PREPARATION OF PEPTIDE LOADED PLGA MICROSPHERES WITH CONTROLLED RELEASE CHARACTERISTICS

Abstract

A novel process for the preparation of a long acting injectable composition based on biodegradable poly(D,L-lactide-co-glycolide) microspheres comprising peptide active pharmaceutical ingredients.

Claims

1. A process for the preparation of a poly(D,L lactide-co-glycolide) polymer microspheres of a peptide, which peptide can also be in the form of a pharmaceutically-acceptable salt, comprising: a. dissolving the peptide, or salt thereof, in at least one organic solvent miscible in water, and optionally containing also water, to form a water phase; b. forming an oil-in-water or water-in-oil-water emulsion in a suitable oil phase comprising an organic solution of the poly(D,L lactide-co-glycolide) polymer, the solution being non-miscible with the water phase; c. evaporating the at least one organic solvent used in step a from the emulsion to form the microspheres by controlling the temperature during the evaporation step and increasing the temperature during the evaporation step.

2. A process according to claim 1 wherein peptide active substance is octreotide acetate.

3. A process according to claim 1 wherein the organic solvent is methanol and water is added.

4. A process according to claim 1 wherein the poly(D,L lactide-co-glycolide) polymer is dissolved in dichloromethane.

5. A process according to claim 4 wherein and the solution in step b is cooled down to 5° C. or below.

6. A process according to claim 1 wherein the evaporation in step c is initiated above 15° C. and during the evaporation step the temperature of the emulsion is raised to greater than 35° C.

7. A process according to claim 1, wherein the temperature is raised over a time period of 20 min to 3 hours.

8. A process according to claim 7 wherein further drying continues for an extended period after the period of temperature elevation.

9. A population of microspheres obtainable by a process as claimed in claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1a: Schematic diagram of the double emulsification process

[0043] FIG. 1b: Schematic diagram of the single emulsification process

[0044] FIG. 2a: Particle size distribution graph for the formulations 1a-1c

[0045] FIG. 2b: Particle size distribution graph for the formulations 2a-2c

[0046] FIG. 3a: SEM and crosssectional SEM images of the formulations 1a-1c

[0047] FIG. 3b: SEM and crosssectional SEM images of the formulations 2a-2c

[0048] FIG. 4a: Comparative dissolution profiles of Sandostatin LAR and formulations 1a-1c

[0049] FIG. 4a: Comparative dissolution profiles of Sandostatin LAR and formulations 2a-2c

[0050] FIG. 5: Plasma concentration profiles in rats of Sandostatin LAR and formulations 1a and 2c

[0051] FIG. 6a: Comparison of the in vivo observed and the back-calculated plasma concentration of Sandostatin LAR

[0052] FIG. 6b: Comparison of the in vivo observed and the back-calculated plasma concentration of formulation 1a

[0053] FIG. 6c: Comparison between the observed and the predicted plasma concentration of formulation 2c

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention provides a sustained release formulation of octreotide acetate with release characteristics controlled by the controlling and increasing the temperature during the solvent evaporation step of the manufacturing process.

[0055] Octreotide (also known as (4R,7S,10S,13R,16S,19R)-19-[(2R)-2-amino-3-phenylpropanamido]-10-(4-aminobutyl)-16-benzyl-N-[(2R,3R)-1,3-dihydroxybutan-2-yl]-7-(1-hydroxyethyl)-13-(1H-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carboxamide, preferably in the form of the acetate salt (or any other pharmaceutically-acceptable salt) or also known as 4D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-((1R,2R)-2-hydroxy-1-(hydroxymethyl)propyl)-L-cysteinamide. cyclic (2-7)-disulfide acetate (salt) or L-Cysteinamide-D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-(2-hydroxy-1-(hydroxymethyl)propyl)-cyclic(2-7)-disulfide (R—(R*,R*)), acetate (salt)) is a synthetic octapeptide (DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol) analogue of the naturally occurring hormone somatostatin, and is approved for use in tumor control in neuroendocrine disorders such as acromegaly and gastroenteropancreatic neuroendocrine tumors. The chemical structure of octreotide acetate is shown below:

##STR00001##

[0056] Suitable commercially obtainable polymers for use in preparing of PLGA microspheres according to the present invention include but are not limited to RESOMER® and LAKESHORE BIOMATERIALS by Evonik Industries AG, Expansorl® by PCAS., PURASORB® by PURAC Biochem BV. The PLGA polymers used in the present invention may have a ratio of lactic acid and glycolic acid in the range of about 50:50 to about 65:35 and a weight average molecular weight (Mw) in the range of 10,000 to 70,000. Preferably the present invention uses PLGA having a monomer ratio of 50:50 and a weight average molecular weight in the range of 30,000-50,000.

[0057] Organic solvents for the PLGA that can be used in the present invention include but not limited to ethylacetate, tetrahydrofurane, acetonitrile, dichloromethane, hexafluoroisopropanol, chloroform and acetone. More preferably, in the present invention dichloromethane is used. The polymer concentration in the organic solvent is 10-40% wt., most preferably 20-30% wt.

[0058] In the present invention octreotide acetate is dissolved in water for injection to result in a concentration of 10-40% wt. and more preferably 25-35% wt. Alternatively octreotide acetate is dissolved in a suitable organic solvent miscible in water, preferably methanol, to result in a concentration of 5-20% wt., more preferably 10% wt. Optionally an organic solvent miscible in water with water is used.

[0059] For the preparation of the dispersed phase, the octreotide acetate solution is dispersed in the polymer solution by using a batch mode high shear disperser operating at a shear rate of 15,000-30,000 s.sup.−1. More preferably, a shear rate of 20,000-25,000 s.sup.−1 is applied. Alternatively, octreotide acetate solution in methanol is added in the polymer solution under stirring. The dispersed phase is controlled at a temperature of lower than 20° C., more preferably 5-10° C.

[0060] In the present invention the continuous phase consists of an aqueous solution with a surfactant, preferably polyvinyl alcohol (PVA). Examples of other surfactants that optionally can be employed include one or more; anionic surfactants (such as, sodium oleate, sodium stearate or sodium lauryl sulfate), non-ionic surfactants (such as, Poloxamers, Tweens), polyvinylpyrrolidone, carboxymethyl cellulose sodium and gelatin, used independently or in combination. PVA, preferably have a weight average molecular weight from about 10,000 to about 150,000 Da that correspond to viscosity range of 3-9 cP when measured as a 4% aqueous solution at 20° C., 85-89% degree of hydrolysis and ester number of 130-150. Selected PVA grades that are used in the present invention include Emprove PVA 4-88 (Mw 25,000-30,000; viscosity 4% in water: 3.4-4.6 cPs), PVA 8-88 (Mw about 65,000; viscosity 4% in water 6.8-9.2 cPs) and PVA 18-88 (Mw about 130,000; viscosity 4% in water) available by Merck KGaA. Amount of the surfactant added to the aqueous phase is preferably up to 5.0% (w/w) relative to mass of the aqueous solution. More preferably the amount of surfactant (optimally the PVA amount) is from about 0.5 to about 2.5% w/w. The continuous phase is thermo stated at a temperature lower than 20° C., more preferably 5-10° C.

[0061] In the present invention the emulsification of the water phase in the continuous oil phase is performed with one of the following means: i) mechanical stirring, ii) batch disperser iii) in line disperser. Preferably, the emulsification process takes place by an in-line disperser MT-3000 available by Kinematica operating at a shear rate of 5,000-20,000 s.sup.−1, most preferably at a range of 10,000-15,000 s.sup.−1 to result in the formation of microspheres of 10-250 μm, most preferably of 20-100 μm. The weight ratio between the dispersed and the continuous phase during is 1:20-1:150, more preferably 1:75-1:100.

[0062] The formed microsphere suspension is transferred (from the outlet of the in-line disperser) into a suitable vessel (preferably insulated to help with temperature control) which is initially controlled at above 15° C., preferably at about 20° C. The temperature during the solvent evaporation is increased from a starting temperature of from 15 to 25° C., preferably about 20° C. Preferably the maximum temperature achieved is up to 35° C. or 38° C. The temperature is raised over a time period of 20 min to 3 hours. Drying may continue for an extended period after the period of temperature elevation. Preferably the rate of temperature increase is 0.1° C./min-1° C./min. The temperature rise may be constant over the period or staged. By staged we mean that each change is a step change in temperature and then that temperature is held for a period before the next change. There can also be a mixture of staged and constant temperature changes during the evaporation stage.

[0063] The evaporation of the solvent from the microsphere takes place according to the applied temperature profile under stirring and a partial vacuum, preferably the partial vacuum is slightly below the vapour pressure of dichloromethane.

[0064] After the evaporation of the solvent the hardened particles are collected from the suspension in a filter dryer under low stirring. Preferably, a filter dryer from PSL (Powder System Limited) is used. The collected particles are washed with water and then dried in the filter dryer by applying a vacuum of about 10 mbar.

[0065] The final microspheres are analysed with respect to particle size, drug loading, polymer molecular weight, residual solvent and in vitro release as described below. The results are summarized in Tables 1 and 2 and also in FIGS. 3-4. Selected formulations (i.e., 1a and 2c) along with the reference product Sandostatin LAR have been tested in rats to establish an in vitro in vivo correlation model. The details of the PK study and the applied methodology for the development and the external are provided below. The in vivo results are provided in FIGS. 5-6.

Particle Size Distribution (PSD) Analysis

[0066] Particle side distribution was measured by laser diffraction using a Malvern Master Sizer 2000 Hydro2000S. The average particle size is expressed as the volume mean diameter in microns.

Determination of Drug Loading

[0067] About 50 mg of microspheres were completely dissolved in 10 ml methylene chloride (30 min sonication). 20 ml of 0.1M acetate buffer (pH 4.0) was added to the solution for extraction of octreotide into an aqueous phase. The two phases were thoroughly mixed by vortexing for 5 min then separated by centrifugation at 4000 rpm for 5 min. The aqueous phase was sampled for HPLC analysis to measure the content of octreotide. The sample was filtered through a 0.45 μm syringe filter before analysis. The HPLC conditions were as follows: gradient separation was performed with an Inertsil ODS3 column (4.6×250 mm, particle size 5 μm); the mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in distilled water (eluent A) and 0.1% TFA in acetonitrile (eluent B) and was run with a linear gradient from 20% to 35% eluent B for 18 min. The flow rate was 1.0 ml/min, the injection volume was 10 μl and the detection wavelength was 210 nm.

Mean Molecular Weight Measurement

[0068] The molecular weight of microspheres was determined by gel permeation chromatography (GPC) using an Agilent Model GPC 50Plus system equipped with 2 columns PLgel 5 μm Mixed-D 300×7.5 mm connected in series and a refractive index (RI) detector. The mobile phase is THF with a flow rate of 1 ml/min and the temperature of the column is 300° C. For the analysis of the samples, 10-15 mg of microspheres are dissolved in 5 mL THF and the solution is left overnight under stirring. 2 ml are withdrawn, filtered through a 40 μm PTFE filters and analysed. The injection volume is 100 μL. The data collection and analysis was performed using Cirrus software. Polystyrene standards with MW range between 162 and 371100 are used for calibration.

In Vitro Release Method

[0069] About 150 mg of the formulated microspheres were placed in a 100 ml bottle and incubated in 30 ml acetate buffer (1 mM, pH 4.0) at 37° C. in a shaking bath (85 rpm). The release medium was sampled at various time points, filtered through a 0.45 μm syringe filter and analyzed via HPLC. The HPLC conditions were as follows: gradient separation was performed with an Inertsil ODS3 column (4.6×250 mm, particle size 5 μm); the mobile phase consisted of 0.1% trifluoroacetic acid (TFA) in distilled water (eluent A) and 0.1% TFA in acetonitrile (eluent B) and was run with a linear gradient from 25% to 35% eluent B for 25 min. The flow rate was 1.0 ml/min, the injection volume was 10 μl and the detection wavelength was 210 nm.

Residual Solvents (Dichloromethane)

[0070] It was investigated whether ethanol and/or methylene chloride are removed from Octreotide micro-particles. Micro-particles are totally dissolved in dimethyl sulfoxide (DMSO). Therefore, concentrations of ethanol and methylene chloride in the organic solution were quantitated by gas chromatography (GC). An amount of 90 mg of fresh micro-particles were dissolved in 5 mL of dimethyl sulfoxide in a GC vial before headspace GC analysis. The residual solvents evaporation was achieved by a 30 min sample incubation at 100° C. in the GC oven. GC analysis was performed with GC-2010 plus (Shimadzu, Japan). An RTX-5 (RESTEK, USA) analysis column using Crossbond 5% diphenyl/95% dimethyl polysiloxane as stationary phase was used. The quantity of solvents was measured using calibration curve standards. The retention time of dichloromethane is 5.75 min

Animal Studies

[0071] Microsphere formulations after mixing with crystalline mannitol (i.e., 17% mannitol with respect to the total weight) were subjected to sterilization by UV radiation at 365 nm for 180 min using a 6 Watt UV light source in order to be used in pharmacokinetic studies in male Sprague-Dawley rats. Rats (six per group) weighing about 240-250 grams and of age 8-12 weeks were injected with the test formulations. Prior to injection, the dry powder samples were suspended in a sterile solvent (vehicle) consisting of water for injection, sodium carboxymethylcellulose 0.5% wt. and mannitol 0.6% wt. Animals were administered one single intramuscular injection in fixed dose of about 60 mg octreotide formulation (microspheres)/rat that corresponds to 3 mg octreotide active substance/rat in a volume of 0.25 ml/rat of a vehicle, into a single site. The suspension was administered via a hypodermic needle of 26G intramuscularly into the rat's quadriceps muscle located on the cranial aspect of the femur. Rat blood samples were collected before drug dosing and thereafter at predetermined time points including 0.5 h, 1 h, 2 h, 6 h, 24 h, Day 3, 7, 14, 18, 21, 28, 35, 42, 49, 56 and Day 70 post-dose. At each time point, approximately 0.5 ml of blood were withdrawn from retro-orbital plexus under light isoflurane anesthesia and transferred into labeled tubes containing 200 mM K.sub.2EDTA as anticoagulant and mixed by manual inversion 4-5 times. The blood samples were kept on wet ice at all times and the plasma was separated by centrifugation within 1 h of sample collection. The plasma samples were stored below −60° C. until bioanalysis using a validated LC-MS/MS method.

In Vitro In Vivo Correlation

[0072] For the development of the IVIVC model, the below procedure was followed: (1) selection of formulations with different release rates including Sandostatin LAR, formulation 1a and formulation 2c, (2) measurement of in vitro dissolution profiles by various dissolution methods, (3) measurement of the PK in vivo plasma concentration profiles of the formulations (i.e., Sandostatin LA, formulation 1a and formulation 2c) after a single intramuscular administration in rats, (4) estimation of the in vivo release profile by the measured plasma concentration profiles using the Wagner-Nelson (one-compartment model) deconvolution technique, (5) calculation of the correlation between the deconvoluted in vivo release profile and the in vitro dissolution profiles using the pooled data of Sandostatin LAR and formulation 1a, (6) selection of the appropriate dissolution method with the higher correlation to in vivo data and establishment of the IVIVC by applying linear regression analysis (6) internal validation of the established model by comparing the back-calculated plasma concentration profiles using Wagner-Nelson convolution model for formulation Sandostatin LAR and formulation 1a and (7) external validation of the established model by comparing the predicted plasma concentration calculated by the in vitro dissolution profile of formulation 2c and the established model and the observed in vivo plasma concentration profile of formulation 2c.

Example 1a-c (Single Emulsion)

[0073] 4 g of poly(D,L-lactide-co-glycolide) with a molar ratio of 50:50 (Mw=41,000 and polydispersity ca 1.65), commercially available from PURAC under the name PURASORB 5004A, was dissolved in 30 g of dichloromethane with magnetic stirring. The polymer solution is cooled down to 5° C. 0.2941 g of octreotide acetate was dissolved in 0.5882 g of water for injection with mixing. The octreotide acetate solution was added to the polymer solution and emulsified by mean of an Ultra Turrax® for 1 minute at 20,000 rpm to form the first emulsion (DP-dispersed phase). 11.5 g of poly(vinyl alcohol) EMROVE® 18-88 by Merck were dissolved in 2307 g of water for injection at 80° C. followed by the addition of 17.46 g of disodium hydrogen phosphate and 4.18 g of potassium dihydrogen phosphate. The solution was cooled down to 5° C. forming the continuous phase (CP). Microspheres of the desired particle size distribution were prepared by delivering the CP at 2.3 L/min and the DP at 15.6 mL/min, into an in-line Kinematica MT 3000 disperser. The microsphere suspension was received in a double-jacketed glass reactor vessel, controlled at 20° C. and with vigorous stirring, in order to remove the solvent. The temperature in the vessel was increased to 38° C. according to predefined time intervals as presented in the following table (Table 1). After 4 hours the microspheres were transferred to a glass filter dryer, washed with an excess of water at room temperature and left at 10 mbar vacuum and under gently stirring for 24 hours to dry.

TABLE-US-00001 TABLE 1 Properties of 1a-1c formulations Time 20- Drug Particle Residual Total 38° C. Loading M.sub.w size DCM Impurities Formulation (min) (%) (g/mol) distribution (%) (%) Dissolution Sandostatin 5.0 64,200 d(0.1) 0.30 3 x.sub.0 (Lag time) LAR ® 36.7  25.4 d(0.5) y.sub.0 (Initial release) 50.7   4.3 d(0.9) a (final release) 69.8  90.0 b (Slope)  6.28 1a 20 4.83 39,600 d(0.1) 0.31 2.23 x.sub.0 (Lag time) 27.56  22.1 d(0.5) y.sub.0 (Initial release) 44.097  0.4 d(0.9) a (final release) 71.158 77.4 b (Slope)  2.6 1b 60 5.15 40,200 d(0.1) 0.26 1.29 x.sub.0 (Lag time) 29.907 25.8 d(0.5) y.sub.0 (Initial release) 51.017 0  d(0.9) a (final release) 84.049 82.0 b (Slope)  2.3 1c 180 4.85 40,850 d(0.1) 0.18 1.95 x.sub.0 (Lag time) 31.148 31.5 d(0.5) y.sub.0 (Initial release) 48.22   0.4 d(0.9) a (final release) 74.301 80.6 b (Slope)  2.2

Example 2a-c (Double Emulsion)

[0074] 4 g of poly(D,L-lactide-co-glycolide) with a molar ratio of 50:50 (Mw=41,000 and polydispersity ca 1.65), commercially available from PURAC under the name PURASORB 5004A, was dissolved in 30 g of dichloromethane with magnetic stirring. The polymer solution is cooled down to 5° C. The polymer solution is sterilized by filtration through a syringe filter and cooled down to 5° C. 0.2941 g of octreotide acetate was dissolved in 2.941 g of methanol with mixing. The octreotide acetate solution was added to the polymer solution and mixed with stirring to form the oil phase (DP-dispersed phase). 11.5 g of poly(vinyl alcohol) EMROVE® 18-88 by Merck were dissolved in 2307 g of water for injection at 80° C. followed by the addition of 17.46 g of disodium hydrogen phosphate and 4.18 g of potassium dihydrogen phosphate. The solution was cooled down to 5° C. forming the continuous phase (CP). Microspheres of the desired particle size distribution were prepared by delivering the CP at 2.3 L/min and the DP at 18.7 mL/min, into an in-line Kinematica MT 3000 disperser. The microsphere suspension was received in a double-jacketed glass reactor vessel, controlled at 20° C. and with vigorous stirring, in order to remove the solvent. The temperature in the vessel was increased to 38° C. according to predefined time intervals as presented in the following table (Table 3). After 4 hours the microspheres were transferred to a glass filter dryer, washed with an excess of water at room temperature and left at 10 mbar vacuum and under gently stirring for 24 hours to dry.

TABLE-US-00002 TABLE 2 Properties of 2a-2c formulations Time 20- Drug Particle Residual Total 38° C. Loading M.sub.w size DCM Impurities Formulation (min) (%) (g/mol) distribution (%) (%) Dissolution Sandostatin 5.0 64,200 d(0.1) 0.30 3 x.sub.0 (Lag time) LAR ® 36.7  26.5 d(0.5) y.sub.0 (Initial release) 50.7   7.4 d(0.9) a (final release) 69.8  84.7 b (Slope)  5.2 2a 20 4.79 41,050 d(0.1) 0.23 2.22 x.sub.0 (Lag time) 26.682 19.4 d(0.5) y.sub.0 (Initial release) 42.577 0  d(0.9) a (final release) 68.198 82.3 b (Slope)  4.2 2b 60 4.8 39,780 d(0.1) 0.38 1.52 x.sub.0 (Lag time) 28.281 23.7 d(0.5) y.sub.0 (Initial release) 46.96  0  d(0.9) a (final release) 76.958 81.5 b (Slope)  3.9 2c 180 5.03 40,230 d(0.1) 0.19 0.85 x.sub.0 (Lag time) 28.644 31.7 d(0.5) y.sub.0 (Initial release) 46.742  5.0 d(0.9) a (final release) 75.041 84.1 b (Slope)  3.9