Injectable long-acting naltrexone microparticle compositions

Abstract

The present disclosure relates to naltrexone sustained release microparticle delivery systems for the treatment of diseases ameliorated by naltrexone. The injectable microparticle delivery system includes naltrexone encapsulated in biodegradable microparticles administered in a pharmaceutically acceptable vehicle.

Claims

1. An injectable microparticle formulation comprising a microparticle including naltrexone and poly(lactide-co-glycolide) with a lactide:glycolide ratio of about 85:15, wherein sustained release of naltrexone is longer than 4 weeks and up to 100 days.

2. The microparticle formulation according to claim 1, wherein sustained release of naltrexone is about 8 weeks to about 12 weeks.

3. The microparticle formulation according to claim 1, wherein the naltrexone is in the form of free base, salt, solvate, cocrystal or combinations thereof.

4. The microparticle formulation according to claim 1, wherein the naltrexone is about 20-40% (w/w) of the microparticle.

5. The microparticle formulation according to claim 1, wherein the poly(lactide-co-glycolide) has a number average molecular weight of 50,000 to 150,000 Daltons.

6. The microparticle formulation according to claim 1, wherein the microparticle is administered in a biocompatible vehicle, including an aqueous-based vehicle, an oil-based vehicle or combination thereof.

7. The microparticle formulation according to claim 6, wherein the aqueous based vehicle comprises a tonicity agent such as sodium chloride, a viscosity enhancing agent such as sodium carboxymethylcellulose, a wetting agent such as polysorbate, or combinations thereof.

8. The microparticle formulation according to claim 6, wherein the oil-based vehicle comprises peanut oil, sesame oil, cottonseed oil, or combinations thereof.

9. The microparticle formulation according to claim 1, wherein the microparticles have particle size in the range of 25 to 125 μm.

10. A method for treating or preventing diseases related to opioid abuse or overdoses, alcohol dependence or pain comprising administering an effective amount of the injectable microparticle formulation according to claim 1 to a subject in need of such a treatment or prevention.

11. A method for preparing the injectable microparticle formulation according to claim 1, the method comprising: (a) mixing a first phase comprising polyvinyl alcohol and a first solvent, and a second phase comprising a biodegradable polymer, naltrexone and a second solvent to prepare a mixture; and (b) performing an extraction process on the mixture with water or an aqueous solution to obtain microparticles.

12. The method according to claim 11, wherein the first solvent comprises at least one selected from the group consisting of water, dichloromethane benzyl alcohol, and ethyl acetate.

13. The method according to claim 11, wherein the second solvent comprises at least one selected from the group consisting of dichloromethane, benzyl alcohol, and ethyl acetate.

14. The method according to claim 11, the aqueous solution comprises at least one selected from the group consisting of polyvinyl alcohol, dichloromethane, benzyl alcohol, and ethyl acetate.

15. The method according to claim 11, wherein the biodegradable polymer comprises a polylactide, poly(lactide-co-glycolide), or combinations thereof, wherein a lactide:glycolide ratio of the biodegradable polymer is 50:50 to 100:0.

16. The method according to claim 11, wherein the method further comprises (c) performing a further extraction process on the microparticles with an ethanolic aqueous solution after the (b) performing the extraction process.

17. The method according to claim 16, wherein the method further comprises drying the microparticles, wherein the drying is performed after the (b) performing the extraction process, after the (c) performing the further extraction process, or after each of the (b) and (c).

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a flow diagram illustrating one embodiment of a method for preparing microparticles.

(2) FIG. 2. illustrates the in vitro release profile (FIG. 2A) and the in vivo pharmacokinetic profiles (FIG. 2B) of formulations from Example 1.

(3) FIG. 3. illustrates the in vitro release of naltrexone from formulations with drug loadings ranging from 37˜40% (w/w) from Example 5.

(4) FIG. 4. illustrates the in vivo pharmacokinetic profiles of Formulations 12-3 (FIG. 4A), 12-4 (FIG. 4B), 12-5 (FIG. 4C), and 12-7 (FIG. 4D) of Table 10 in Example 12.

(5) The following examples are provided by way of illustration and should not be construed by way of limitation.

EXAMPLES

Example 1

(6) Effect of PLGA 75:25 Processing

(7) The continuous phase was prepared by weighing 40 g of poly(vinyl alcohol) (PVA) (Mowiol 40-88, Sigma Aldrich, St. Louis, Mo.) and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 75:25 (Resomer RG756S) and 267 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 1.333 g of dichloromethane (DCM) (Fisher Scientific, Fair Lawn, N.J.) and 623 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J.) in a 20 mL scintillation vial or 500 mg of PLGA 75:25 (Resomer RG756S) and 294 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 1.333 g of dichloromethane (DCM) (Fisher Scientific, Fair Lawn, N.J.) and 623 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J.) in a 20 mL scintillation vial. 10 mL of the continuous phase was added to the top of the organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 380 mL of 1% (w/v) PVA in water and stirred at 4° C. for 8 h. Microparticles were then collected with a 25 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then collected on a 25 μm sieve and vacuum dried for 48 h.

(8) These two formulations were evaluated in pharmacokinetic studies with Sprague-Dawley rats at a dose of 38 mg/kg

(9) The resulting drug loading and residual benzyl alcohol content are shown in Table 1. Both formulations provided about 90% encapsulation efficiencies but with release of only approximately 4-5 weeks, with the higher drug loading resulting in faster kinetics. A level A in vitro-in vivo correlation is noted in FIG. 2 for F.1-1, whereas the in vitro test predicts a faster in vivo response than what is observed for F.1-2.

(10) TABLE-US-00001 TABLE 1 Formulation summary for Example 1 Starting Drug Formulation Naltrexone Weight Loading Residual BA EE (F) (mg) (% w/w) (% w/w) (%) F.1-1 267 31.2 1.31 89.7 F.1-2 294 33.5 1.51 90.6

(11) Drug loading (% w/w) indicates the amount of naltrexone contained in the microparticles, and EE (%) indicates the final amount of remaining naltrexone as compared to the starting naltrexone when preparing the microparticles.

Example 2

(12) Effect of PLGA 75:25 Molecular Weight and Supplier

(13) The continuous phase was prepared by weighing 40 g of poly(vinyl alcohol)(PVA) (Mowiol 40-88, Sigma Aldrich, St. Louis, Mo.) and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 75:25 and 267 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 1.333 g of dichloromethane (DCM) (Fisher Scientific, Fair Lawn, N.J.) and 623 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J.) in a 20 mL scintillation vial. 10 mL of the continuous phase was added to the top of the organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 380 mL of deionized water and stirred at 4° C. for 8 h. Microparticles were then collected with a 25 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then collected on a 25 μm sieve and vacuum dried for 48 h.

(14) The resulting drug loading and residual benzyl alcohol content are shown in Table 2.

(15) Resomer RG 750 and 756 resulted in similar drug loadings and release profiles, likely due to the similar inherent viscosity, with RG 750 having a wider range. Resomer RG 755 and Lactel B6007-1, with nearly similar inherent viscosity specifications, also resulted in similar drug loadings and release profiles. While resulting in similar drug loadings as the other PLGAs tested, Lactel B6007-2 resulted in a release profile with much faster kinetics. Formulations F.2-1 to F.2-5 provided drug release profiles about 30-35 days.

(16) TABLE-US-00002 TABLE 2 Formulation summary for formulations made with 75:25 PLGA Inherent Drug Residual Formulation Viscosity Loading BA EE (F) PLGA (dL/g) (% w/w) (% w/w) (%) F.2-1 Resomer RG 0.8-1.2 29.2 2.86 83.8 750 F.2-2 Resomer RG 0.5-0.7 28.9 1.73 83.0 755 F.2-3 Resomer RG 0.71-1.0  29.6 2.73 85.0 756 F.2-4 Lactel B6007-1 0.55-0.75 29.3 1.95 84.2 F.2-5 Lactel B6007-2 0.8-1.2 29.6 1.70 85.0

Example 3

(17) Preparation of Naltrexone Microparticles: Formulation 3

(18) The continuous phase was prepared by weighing 40 g of poly(vinyl alcohol) (PVA) (Mowiol 40-88, Sigma Aldrich, St. Louis, Mo.) and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG 858S Evonik Cyro, Parsippany, N.J.) and 294 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 2.0 g of dichloromethane (DCM) (Fisher Scientific, Fair Lawn, N.J.) and 467 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J.) in a 20 mL scintillation vial. The continuous phase was further mixed with DCM, and 10 mL of the continuous phase with 1.8% (w/v) DCM was added to the top of the organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 380 mL of the water with 0.5% w/v DCM and stirred at 4° C. for 8 h. Microparticles were collected with a 25 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then collected on a 25 μm sieve and vacuum dried for 48 h.

(19) The resulting drug loadings was 28.1% (75.9% EE) and residual benzyl alcohol was 0.55%. The in vitro drug release profile resulted in near zero order release kinetics for approximately 50 days.

Example 4

(20) Preparation of Naltrexone Microparticles: Formulation 4

(21) The continuous phase was prepared by weighing 40 g of PVA and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG 858S) and 294 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 2.0 g of DCM and 623 mg of benzyl alcohol in a 20 mL scintillation vial. The continuous phase was further mixed with DCM, and 10 mL of the continuous phase with 1.8% (w/v) DCM was added to the top of organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 380 mL of water with 0.66% (w/v) DCM at 4° C. Microparticles were then allowed to stir for 8 h and then collected with 23 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then passed through a 125 μm sieve, collected on a 23 μm sieve, and vacuum dried for 48 h.

(22) The resulting drug loading was 21.7% (58.6% EE) and residual benzyl alcohol was 0.83%. A greater amount (0.66% (w/v) of DCM in the extraction phase and higher starting benzyl alcohol contents in the organic phase greatly decreases the drug loading of the formulation. The in vitro drug release profile resulted in near zero order release kinetics for approximately 60 days.

Example 5

(23) Preparation of Naltrexone Microparticles: Effect of Solvent in Emulsification Media

(24) The continuous phase was prepared by weighing 40 g of PVA and mixing with 4 L of deionized water. The organic phase consisted of 1.0 g of PLGA 85:15 (Resomer RG 858S) and 818 mg of naltrexone free base (SpecGx, LLC) dissolved in 4.0 g of DCM and 1.908 g of benzyl alcohol in a 20 mL scintillation vial or 1.0 g of PLGA 85:15 (Resomer RG 858S) and 818 mg of naltrexone free base (SpecGx, LLC) dissolved in 4.0 g of DCM and 1.04 g of benzyl alcohol in a 20 mL scintillation vial. 15 mL of the continuous phase or the continuous phase further mixed with DCM or benzyl alcohol (BA) (described in Table 3) was added to the top of the organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into 760 mL of an extraction phase (described in Table 3) and stirred at 4° C. for 4 h. Microparticles were then collected with a 25 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 400 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then passed through a 125 μm sieve and collected on a 23 μm sieve and vacuum dried for 48 h.

(25) The resulting drug loading and residual benzyl alcohol content can be found in Table 3. An increase in residual benzyl alcohol is seen with a greater starting amount of benzyl alcohol. Encapsulation efficiencies of 80˜90% can be obtained. Dichloromethane in the extraction media results in the lowest residual benzyl alcohol contents for this example, although naltrexone is also lost in this process. The drug release curves do not appear to show a difference in release between 3.3% BA (benzyl alcohol) in 1% PVA vs 1% PVA alone. The 1.8% DCM in 1% PVA shows a slightly lower release rate initially, potentially due to the lower drug load.

(26) TABLE-US-00003 TABLE 3 Effects of the continuous phase and extraction phase compositions on the resultant drug loading, residual benzyl alcohol (BA), and encapsulation efficiency (EE). Re- Continuous Phase Starting Drug sidual Formula- (w/v)/Extraction BA Loading BA EE tion (F) Phase (w/v) (g) (% w/w) (% w/w) (%) F.5-1 1.8% DCM in 1% 1.908 37.0 1.44 82.2 PVA in water/ 0.5% DCM in water F.5-2 3.3% BA in 1% 1.908 39.5 2.43 87.8 PVA in water/ H.sub.2O F.5-3 1% PVA in water/ 1.908 39.8 2.26 88.5 H.sub.2O F.5-4 1.8% DCM in 1% 1.04 37.8 0.96 84.0 PVA in water/ 0.5% DCM in water F.5-5 3.3% BA in 1% 1.04 39.4 1.92 87.6 PVA in water/ H.sub.2O F.5-6 1% PVA in water/ 1.04 39.8 1.02 88.5 H.sub.2O

Example 6

(27) Preparation of Naltrexone Microparticles: Effect of Oil/Water Ratio

(28) The continuous phase was prepared by weighing 40 g of PVA and mixing with 4 L of deionized water. The organic phase consisted of 0.630 g of PLGA 85:15 (Resomer RG 858S) and 370 mg of naltrexone free base (SpecGx, LLC) dissolved in 3.15 g of ethyl acetate (Fisher Scientific, Fair Lawn, N.J.) and 1.17 g of benzyl alcohol in a 20 mL scintillation vial. The continuous phase was further mixed with ethyl acetate, and 20 mL of the continuous phase with 6.525% (w/v) ethyl acetate was emulsified with the organic phase at 4,000 or 7,000 RPM for 30 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 250 mL of 2.5% (w/v) ethyl acetate in water and stirred at 4° C. for 2 or 4 h. The microparticles were then collected with a 10 μm sieve and dried at 4° C. under vacuum for about 16 h. Microparticles were then suspended and washed in 150 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then passed through a 150 μm sieve and collected on a 10 μm sieve and vacuum dried for 48 h.

(29) The resulting drug loading and residual benzyl alcohol content can be found in Table 4.

(30) The encapsulation efficiencies appear to be lower than formulations prepared under similar conditions with dichloromethane. Minimal differences are observed in the in vitro release profiles of the three formulations, with all formulations lasting slightly longer than approximately 55 days.

(31) TABLE-US-00004 TABLE 4 Effects of O/W ratio, homogenization speed, and solvent extraction time on the resultant drug loading, residual benzyl alcohol (BA), and encapsulation efficiency (EE). O/W Solvent Drug Residual Formulation ratio Homogenization Extraction Loading BA (F) (v/v) Speed (RPM) Time (hr) (% w/w) (% w/w) EE (%) F.6-1 1/2.82 4,000 4 27.5 0.72 74.3 F.6-2 1/4.35 4,000 2 25.0 0.43 67.6 F.6-3 1/4.35 7,000 4 23.2 0.17 62.7

Example 7

(32) Determination of PLGA Molecular Weight with a Gel-Permeation Chromatography Quaternary Detector

(33) Samples of Resomer RG 858S and Lactel B6006-2P were dissolved in acetone to a concentration of approximately 2.5 mg/mL, filtered through a 0.22 μm PTFE filter, and collected into an HPLC auto-sampler vial for injection. The samples were analyzed using GPC-4D. The GPC-4D system consisted of an Agilent 1260 Infinity II HPLC connected to Dawn Heleos II (MALLS) coupled to Dynapro Nanostar DLS via optical cable, Optilab T-rEX (RI detector) and Viscostar III viscometer operated by Astra 7 software. GPC analysis was performed by injecting 50.0 μl of the polymer solution. Separation was performed with a linear gradient column (Tosoh Bioscience LLC, TSKgel GMHHR-L, 7.8 mm×30 cm) at 0.6 ml/min flow of acetone with a 60-minute run time. The molecular weights of the polymer samples are shown in Table 5.

(34) TABLE-US-00005 TABLE 5 M.sub.n (number average molecular weight), M.sub.w (weight average molecular weight), M.sub.z (higher average molecular weight), M.sub.avg (average molecular weight), and polydispersity Excipient M.sub.n M.sub.w M.sub.z M.sub.avg Polydispersity (E) Polymer Lot Number (kDa) (kDa) (kDa) (kDa) (M.sub.w/M.sub.n) E.7-1 Resomer D161000568 120.15 141.07 171.75 121.95 1.17 RG 858S E.7-2 Lactel A17-028 66.01 78.19 94.40 65.41 1.19 B6006-2P

Example 8

(35) Preparation of Naltrexone Microparticles: Effect of 85:15 Molecular Weight and Extraction Time

(36) The continuous phase was prepared by weighing 40 g of PVA and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG 858S (IV 1.3-1.7 dL/g, Lot Number D161000568, Evonik Cyro, Parsippany, N.J.) or Lactel B6006-2P (IV 0.76-0.85 dL/g, Lot Number A17-068, Durect, Cupertino, Calif.)) and 267 mg of naltrexone free base (SpecGx, LLC) dissolved in 2.0 g of DCM and 623 mg of benzyl alcohol in a 20 mL scintillation vial. The continuous phase was further mixed with DCM, and 10 mL of the continuous phase containing 1.8% (w/v) DCM was added to the organic phase and emulsified at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase (380 mL of 0.5% (w/v) dichloromethane in water) and stirred at 4° C. for 2, 4, or 7 h. The microparticles were then collected with a 25 μm sieve and dried at 4° C. under vacuum for about 16 h. Microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove PVA and any residual solvents from the microparticles. The washed microparticles were then passed through a 125 μm sieve and collected on a 25 μm sieve and vacuum dried for 48 h.

(37) The resultant drug loadings, residual benzyl alcohol content, and encapsulation efficiency can be found in Table 6. Minimal effects of extraction time were noted between the two types of PLGA and their respective drug loading. The residual benzyl alcohol appears to be slightly higher for the Lactel batches vs the Resomer batches. The extraction time does not appear to have an effect on the release rate, whereas differences in the release profile between the two polymers is evident. The Resomer batches appear to have a shorter lag phase vs the Lactel batches, resulting in a more steady-state release over 60 days in vitro compared to the Lactel batches.

(38) TABLE-US-00006 TABLE 6 Effects of PLGA and extraction time on the resultant drug loading, residual benzyl alcohol (BA), and encapsulation efficiency (EE). Extraction Drug Residual Formulation Time Loading BA EE (F) PLGA (hr) (% w/w) (% w/w) (%) F.8-1 Resomer RG 2 28.0 0.88 80.5 F.8-2 858S 4 27.7 0.88 79.6 F.8-3 7 28.6 0.89 82.2 F.8-4 Lactel B6006-2 2 27.5 1.16 79.0 F.8-5 4 28.1 1.06 80.7 F.8-6 7 28.5 1.12 81.9

Example 9

(39) Preparation of Naltrexone Microparticles: Effect of No Ethanol Wash Vs 25% Ethanol Wash

(40) The continuous phase was prepared by weighing 40 g of PVA and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG 858S) and 267 mg of naltrexone free base (SpecGx, LLC) dissolved in 2.0 g of DCM and 623 mg of benzyl alcohol in a 20 mL scintillation vial. The continuous phase was further mixed with DCM, and 10 mL of the continuous phase containing 1.8% (w/v) DCM was added to the organic phase and emulsified at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase (380 mL of 0.5% (w/v) DCM in water) and stirred at 4° C. for 2 h. Microparticles were then collected with a 25 μm sieve and dried at 4° C. under vacuum for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove PVA and any residual solvents from the microparticles or passed through a 125 μm sieve and vacuum dried for 48 h. The washed microparticles were then passed through a 125 μm sieve and collected on a 25 μm sieve and vacuum dried for 48 h.

(41) The resultant drug loadings, residual benzyl alcohol content, and encapsulation efficiency can be found in Table 7. The ethanol wash resulted in microparticles that had a lower residual benzyl alcohol content, and higher drug load—likely due to the benzyl alcohol extraction. The initial 10 days of release is similar between the washed and unwashed particles, but the rate post-10 days is faster for the unwashed particles vs. washed particles.

(42) TABLE-US-00007 TABLE 7 Effects of PLGA and extraction time on the resultant drug loading, residual benzyl alcohol (BA), and encapsulation efficiency (EE). Formulation Drug Loading Residual BA EE (F) Process (% w/w) (% w/w) (%) F.9-1 No Ethanol Wash 27.9 1.71 80.2 F.9-2 25% Ethanol Wash 28.3 0.86 81.3

Example 10

(43) Preparation of Naltrexone Microparticles: Effect of Ethanol Wash Concentration

(44) The continuous phase was prepared by weighing 40 g of PVA and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG 858S) and 305 mg of naltrexone free base (SpecGx, LLC) dissolved in 2.0 g of DCM and 467 mg of benzyl alcohol in a 20 mL scintillation vial. The continuous phase was further mixed with DCM, and 10 mL of the continuous phase with 1.8% (w/v) DCM was added to the organic phase and emulsified at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase (380 mL with 0.33% (w/v) DCM in water) and stirred at 4° C. for 8 h. The microparticles were then collected with a 25 μm sieve and dried at 4° C. under vacuum for about 16 h. Microparticles were then suspended and washed in 200 mL of a 6.25, 12.5, 25, or 50% (v/v) ethanol solution for 8 h at 22° C. to remove PVA and any residual solvents from the microparticles. The washed microparticles were then passed through a 125 μm sieve and collected on a 25 μm sieve and vacuum dried for 48 h.

(45) The resulting drug loadings, residual benzyl alcohol content, and encapsulation efficiency can be found in Table 8. The drug loadings for a 6.25, 12.5, and 25% (v/v) did not result in any significant differences or trend. The residual benzyl alcohol content decreases with an increase in ethanol wash concentration for this condition. At 50% (v/v) a significant amount of naltrexone is lost during the wash and naltrexone release was much faster relative to the 6.25, 12.5, and 25% condition.

(46) TABLE-US-00008 TABLE 8 Effects of ethanol wash concentration on the resultant drug loading, residual benzyl alcohol (BA), and encapsulation efficiency (EE). Ethanol Wash Formulation Concentration Drug Loading Residual BA EE (F) (% v/v) (% w/w) (% w/w) (%) F.10-1 6.25 29.5 0.77 77.8 F.10-2 12.5 29.2 0.67 77.0 F.10-3 25 29.4 0.41 77.5 F.10-4 50 23.9 0.00 63.1

Example 11

(47) Preparation of Naltrexone Microparticles: Effect of Ethanol Wash Temperature

(48) The continuous phase was prepared by weighing 40 g of PVA and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG 858S) and 294 mg of naltrexone free base (SpecGx, LLC) dissolved in 2.0 g of DCM and 467 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J.) in a 20 mL scintillation vial. The continuous phase was further mixed with dichloromethane, and 10 mL of the continuous phase with 1.8% (w/v) dichloromethane was added to the organic phase and emulsified at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase (380 mL of 1% (w/v) PVA in water) and stirred at 4° C. for 8 h. Microparticles were then collected with a 25 μm sieve and dried at 4° C. under vacuum for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 4° C. or 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then passed through a 125 μm sieve and collected on a 25 μm sieve and vacuum dried for 48 h. The resulting drug loadings, residual benzyl alcohol content, and encapsulation efficiency can be found in Table 9.

(49) The ethanol wash at 22° C. resulted in microparticles that had a lower residual benzyl alcohol content, and higher drug load—likely due to the benzyl alcohol extraction. The release of naltrexone from microparticles washed at 22° C. resulted in a quicker release rate until 10 days. The release of naltrexone from microparticles at 4° C. resulted in a quicker release rate after the initial 10 days and only lasted approximately 35 days in vitro, whereas the 22° C. washed particles provided naltrexone release for approximately 50 days.

(50) TABLE-US-00009 TABLE 9 Effects of ethanol wash concentration on the resultant drug loading, residual benzyl alcohol (BA), and encapsulation efficiency (EE). Formulation Ethanol Wash Drug Loading Residual BA EE (F) Temperature (° C.) (% w/w) (% w/w) (%) F.11-1 4 32.1 1.61 86.6 F.11-2 22 32.9 0.76 88.9

Example 12

(51) In Vivo Response of Formulations

(52) Seven formulations with naltrexone (free base) loadings from ˜20% to ˜40% were evaluated in pharmacokinetic studies with Sprague-Dawley rats. Among the seven formulations, each of F.12-1, F.12-2, F.12-4, and F.12-6 was prepared in the same manner as F.3-5, F.4-1, F.8-6, and F.5-4, whereas F.12-3, F.12-5, and F.12-7 were prepared as described below.

(53) F.12-3

(54) The continuous phase was prepared by weighing 40 g of poly(vinyl alcohol) (PVA) (Mowiol 40-88, Sigma Aldrich, St. Louis, Mo.) and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG858S) and 294 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 2.0 g of dichloromethane (DCM) (Fisher Scientific, Fair Lawn, N.J.) and 467 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J. 10 mL of the continuous phase was added to the top of the organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 380 mL of 1% (w/v) PVA in water and stirred at 4° C. for 8 h. Microparticles were then collected with a 25 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then collected on a 25 μm sieve and vacuum dried for 48 h.

(55) F.12-5

(56) The continuous phase was prepared by weighing 40 g of poly(vinyl alcohol) (PVA) (Mowiol 40-88, Sigma Aldrich, St. Louis, Mo.) and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG858S) and 409 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 2.0 g of dichloromethane (DCM) (Fisher Scientific, Fair Lawn, N.J.) and 623 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J.) in a 20 mL scintillation vial. The continuous phase was further mixed with dichloromethane, and 10 mL of the continuous phase with 1.8% (w/v) DCM was added to the top of the organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 380 mL of 0.5% (w/v) DCM in water and stirred at 4° C. for 4 h. Microparticles were then collected with a 25 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then collected on a 25 μm sieve and vacuum dried for 48 h.

(57) F.12-7

(58) The continuous phase was prepared by weighing 40 g of poly(vinyl alcohol) (PVA) (Mowiol 40-88, Sigma Aldrich, St. Louis, Mo.) and mixing with 4 L of deionized water. The organic phase consisted of 500 mg of PLGA 85:15 (Resomer RG858S) and 267 mg of naltrexone free base (Tecoland Corporation, Irvine, Calif.) dissolved in 2.0 g of dichloromethane (DCM) (Fisher Scientific, Fair Lawn, N.J.) and 623 mg of benzyl alcohol (Fisher Scientific, Fair Lawn, N.J.) in a 20 mL scintillation vial. The continuous phase was further mixed with dichloromethane, and 10 mL of the continuous phase with 1.8% (w/v) DCM was added to the top of the organic phase and homogenized at 7,000 RPM for 60 sec with an IKA T25 homogenizer with S25N-10G generator (IKA Works, Inc. Wilmington, N.C.). The mixture was then transferred into an extraction phase, which is 380 mL of 0.5% (w/v) DCM in water and stirred at 4° C. for 7 h. Microparticles were then collected with a 25 μm sieve. The product retained by the sieve was dewatered for 15 min at 22° C. and vacuum dried for about 16 h. The microparticles were then suspended and washed in 200 mL of a 25% (v/v) ethanol solution for 8 h at 22° C. to remove the emulsifying agent (PVA) and any residual solvents from the microparticles. The washed microparticles were then collected on a 25 μm sieve and vacuum dried for 48 h.

(59) The effect of drug loading, solvent system, and solvent concentration on naltrexone release was evaluated. The lowest drug loading sample (F.12-2), resulted in what appears to be the largest burst release, relative to the formulations steady state release. As the drug load increased from approximately 28% to 38%, higher concentrations from around day 2 to day 20 were observed, but the formulations observed similar steady state plasma concentrations after day 20 of between approximately 1.5 to 4 ng/mL until approximately day 60. The naltrexone concentration of 0.5 ng/mL was maintained for about 100 days.

(60) TABLE-US-00010 TABLE 10 Formulation summary for formulations characterized in vivo PLGA Naltrexone Organic Concentration Concentration Phase Dose Drug in DCM or in BA (% Solvent Level Loading (% Formulation (F) EA (% w/w) w/w) System (mg/kg) w/w) EE (%) F.12-1 20.0 38.6 DCM/BA 76 28.1 80.7 F.12-2 20.0 32.1 DCM/BA 76 21.7 58.6 F.12-3 20.0 38.6 DCM/BA 50 34.2 92.4 F.12-4 15.0 24.0 EA/BA 50 28.5 77.0 F.12-5 20.0 39.6 DCM/BA 50 36.1 80.2 F.12-6 20.0 44.0 DCM/BA 50 37.8 84.0 F.12-7 20.0 30.0 DCM/BA 50 27.8 79.9 Drug Loading (% w/w) is based on the microparticle weight.

(61) Test Procedures

(62) In Vitro Release of Naltrexone from Test Formulations

(63) The medium, 20 mL of pH 7.4 phosphate buffered saline with 0.05% Tween 20 (Sigma Aldrich, St. Louis, Mo.) and 0.0625% (w/v) sodium ascorbate (Sigma Aldrich, St. Louis, Mo.) and approximately 5 mg of test article were placed in a stoppered 50 mL Erlenmeyer flask and placed in a 37° C. water bath at 100 RPM. Samples were taken at various time points and replaced with fresh release medium. Naltrexone content in buffer was measured via High Performance Liquid Chromatography (HPLC).

(64) Reversed Phase HPLC for the Quantitation of Naltrexone

(65) The HPLC had the following conditions: Mobile Phase: 65:35 methanol:potassium phosphate buffer, pH 6.6; flow rate: 1.0 ml/min; autosampler temperature:room temperature; column temperature: 30° C.; detection: 210 nm (UV); total run time: 7 min; injection volume: 10 μL; column: Zorbax SB-C18 150×4.6 mm, 5 μm; approximate retention time of naltrexone: 4.8 min.

(66) In Vivo Pharmacokinetic Studies

(67) All rat preclinical studies were conducted in Sprague-Dawley rats. Three to five rats per test formulation were injected subcutaneously in the scruff behind the neck or in the scapular region with a dosage of naltrexone ranging from 50 mg/kg to 100 mg/kg in 1 ml of an aqueous-based vehicle, composed of 0.9% sodium chloride, 0.02% Tween 20, and 0.5% sodium carboxymethylcellulose.

(68) During the course of the study, the animals were observed for overt toxicity and any existing test site abnormalities, including redness, swelling, bleeding, discharge, and bruising at the injection site. In addition, body weights were taken and recorded at administration and at the conclusion of the study.

(69) At selected time points, rats were anesthetized and bled (approximately 250 μL) via the tail or submandibular vein. Blood was collected in labeled potassium ethylenediaminetetraacetic acid tubes. The blood was centrifuged for 10 min at 4,000 rpm at 4° C. The plasma fraction was transferred to labeled 1 mL plastic tubes and stored at −80° C. prior to analysis.