disorder, or condition is an autoimmune disease.

In certain embodiments, the disease, disorder, or condition is diabetes mellitus.

In certain embodiments, the disease, disorder, or condition is transplant rejection.

In certain embodiments, the disease, disorder, or condition is allergy’.

In certain embodiments, the disease, disorder, or condition is asthma.

In certain embodiments, the disease, disorder, or condition is anemia.

In certain embodiments, the disease, disorder, or condition is glaucoma. In certain embodiments, the disease, disorder, or condition is benign prostatic hypertrophy.

In certain embodiments, the disease, disorder, or condition is addiction.

In certain embodiments, the disease, disorder, or condition is viral infection.

In certain embodiments, the disease, disorder, or condition is bacterial infection.

In certain embodiments, the disease, disorder, or condition is fungal infection.

In certain embodiments, the disease, disorder, or condition is a genetic disorder.

In certain embodiments, the disease, disorder, or condition is hypertension.

In certain embodiments, the disease, disorder, or condition is infertility.

In certain embodiments, the disease, disorder, or condition is pregnancy.

As used herein, in certain embodiments a ‘‘subject” refers to an animal. In certain embodiments a “subject” refers to a mammal, including but not limited to mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats, sheep, horses, cows, non-human primates, and humans. In certain embodiments, a “subject” is a human.

In certain embodiment a “subject” refers to a plant.

EXAMPLE

The present invention is further illustrated by the following non-limiting example.

Example 1: Microsphere preparation with glycofurol.

Materials

PLGA with lactide:glycolide ratio of 75:25 and molecular weight of 66,000-107,000 Daltons was obtained from Sigma Aldrich, Product # P1941). The solvent, Tetraglycol BioXtra (glycofurol) was obtained from Sigma Aldrich, Product #T3396). Dexamethasone, 98% was obtained from Alfa Aesar, Product #A17590.

Microsphere preparation with glycofurol

PLGA spheres were prepared by a phase inversion technique using an air-driven droplet generator device (FIG. 1). PLGA was suspended in glycofurol at concentrations of either 1, 5, or 20% w/v and placed on a shaker at 150 rpm for 24 h at room temperature to ensure complete dissolution. Dexamethasone was added to the polymer solution at concentrations of 1% and 20% (wt/wt PLGA). The PLGA-gly cofurol solution was pumped via Hamilton 100 pL syringe, through a Hamilton syringe pump, to a droplet generator (made from solution dropped from a needle surrounded by an air jacket into distilled deionized water to allow phase inversion which quickly resulted in the formation of PLGA compositions.

PLGA spheres remained in water for 30 min to ensure complete phase inversion. After collected by fdtration, spheres were washed twice with distilled, deionized water, and lyophilized and then stored at -20°C prior to use.

Alternatively, PLGA compositions can be created by spraying the polymer solution containing the API into aqueous solution to form smaller microspheres and nanospheres. The spraying mechanism can be mechanical or can be facilitated using an electrostatic field to create polymer jets.

Electrospray Procedure

PLGA was first dissolved in glycofurol at concentration of either 1%, 5% or 20% w/v. and placed on a shaker at 150 rpm for 24 hours, at room temperature to ensure complete dissolution. DEX was added to the polymer solution at concentrations of 1% and 20% (wt./wt. PLGA). For the electrospray procedure, the applied voltage used was 10 kV, nozzle internal diameter 0.25 mm, the collecting distance and flow rate were fixed at 15 cm and 0.1 ml/h. respectively. The PLGA/drug solution was pumped using syringe pump. The positive electrode was connected to needle and collector was connected to earth. This results in a cone jet that appears, followed by evaporation of the solvent during the jet process and formation of PLGA microspheres at the collector.

Furthermore, the PLGA/ API constructs can be made into shapes other than spheres by placing the polymer solution containing the API into molds and immersing molds in the aqueous solution to form various shapes and sizes of the drug-laden polymer.

PLGA was first dissolved in Glycofurol at concentration of either 1%, 5% or 20% w/v. and placed on a shaker at 150 rpm for 24 hours, at room temperature to ensure complete dissolution. DEX was added to the polymer solution at concentrations of 1% and 20% (wt./wt. PLGA). The PLGA/drug solution is placed inside a 5 ml dispenser syringe and expelled from the tip onto a silicone mold (size/shape customizable, usually rod-shaped molds between 5 and 20 mm). The mold is then immersed in an aqueous solution to facilitate phase inversion. After 5 min. The formed polymer is removed from the mold and is allowed to sit in the aqueous solution for another 5 min to complete the phase inversion process resulting in a polymer scaffold.

In vitro release of dexamethasone from compositions

In vitro release studies were performed in phosphate buffered saline (PBS). PLGA compositions were placed in tubes containing 500 pL of PBS (n=4, 1 spheres per tube) and incubated at 37°C. At set time intervals. PBS was removed for drug determination and carefully replaced with fresh PBS. Using a SPECTRAmax 340PC 384 Microplate Reader, standards were run along with the samples at 242 nm to determine the amount of drug being released from the spheres. Three batches of compositions were investigated and the means and standard deviations reported.

Microsphere preparation with dichloromethane

Dexamethasone-loaded microsphere formulations were prepared using an oil-in-water (o/w) emulsion solvent extraction/ evaporation technique. The PLGA polymer was dissolved in dichloromethane at 20% w/v and dexamethasone was dispersed in this solution at 20% w/w. This organic phase was then slowly added to 10 mL of PVA solution (1% (w/v), average MW 30-70 kDa) under constant mechanical stirring at 250 rpm. The emulsion was then transferred to 125 mL of an aqueous polyvinyl alcohol (PVA) solution (0.1% (w/v), MW 30-70 kDa) and stirred at 250 rpm under vacuum for 2.5 hours to evaporate the solvent and harden the compositions. The compositions were then washed three times with 10 mL deionized water, collected by centrifugation, lyophilized and stored at 4°C until further use.

Cytotoxicity of PLGA compositions on fibroblasts and splenocytes

C57BL/6 mouse spleen cells (1 x 10⁵), isolated using Ficoll-Paque density gradient and mixed with Con A 2 pg/mL, or C57BL/6 mouse embryonic fibroblasts (1 x 10⁵), served as target cells. Each were individually incubated with PLGA compositions at 0.5. 1, 2.5 and 5 mg/mL in complete RPMI and DMEM respectively. After 2 days of incubation, cell proliferation was assessed by Alamar Blue immunofluorescent assay.

Measurement of drug load and encapsulation efficiency

To determine the mass of dexamethasone within spheres, PLGA compositions (10 mg, 3 batches) were dissolved in 0.9 mL dimethylsulfoxide (DMSO) in a glass tube for 1 hour, followed by the addition of 3 mL of 0.05 M HC1 for another 1 hour. Drug content was determined using spectrophotometric analysis. The encapsulation efficiency was calculated. The theoretical drug load is the maximum drug load of the PLGA. i r Total mass of druq in spheres (m)

Actual Drug Load % (DE) = - Total m ¹ -ass o -f - sph -e -res — x 100%

Encapsulation efficiency % (EE) = ^{actual drua load} — x 100% theoretical drug load

Determination of PLGA microsphere size, density and porosity

The average diameters of microsphere shaped compositions were analyzed by software image measurement (NIH, ImageJ) (29/27).

The density and porosity values of the PLGA spheres were measured in triplicate by a liquid displacement methodology. Forty PLGA spheres were immersed in a volume measuring device containing a known volume (VI) of water. The sample was allowed to stand for 10 min and the new volume was then recorded as V2. The volume difference. (V2-V 1), represented the total volume of the PLGA spheres.

The density of the PLGA (p_P) was expressed as p_P = W/ (V2 - VI).

The porosity of the PLGA (tp) expressed as percentage (%) was calculated by:

Osolid - Op

(p (%) = - 100% psolid where p_soiid = is the density of the solid native PLGA.

Assessment of water diffusion into PLGA compositions

Lyophilized PLGA compositions were weighed to obtain the dry weight (wl). The spheres were placed in tubes containing 500 pL of ddFLO (n=4, 10 mg per tube) and incubated at 37°C. At set time intervals the spheres were removed from the water, blotted to remove excess water, and weighed to obtain the wet weight (w2). Following this, the spheres were lyophilized to obtain the dry weight (wl) of the spheres at each time interval. The % water diffusion was then calculated using the formula w2 — wl

% Water Diffusion = - X 100 wl

Assessment of sphere hardness (compressive modulus)

The method to assess hardness was similar to that described by Rodriguez et al. in which PLGA spheres are placed on a stage and force is exerted on top of the spheres until the sphere deforms. The original diameter (d) and cross-sectional area (A) of the sphere were noted. The force applied at which the sphere deforms by 50% was noted, this is the change in diameter of the sphere. From this the compressive modulus (E) is determined as:

Force > Area _

Change in diameter Original Diameter

The solidification time (ti), the time required for phase inversion to complete, was assessed and compared in 20% PLGA, 10% PLGA, 5% PLGA and 1% PLGA spheres (n=12/group) not containing drug by determining the required time for spheres to turn opaque once the generated droplet entered the aqueous phase. To confirm that phase inversion was complete, some PLGA spheres were taken out of the aqueous phase immediately after turning opaque and some were maintained in the aqueous phase for another 5, 10, or 15 min. Following routine processing, sphere hardness was compared in all groups.

Residual glycofurol quantification

Residual glycofurol content was analyzed in blank compositions. Spheres (20 mg) were dissolved in 1 mL acetone, re-precipitated in 4 mL of distilled water, and centrifuged at 6000 rpm for 30 min. The supernatant was then withdrawn and analyzed. Four mL of ammonium cobalt thiocyanate solution and 4 mL methylene chloride were added to 2 mL of sample. The mixture was centrifuged at 3000 rpm for 10 min and the methylene chloride phase was obtained and analyzed spectrophotometrically for residual glycofurol content on a SPECTRAmax Microplate Reader at 620 nm. The glycofurol calibration curve was linear from 0.1 to 0.75 g/100 g (f-=Q.916A). All experiments were made in triplicate.

Assessment of dexamethasone breakdown by HPLC

Dexamethasone-loaded PLGA compositions were added to tubes containing 500 pL of PBS (n=4, 15 spheres per tube) and incubated at 37°C. At daily intervals. PBS was withdrawn for HPLC analysis of dexamethasone breakdown and replaced with fresh PBS. HPLC system (Waters 600 pump; Waters, Milford, MA), equipped with a UV detector (RAININ, Dynamax, Absorbance Detector Model UV-C) and a Zobax C18 (4.6 mm x 15 cm, Agilent, Santa Clara, CA) analytical column were used. All samples were analyzed by isocratic method with a mobile phase containing acetonitrile/water/phosphonc acid (35/70/0.5, v/v/v) at a flow rate of 1 mL/min with an injection volume of 20 pL. The detector was set at a wavelength of 240 nm.

Assessment of kinetic models of drug release Models tested included the zero-order, first-order, Higuchi, Korsmeyer-Peppas, and Weibull with time lag parameter. In order to determine the kinetic model that best described the dissolution profile of the drug loaded PLGA system, the goodness of fit was established using the model selection criteria (MSC), Akaike’s Information Criteria (AKC), mean squared error, and adjusted R2. The model parameters were calculated using the DDSolver program.

Statistical analyses

Means were evaluated by Student’s t-test or ANOVA analysis; p < 0.05 was considered statistically significant. Drug release profiles were analyzed using DD Solver, an Excel addin package. All error bars represent standard deviations unless otherwise noted. Parameters were estimated on each replication, and mean dissolution models were built using the mean of the parameters. Goodness of fit was evaluated using the model selection criteria (MSC), Akaike’s Information Criteria (AIC), mean squared error, and adjusted R2. The comparison of release rates will be done using model-dependent and independent techniques. A p<0.05 was considered significant.

Example 2: Results

Spheres made with 20% PLGA solidified almost instantaneously. The solidification time inversely correlated to the PLGA concentration (Table 2). The morphology’ and sizes of spheres resulting from the modifications of (modifying or altering or adjusting) the following device parameters: needle gauge, distance of needle exposed past below the air tube, distance of needle to aqueous phase, and air flow rates is found in Table 2. Certain parameters yielded spindle like rods of PLGA which were not uniform and not utilized in further experiments.

Table 2: Device parameters and resultant PLGA spheres morphology

PLGA spheres of varying sizes and morphology’ resulting from various polymer concentration, polymer-drug mixture flow rate, air flow rate, and d2. *indicates the parameters selected for preparing PLGA spheres for the drug elution experiments.

The solidification time and compression modulus of 1%, 5%, 10%, and 20% spheres was assessed (Table 3). PLGA concentration of spheres correlated with compression modulus and inversely correlated the solidification times. Mean solidification time and mean compressive modulus were significantly different between PLGA groups (p< 0.001 and p < 0.005 respectively). To confirm that phase inversion was complete at measured solidification times, some PLGA spheres were taken out of the aqueous phase immediately after turning opaque and some remained in the aqueous phase after another 5, 10 and 15 min. Following routine processing, mean sphere hardness was compared in each group over time and were found not significantly different from each other (data not shown).

The mean time taken for the spheres to turn opaque following immersion of the “organic’’ phase droplet into the aqueous phase was compared in 20% PLGA, 10% PLGA, 5% PLGA and 1% PLGA sphere (n=12). Mean sphere hardness w as also assessed (n=30/group). Mean solidification time and mean compressive modulus were significantly different between groups (p< 0.001 and p <0.005 respectively) (20% vs 10% PLGA- p=0.000021; 10% vs 5% PLGA- p=0.00044; 5% vs 1% PLGA p= 0.00078). Table 3: Effect of PLGA concentration on the solidification time and hardness (compressive modulus) of PLGA compositions constructed with glycofurol.

Physical Properties

Utilizing 20% PLGA and the following device parameters: flow rate of polymer and drug through the needle of 5 pL min'¹, air flow rate set to 30 LPM, dl at 2.5 mm and d2 at 25 mm, PLGA compositions were spherical (FIG. 2A) and had a mean diameter of 397.38 pm (±32.43 SE) (FIG. IB).

The density, porosity, drug load, encapsulation efficiency, and drug burst of glycofurol- and dichloromethane (DCM)-constructed spheres was assessed and compared (Table 4). Glycofurol spheres prepared with 1% dexamethasone had a significantly higher encapsulation efficiency than 20% dexamethasone spheres, 86.21% and 80.45%, respectively, resulting in a final drug load of 0.86% and 16.09% (p<0.01), respectively.

The drug encapsulation efficiency within the glycofurol spheres was significantly greater than that in DCM spheres, 80.45% and 72.25% respectively. The burst over the first day was also significantly lower in the glycofurol spheres than in DCM spheres.

Table 4: Density’, porosity, drug load (DL) %, encapsulation efficiency% and drug burst (1^st week) of PLGA spheres constructed with glycofurol and dichloromethane (DCM). f, f f, f are pairwise statistical comparison between groups. ** indicates p<0.01, * indicates p<0.05.

The hardness of the DCM and glycofurol PLGA spheres (n= 30) made with 20% PLGA were compared by measuring the compressive modulus. The mean compressive modulus of DCM spheres 21.16 (1.00) E (x IO^-3 MPa) was greater than glycofurol-constructed spheres 18.44 (0.73) E (x 10³ MPa) P < 0.003.

The in vitro diffusion of water into DCM and glycofurol compositions over 28 days was compared (FIG. 8). The % water diffusion was significantly greater into DCM spheres than into glycofurol-made spheres at even,' time point and was more than twice as much from Day 7 to the end of the study at Day 28.

Integrity of Dexamethasone (DEX) molecule released from PLGA compositions constructed with glycofurol

High Performance Liquid Chromatography (HPLC) was performed to determine if the dexamethasone molecule released from the spheres is denatured over time (FIG. 3). Supernatant from incubated compositions prepared with 20 % dexamethasone (DEX) and 20% PLGA within glycofurol was analyzed weekly by HPLC. Dexamethasone had a retention time of 9.99 min. There was a sharp DEX peak at 1 w eek that was not altered over time. There was no secondary breakdown peak(s) even by 5 weeks.

Glycofurol content of glycofurol-constructed PLGA compositions

The mean glycofurol content of compositions made from 20% PLGA was 6.26 mg (± 1.78 SE) per 100 mg of PLGA compositions.

In vitro cytotoxicity of PLGA compositions

The potential toxicity of compositions prepared with dichloromethane or glycofurol was assessed and compared by measuring the effect of different quantities of each type of spheres has on the proliferation (or viability) of fibroblasts or Con A-stimulated mononuclear spleen cells (FIG. 4). The proliferation of spleen cells and fibroblasts decreased dose- dependently with the number of incubated DCM-constructed spheres (FIG. 4). In contrast, all doses of glycofurol-constructed PLGA spheres caused no decrease in viability of fibroblasts and spleen cells. Spheres with 20% PLGA had lower mean cumulative dexamethasone release at every time point. The duration of drug release was also longer in the 20 % PLGA than 5% PLGA spheres (FIG. 5).

The effect of dexamethasone concentration on drug release from 20% PLGA compositions is depicted in FIG. 6. The drug bursts in the 20% dexamethasone and 1 % dexamethasone compositions were similar, 5.99% and 4.91%, respectively. In both the 1% and the 20% dexamethasone-made spheres, there was no lag phase after the drug burst.

After the burst, the drug release patterns of the 20% and 1% dexamethasone-constructed spheres were very different. Spheres constructed with 1% dexamethasone had an overall faster accumulated drug release than the 20% dexamethasone spheres.

In the 1% dexamethasone spheres, there was a significantly shorter time to release 25%, 50%. and 75% of the releasable drug as compared to the 20% dexamethasone spheres (Table 5). Both the 1% and the 20% dexamethasone spheres had a sustained release rate over 6 months.

Table 5: Comparision of time to 25%, 50%, and 75% release of spheres made from 20% PLGA in glycofurol with 20% and 1% dexamethasone (DEX). * indicates p<0.001.

The pattern of drug release included a small decline in drug release rate from 48-68 days in the 20% dexamethasone spheres and a smaller decline of drug release from 60-75 days in the 1% dexamethasone spheres.

After the burst until halfway through the study at 110 days, the 1% dexamethasone spheres exhibited a zero-order fit while the 20% dexamethasone spheres did not, with a mean R² of 0.971 (0.015) (range 0.96-0.98) vs 0.884 (.033) (range 0.85-0.93) (p<0.0002).

From 110 days to the end of the study at 210 days, the 1% dexamethasone and 10% dexamethasone compositions exhibited similar zero-order kinetics with an R² of 0.920 (0.023) (range 0.90-0.95) and 0.941(0.052) (range 0.94-0.99). The assessment of different mathematical models of drug release of 1% and 20% dexamethasone spheres for the entire length of the experiment (0-210 days) is presented in Table 6

Table 6: Comparison of kinetic models of release for 20% dexamethasone (DEX) vs 1% DEX

f and J are the best fit models for 20% Dex and 1% Dex respectively. * and ** are p<0.005 and p<0.008, respectively, when compared to the best fit model(s).

The accumulated dexamethasone release over time was compared in 320-380 pm in diameter PLGA microsphere made with glycofurol and DCM. The mean drug burst release at

24 h was greater in the DCM spheres than the glycofurol spheres (p< 0.05) (Table 7).

Table 7: Comparision of time to 25%, 50%, 75% and 80% release of spheres made from DCM and glycofurol with 20% dexamethasone drug load and 20% PLGA concentration.

* indicates pO.OOl

The time to 25, 50, and 75% release of releasable drug was faster in the DCM-prepared spheres (Table 8). For example, 50% of releasable drug was release within 12 days in DCM spheres as compared to 71 days in the Tetraglycol spheres. The DCM prepared spheres released all of its releasable drug within 42 days as compared to >110 days in the Tetraglycol spheres when the experiment was terminated.

Table 8: Comparision of time to 25%, 50%, and 75% release of spheres made from 5% PLGA and 20% PLGA in glycofurol having a dexamethasone drug load of 20%.

* indicates p<0.001

Discussion

A method to construct dexamethasone laden PLGA spheres with the low toxicity solvent, glycofurol, that release drug over an extended period of time was developed and characterized.

Utilizing an air-driven droplet generator device to administer the inner phase into the outer aqueous phase, the optimal device settings were determined to construct PLGA compositions wi th 20% PLGA were: a solvent/drug (pump) flow rate of 5 pL min'¹, a 35 gauge needle, air flow rate of 30 LPM, a dl of 2.5 mm and a d2 of 25 mm.

Very small spheres were not attainable with this exact method, though modifications of our procedure such as spraying solvent drug mixture using acoustic excitation or non-solvent carrier stream may allow this.

Previous reports show that PLGA compositions made with dichloromethane emulsionevaporation techniques or with electrostatic methodologies commonly have a wide distribution of sizes with one standard deviation of distribution frequently equaling 50%, making the procedure less amenable for clinical use. However, spheres prepared with the methodology disclosed herein possessed a much tighter distribution of sphere sizes with one standard deviation of the distribution equaling only 10% of the mean sphere diameter.

The encapsulation efficiency of dexamethasone within the spheres was very high (80%), resulting in much greater dexamethasone load of 16% than previously described of 1- 8%. These higher drug loads can potentially provide a much larger depot of drug per PLGA mass within the body to allow the potential release of drug over a longer period of time.

One of the issues with PLGA spheres prepared with organic solvents such as dimethylchloride (DCM) has been toxicity on the surrounding tissue and the degrading effect on the drug payload. Similarly, results disclosed herein also indicated that PLGA compositions made with DCM were toxic to spleen cells and fibroblasts in vitro. In contrast, PLGA spheres made with glycofurol were non-toxic, having no inhibitory effect on either of these ty pes of target cell, a significant advantage for using glycofurol-constructed spheres disclosed herein.

Hickey observed the dexamethasone molecule released from PLGA spheres constructed with DCM degraded within just a week of in vitro incubation. Their HPLC studies revealed a second peak, representing degraded dexamethasone that increased over time. In contrast, the glycofurol-constructed spheres caused no dexamethasone degradation even after 5 weeks of incubation.

Even though glycofurol is considered non-toxic^ it seems reasonable to keep the amount of solvent within the spheres as low as possible. The residual amount of glycofurol within the spheres presented here was 5.71 mg/ 100 mg, far less than the 14-16.9 mg/100 mg found in previously described glycofurol PLGA spheres prepared by methods different from the method disclosed herein. This surprisingly low residual amount may be further reduced by utilizing standard methods such as dialysis.

There are few previous reports describing the release of drug from PLGA spheres constructed with glycofurol without the addition of the volatile organic solvents. All of these previous methodologies differed from the method disclosed herein. Further, these previously described PLGA spheres were unable to release drug over an extended period of time.

Microspheres in the art were constructed with glycofurol using an emulsion extraction method and incorporated three substances, Ritonavir, Lopinavir, and Sudan III, with a reported maximal duration of release of only 4 h, 20 h, and 18 days, respectively. PLGA nanospheres in the art were made using interfacial polymer deposition method with glycofurol and then loaded Paclitaxel by adsorption on to the spheres. However, it was reported that all drug was released by' 7 days. Using a phase separation method, nanospheres were constructed in the art with glycofurol incorporating lysozy me or TGF-P w ith a total duration of drug release of only 10 days and 20 days respectively. Conversely, the duration of drug (dexamethasone) release from compositions prepared with glycofurol according to the method disclosed here exceeded 6 months, making these spheres more amenable for long-term therapy. There are, however, no previous reports of dexamethasone-laden compositions made with glycofurol.

Drug release from PLGA spheres are described to generally occur in three phases. An initial short drug burst over the first 24 hours is thought to be due to drug release from the surface or just below the surface of the microsphere. Then, a minimal drug release for 2-3 or more weeks usually occurs, called a lag phase, which is thought to be due to drug diffusing from the core of the sphere to the surface. Spheres with very short duration of drug release (less than 1-2 weeks) may not demonstrate this lag phase. The lag phase is frequently followed by a third phase of a more rapid rate of drug release sometimes occurring at a constant (zeroorder kinetics) induced by the (hydrolytic) breakdown of the PLGA.

The kinetics of drug release from dexamethasone-laden spheres prepared with glycofurol as described above are very different from previous reports using DCM. First, glycofurol-constructed spheres have a relatively small bursts of 5% to 6% as compared to the 18% to 65% burst in previously reported dexamethasone spheres constructed with dichloromethane. This small burst may be clinically helpful to avoid a large bolus of drug. The small burst that we observed could be due to a small amount of drug on the sphere surface (which may be related to the speed these spheres formed) and/or a very slow time for the drug near the surface of the spheres to diffuse through the pores to the surface. Without intending to be bound by scientific theory, the latter explanation is not expected since tiny pores below the surface would most likely result in a lag phase after the burst which did not occur our spheres. Gel coating of spheres described herein may be able to further decrease the burst phase.

The absence of a lag phase soon after the drug burst in glycofurol spheres described herein is rarely found in PLGA spheres which release dexamethasone or other drugs over an extended period of time and that are not coated with gels. The absence of the lag phase following the drug burst that was observed indicates that drug flows more freely through the sphere to the surface than previously reported and/or surface erosion is initially playing a greater role in PLGA degradation than previously described versus bulk erosion, which previously has been shown to play a major larger role in PLGA sphere degradation. Small declines in rates of drug release occurred from 60-75 days and 48-68 days in the 1% and 20% Dex spheres respectively. These short intervals of decline could be due the lack of releasable drug near the sphere surface and the time needed transport drug from the core of the sphere towards the surface that is a lag phase.

Alternatively, the increased dexamethasone release after the slowdown may not be due to dexamethasone transfer from the core but solely due to hydrolytic degradation of PLGA which then starts the last phase of drug release, a phase of brisk extended drug release at zeroorder kinetics in both the 1% and 20% dexamethasone spheres.

After the burst, drug release patterns of the 20% and 1% dexamethasone-containing spheres were very different. The rate of % accumulated drug release was slower from the 20% dexamethasone spheres than from the 1 % dexamethasone spheres. This was not unexpected since slower rates of drug release have been previously observed in PLGA spheres with higher loads of hydrophobic drug which is thought to be due the crystallized or aggregated drug that frequently occurs in higher hydrophobic drug loads than in the more dispersed drug found in spheres with lower hydrophobic drug loads.

The kinetics of dexamethasone release for the first half of the experiment, until 110 days, differed between the dexamethasone 1% and 20% spheres with the 1% dexamethasone spheres exhibiting drug release with zero-order kinetics. However, from 110 days to the end of the experiment, drug release followed the zero-order kinetics for both the 1 % dexamethasone and 20% dexamethasone. This zero-order kinetics of dexamethasone release for over 110 days duration is the longest ever reported from PLGA spheres. Further, this zero-order kinetics drug release, that is, a constant rate of drug release over time, is frequently clinically advantageous.

When assessing the entire time of the experiment (0-210 days) using the three criteria of R², MSC, and AIC, the best fit mathematical models of drug release were similar in the 1% and 20% dexamethasone-containing spheres, with the Weilbull and Korsmeyer-Peppas models equally fitting drug release from the 1% dexamethasone spheres and the Weilbull model for the 20% dexamethasone spheres.

The release/diffusion mechanism was assessed by calculating the diffusion exponent A and erosion exponent B derived from the nonlinear-fitted Kopchas model. The calculated value of A and B indicated that both factors, diffusion and erosion, are responsible for drug release. The higher value of A/B 5.6 and 8.2 for the 1% and 20% dexamethasone spheres constructed with glycofurol respectively indicated that the predominant mechanism of drug release is from bulk diffusion.

An extended duration of drug release from implanted compositions may be critical in treating chronic inflammation and disease such as cancer, chronic inflammatory bowel disease, arthritis, chronic abscess, and AIDS. The duration of dexamethasone release from our 1 % and 20% dexamethasone compositions was approximately 6 months, far longer than the duration of active release of previously reported dexamethasone-laden PLGA spheres. Without intending to be bound by scientific theory, alterations to the methodology to prepare spheres described herein, such as encasing the compositions in gels, may further improve and extend the release of drug.

The prolonged drug release from our dexamethasone PLGA compositions as compared to DCM spheres may be related to the rapid speed that the more polar solvent, glycofurol vs DCM, diffuses out of the polymer (PLGA) into the outer aqueous phase during sphere preparation. This in turn causes rapid formation of the spheres, which has been shown to decrease water diffusion into PLGA spheres. Such decrease in water diffusion can in turn slow PLGA degradation and lengthen the duration of drug release. This explanation is further supported by our observation that water diffusion into the PLGA spheres was much slower than the water diffusion into the DCM-made PLGA spheres.

This slowed water diffusion into glycofurol -constructed spheres was not due to an increase in porosity, density, or innate sphere hardness since we found the porosity, density, and hardness were in fact greater in DCM-constructed spheres.

Since larger-sized PLGA spheres, having smaller surface area to volume ratios, may have longer durations of drug release as compared to smaller spheres, we explored if the relatively large size of our compositions could account for the very long drug release that we observed. Thus, we assessed the dexamethasone release rates in glycofurol- and dichloromethane-constructed PLGA spheres having similar diameters, PLGA types, PLGA molecular weight, PL A and PLG proportions, and concentrations of PLGA and dexamethasone during sphere formation. It was determined that the duration of dexamethasone release from the DCM spheres was similar to that previously reported. Further, the spheres made with DCM had far shorter duration of drug release than spheres made with glycofurol. For example, the time to 50% drug release was 12 days vs 71 days in DCM and glycofurol spheres, respectively (p < 0.01 ). Thus, it does not appear that sphere size was the main reason for the long duration of drug release from our spheres. Similarly, Panyam found that a 10-fold increase in PLGA sphere size may have no effect on rates of sphere degradation. Panyam J et al., J Control Release 92(1 -2): 173 (2003).

By understanding the effect of PLGA concentration and drug load has on the kinetics of dexamethasone release from our glycofurol-constructed PLGA spheres as disclosed herein, one can tune the rate of drug release by utilizing the appropriate combination of spheres made with different loads and PLGA content. This tuning procedure may be further improved by determining the effects that different molecular weights of PLGA and proportions of PLA to PGA have on the release rate of drug.

In conclusion, we describe a method to prepare PLGA compositions constructed with glycofurol, a solvent is much less toxic than the solvents presently being utilized to construct PLGA compositions. Spheres generated with glycofurol were not toxic to target cells in vitro as were spheres made with DCM emulsion methodology. The dexamethasone molecule was also not denatured within these spheres as previous found with DCM-prepared spheres, and the duration of dexamethasone release was far longer than for previously described spheres. This unexpected advantage may be related to decreased diffusion of water into these spheres.

Further, the kinetics of release are also more clinically advantageous with a unique lack of a lag phase after the burst, and a prolonged constant drug release rate particularly in the spheres made with 1 % dexamethasone.

Claims

CLAIMS What is claimed is:

1. A drug delivery composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API), wherein the API is released over an extended period of time.

2. The composition of claim 1 , wherein the composition comprises substantially spherical components.

3. The composition of claim 1 or 2, wherein the spherical components are a plurality of microspheres, each having a diameter of about 1 micrometer to about 1000 micrometers (pm).

4. The composition of claim 1 or 2, wherein the PLGA comprises a lactide:glycolide ratio of about 95:5.

5. The composition of claim 1 or 2, wherein the PLGA comprises lactide:glycolide ratio of about 85: 15.

6. The composition of claim 1 or 2, wherein the PLGA comprises lactide:glycolide ratio of about 40:60.

7. The composition of any one of claims 1-6. wherein the PLGA has a molecular weight of about 2 kDa to about 2,000 kDa.

8. The composition of any one of claims 1-6, wherein the API is hydrophobic or hydrophilic.

9. The composition of any one of claims 1-8, wherein the API is selected from the group consisting of anti-inflammatory agents, anti-angiogenic agents, anti-cancer agents, antiviral agents, anti-bacterial agents, anti-fungal agents, anti-hypertensive agents, hormones, insulin, clotting factors, cytokines, growth factors, enzymes, other polypeptides, and any combination thereof.

10. A pharmaceutical composition comprising the composition of any one of claims 1-8 and a pharmaceutically acceptable carrier.

11. A method of making a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API) comprising: suspending PLGA in glycofurol, thereby providing a polymer solution; combining at least one API with the polymer solution, thereby providing an API-polymer solution; and combining the API-polymer solution with an aqueous solution, thereby forming the composition.

12. A method of making a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API) comprising: suspending PLGA in glycofurol, thereby providing a polymer solution; combining at least one API with the polymer solution, thereby providing an API-polymer solution; and pumping the API-polymer solution through a droplet generator into aqueous solution, wherein the droplet generator comprises a fine needle surrounded by an air jacket, thereby forming the composition.

13. The method of claim 12, wherein the fine needle consists of a 10s - 34s gauge needle.

14. A method of making a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API) comprising: suspending PLGA in glycofurol, thereby providing a polymer solution; combining at least one API with the polymer solution, thereby providing an API-polymer solution; placing the API-polymer solution into a mold having a desired shape and size; and placing the mold containing the API-polymer solution into an aqueous phase, thereby forming a composition having the desired shape and size of the mold.

15. The method of any one of claims 11-14, wherein the PLGA accounts for about 0.1% weight to about 50% weight of the polymer solution.

16. The method of any one of claims 11-14, wherein the API accounts for about 0. 1 % weight to about 50 % weight of the polymer solution.

17. A method of treating or preventing a disease, disorder, or condition, comprising administering to a subject in need thereof an effective amount of a composition comprising poly(D,L-lactide-co-glycolide) copolymer (PLGA), glycofurol, and at least one active pharmaceutical ingredient (API), wherein the API is therapeutically effective for treating or preventing the disease, disorder, or condition.

18. The method of claim 17, wherein the disease, disorder, or condition is selected from the group consisting of cancer, autoimmune disease, transplant rejection, allergy, asthma, anemia, glaucoma, benign prostatic hypertrophy, addiction, viral infection, bacterial infection, fungal infection, genetic disorder, infertility, pregnancy, and any combination thereof.

19. The method of claim 17 or 18, wherein the subject is an animal.

20. The method of claim 17 or 18, wherein the subject is a human.

21. The method of claim 17 or 18, wherein the subject is a plant and the disease, disorder, or condition is selected from the group consisting of tumor, viral infection, bacterial infection, fungal infection, genetic disorder, and any combination thereof.

22. The method of claim 11, wherein the API-polymer is sprayed into the aqueous solution.

23. The composition of any one of claims 1-22, wherein the composition is substantially free of dichloromethane.