Biomacromolecules 2003, 4, 1316-1320
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Heterostereocomplexes Prepared from D-PLA and L-PLA and Leuprolide. II. Release of Leuprolide Joram Slager and Abraham J. Domb* Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel Received March 25, 2003; Revised Manuscript Received June 10, 2003
Reversible stereoselective complexes were spontaneously obtained from mixing acetonitrile solutions of enatiomeric D-poly(lactic acid) (D-PLA), L-poly(lactic acid) (L-PLA), and leuprolide, a L-configured nonapeptide LHRH analogue. The complex spontaneously aggregated and precipitated in high yields (>90%) from acetonitrile solution, forming uniform, porous microparticles. The stereocomplex microparticles showed a continuous release of the interlocked peptide for a period of one to three months under physiological conditions. Various factors, including method of complex formation, molecular weight of PLA, leuprolide: polymer and D-PLA:L-PLA complex ratios, and additives, influenced the release pattern of leuprolide from the stereocomplexes. Continuous release of leuprolide for over 100 days was observed for certain stereocomplex compositions. In vivo evaluation of the leuprolide loaded stereocomplexes in rats by monitoring testosterone levels in the blood of rats after subcutaneous injection showed low testosterone levels for over 42 days. Introduction For more than three decades, polyesters of lactic and glycolic acid have been used for a variety of medical applications, including drug delivery, because of their biocompatibility and biodegradability.1-4 Until today, however, only very few examples of clinically available slow release systems for peptides are known. The list includes lactide based matrix microspheres for the delivery of leuprolide hormones, somatostatin hormone, and growth hormone.5,6 These peptide delivery systems are generally based on dispersion of the drug powder in the biodegradable polymer matrix. The controlled release from these systems is mainly dependent on the rate of diffusion of the peptide from the matrix, aided by the polymer degradation. The main disadvantages of this matrix type delivery system for peptides are fast initial release of the bioactive agent from the polymer matrix (‘burst release’) and loss of bioactivity of the peptide drug.7,8 In recent publications, we reported on a novel reversible stereoselective complex, formed between naturally L-configured peptides and D-configured poly(lactic acid) (DPLA),9,10 that offered an alternative approach for peptide delivery. The controlled release of peptide was thought to rely on complex disruption, aided by polymer degradation, rather than diffusion from the polymer matrix.9 The release of leuprolide from the heterostereocomplex with D-PLA has been reported.11 It is expected that by adding L-PLA to the D-PLA/leuprolide reaction mixture stereocomplex precipitates may be formed and a change in peptide release is expected * To whom correspondence should be addressed. Dept of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University, Campus Ein Karem, Jerusalem 91120, Israel. Phone: ++972-2-6757573. Fax: ++972-2-6758959. E-mail:
[email protected].
to be observed. This article focuses on the addition of L-PLA to the complexation reaction mixture of leuprolide and D-PLA and its effect on the controlled release of the peptide. Materials Chemicals and solvents were purchased from SigmaAldrich Israel or from Mallinckrodt-J. T.Baker BV, Deventer, Holland. D- and L-Lactides were obtained from Purac BV, Gorinchem, Holland. Stannous(II)bis-2-ethylhexanoate (Sn(Oct)2) was obtained from Sigma-Aldrich, Israel. Leuprolide acetate was purchased from Novetide Ltd., Israel. Thermal analysis was determined on a Mettler TA 4000DSC differential scanning calorimeter (DSC), calibrated with zinc and indium standards, at a heating rate of 10 °C/min. Molecular weights were estimated using a gel permeation chromatography (GPC) system consisting of a Spectra Physics (Darmstadt, Germany) P1000 pump and equipped with a refractive index detector ERC-7510, ERMA Inc., a Rheodyne (Coatati, CA) injection valve with a 20 µL loop, and a Spectra Physics Data Jet integrator connected to a computer. Samples were eluted with CHCl3 through a linear Styrogel column (Waters, 10 µm pore size) at a flow rate of 1 mL/min. The molecular weights were determined relative to polystyrene standards (Polyscience, Warrington, PA). Sonication was performed in an ultra-sonic bath Transsonic T 460 (Elma, Germany). Particle size was determined by a Coulter N4 SD Submicron Particle Size Analyzer, Coulter Electronics, U.S.A. HPLC analysis was performed on a HP1100 system (Hewlett-Packard, U.S.A.), column: RP C-18 5 × 250 mm equipped with guard column C-8 LichroCard, (Merck, Germany). Testosterone levels were determined by a competitive immunoassay using an automated chemiluminescence system ACS 180 (Chiron Diagnostics Corp., USA).
10.1021/bm0300249 CCC: $25.00 © 2003 American Chemical Society Published on Web 07/24/2003
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Figure 1. DSC thermoscans of leuprolide and stereocoplexes. A. leuprolide; B. D-PLA 120 kDa; C. Stereocomplex of leuprolide and D-PLA and L-PLA (120 kDa), obtained by precipitation from an acetonitrile solution at 60 °C. DSC was determined on a 5 mg dry powder of the complex at a heat rate of 10 °C/min.
Figure 2. Release of leuprolide from leuprolide/D-PLA/L-PLA complex, effect of addition of L-PLA. Complexes of leuprolide, ( (diamond) 2% w/w of total polymer, 9 (square) 5% w/w or 2 (triangle) 10% w/w, and D-PLA (100 kDa) with added L-PLA (30 kDa, amount equal to D-PLA). Complexes were obtained by spontaneous precipitation from solution in acetonitrile while stirring at 60 °C for 3 days. 9 (gray square): addition of PEG400 (5% v/v) to the reaction mixture of D-PLA, L-PLA (100 kDa) and leuprolide (4% w/w). The release of leuprolide was conducted in 0.1 M phosphate buffer solution pH 7.4, at 37 °C. Leuprolide concentration in the releasing medium was determined by HPLC. Release (%) was determined based on the total leuprolide content in the particles.
Formation of a PLA Stereocomplex Containing Leuprolide. Stereoselective and racemic PLA were synthesized by ring opening polymerization of lactide using stannous 2-ethylhexanoate (Sn(Oct)2) as catalyst and different alcohols as cocatalysts as discribed earlier.12 Stereocomplexes were prepared by mixing D-PLA (19 mg), L-PLA (19 mg), and leuprolide (2 mg, 5% w/w) in acetonitrile (1 mL) in glass ampules equipped with a micro stirrer. After sealing the ampule, the mixture was stirred at 60 °C for 3 days. During the reaction, the solution became turbid, and a white precipitate was formed, which was isolated by
Figure 3. Release of leuprolide from leuprolide/D-PLA/L-PLA complex, effect of L-PLA/D-PLA ratio. Complexes of leuprolide (5% or 9% w/w with D-PLA (100 kDa) and with added L-PLA (100 kDa) in different ratios, were obtained by precipitation from acetonitrile (2 mL) at 60 °C, after stirring for 3 days. Gray line: leuprolide 5% w/w and D and L-PLA added in 1:1 ratio. Black symbols: complexes with 5% w/w leuprolide, ( (diamond) D-PLA:L-PLA, 2:1 ratio, 2(triangle) 2:3 ratio, Open symbols: complexes with 9% w/w leuprolide, ) (diamond) D-PLA:L-PLA, 2:1 ratio, 4(triangle) 1:2 ratio. Release studies were conducted as described in Figure 2.
filtration or centrifugation. The precipitate was dried overnight in a vacuum over P2O5. The obtained precipitates were characterized by differential scanning calorimetry (DSC). The peptide content of the precipitate and remaining supernatant were determined by HPLC. Stereocomplex Formation by Spray Freezing. An aqueous leuprolide solution (40 µL, 5% w/v, 2 mg) was dispersed in a dichloromethane solution (2 mL) containing equal amounts of D- and L-PLA (19 mg of each, 3.8% w/v). The
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Figure 4. Release of leuprolide from leuprolide/D-PLA/L-PLA complex, effect of PLA molecular weight. O (open circle) leuprolide (1 mg, 2.5% w/w) was reacted with D-PLA (3.5kDa, 19 mg). After 5 days, L-PLA (30 kDa, 19 mg) was added to the still clear solution and stirred for an additional 3 days at 60 °C. b (closed circle) leuprolide (2 mg, 5% w/w) was reacted with D-PLA 10kDa (18 mg). After 5 days, L-PLA 30k (18 mg) was added to the clear solution and stirred for an additional 3 days at 60 °C. The precipitated complex was dried overnight over P2O5 and used for the release study. Release study was conducted as described in Figure 2.
dispersion was thoroughly mixed and subsequently sprayed into liquid nitrogen (300 mL) containing frozen petroleum ether (100 mL) after which the mixture was put at -4 °C until all liquid nitrogen had evaporated.10 The microparticles were isolated by centrifugation and dried in a vacuum, and their thermal behavior was analyzed using DSC in order to ascertain stereocomplex formation. In Vitro Release of Leuprolide. Peptide release from the obtained stereocomplexes was measured by suspending the dry complex (20 mg) in 0.1 M phosphate buffer pH 7.4 (1 mL) in disposable syringes, equipped with stoppered 0.45 µm poly(tetrafluoroethylene) (PTFE) syringe filters, while continuously shaking on an orbital shaker at 100 rpm at 37 °C. At specific time intervals the buffer was removed from the syringe via the filter. Fresh buffer was returned into the syringe via the same filter in order to release stuck particles. Phosphoric acid (50 µL) was added to the samples, and leuprolide concentrations were determined by reverse phase HPLC, at a flow of 1 mL/min of 30% acetonitrile/70% 0.01M TEAP-buffer pH ) 3 and UV detection at 278 nm. In Vivo Studies. The in vivo activity of the leuprolide/ D-PLA heterostereocomplex was determined in male Wistar rats weighing 250 g (n ) 5). Formulations containing heterostereocomplex and drug-free control formulations were prepared as described above. The following formulations were prepared: A. D-PLA (300 mg) and leuprolide (30 mg, 5% w/w) were dissolved in acetonitrile (40 mL) at 60 °C. L-PLA (300 mg) was added, and the solution was stirred for 3 days. The yield was 580.8 mg (92%). Particle size is 2.8 µm.
Slager and Domb
Figure 5. Release of leuprolide from leuprolide/D-PLA/L-PLA microparticles, obtained by spray-freezing in liquid nitrogen. Leuprolide (2 mg, 5% w/v) in water (40 µL) was dispersed in dichloromethane (2 mL) with L-PLA (19 mg, 100 kDa) and D-PLA (19 mg) of the following molecular weights, 3 kDa (open diamond), 10 kDa (open square) or 120 kDa (open triangle), and sprayed in a mixture of liquid nitrogen (300 mL) and petroleum ether (100 mL). The particles were isolated and dried in a vacuum overnight. Release study was conducted as described in Figure 2.
B. D-PLA (100 mg) and leuprolide (30 mg, 10% w/w) were dissolved in acetonitrile (40 mL) at 60 °C. L-PLA (200 mg) was added and the solution stirred for 3 days. The yield was 230 mg (70%). The complexes were resuspended in a 5% glucose solution, vortexed thoroughly, sonicated in a sonication bath, and lyophilized. Prior to injection, the complexes were resuspended in deionized water and injected subcutaneously into the rats. The injected amount of formulation contained a total of 1.25 mg leuprolide per kg rat to be released in a sustained manner. Blood was collected from the tail-artery once every week over a period of five weeks postinjection. The samples were centrifuged for 10 min at 3000 rpm, and the serum was isolated. Testosterone levels were determined by a competitive immunoassay. Serum (15 µL) and steroid releasing agent (50 µL, 0.1 µg/mL) were mixed in order to release testosterone from endogenous binding proteins in the serum. A fixed amound of acridinium ester-labeled testosterone (50 µL, ∼3.2 ng/mL) was added to the serum, which was incubated with 300 µL polyclonal rabbit anti-testosterone antibodies (33 ng/mL) for 5.0 min in a competitive binding assay. The rabbit anti-testosterone antibody was bound to a mouse anti-rabbit antibody which was coupled to paramagnetic particles in a solid phase. The amount of chemical luminescence was inversly related to the amount of testosterone present in the sample. Results and Discussion Complex Formation. The PLA polymers used in this study were synthesized and characterized as described in Part I. Various complexes were prepared containing leuprolide and D-PLA to which L-PLA was added as co-complexing
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Figure 6. In vivo activity of leuprolide stereocomplex. 9 (square) leuprolide (5% w/w) and D- and L-PLA (120 kDa) or 2 (triangle) leuprolide (10% w/w) and D- and L-PLA (120 kDa). b (circle) control group. Particles were suspended in isotonic glucose solution and injected subcutaneously in male Wistar rats (n ) 5). Testosterone levels were determined by drawing blood from the rat tail artery at specific time intervals. Testosterone blood concentrations were determined by chemiluminescence, using an automated competitive immunoassay with acridinium ester-labeled testosterone.
agent. Stereocomplex microparticles were obtained in high yields (>90%) by spontaneous precipitation from an acetonitrile solution of D-PLA, L-PLA, and leuprolide, which was stirred at 60 °C for 3 days. The DSC scans of the precipitated microparticles showed a transition state at 230 °C, characteristic of the D-PLA/L-PLA homostereocomplex,13 and an additional smaller endotherm at 209 °C (Figure 1). The amount of free leuprolide remaining in the complexation solution after isolating the precipitate remained low (less than 2%). Recovery of leuprolide from the various PLA stereocomplexes was performed by suspending the obtained precipitates in acetonitrile, containing Span 80 (5% w/w) and stirred overnight at 60 °C. Values close to 100% recovery of the peptide were detected by HPLC, which indicate that the complexation can be reversed by surfactants that disrupt the interaction between the peptide and the polymers. A microparticulate system was also achieved by sprayfreezing a suspension of a concentrated aqueous leuprolide solution in dichloromethane containing D- and L-PLA. DSC analysis confirmed the formation of the PLA homostereocomplex. Release Studies. The effects on the release profile of leuprolide by adding L-PLA to the complexation reaction of D-PLA and leuprolide was studied in various different formulations. Stereocomplex microparticles containing 5% w/w leuprolide, released 40% of the leuprolide content in 30 days at a release rate of 0.34 µg/day/mg complex, between day 6 and 29. That was very similar to those found for heterostereocomplexes of D-PLA and leuprolide.11 Complexes with different leuprolide content (2 or 10% w/w) showed a similar leuprolide release of 40% after a period of 30 days, in a similar release pattern (Figure 2). Adding PEG400 (5% v/v) to the complexation reaction of leuprolide and D- and L-PLA, the release rate of leuprolide from the obtained particles was twice as much compared to
the release from complexes without added PEG400. About 80% of the leuprolide content was released from the complex over a period of 30 days (at 0.74 µg/day/mg complex between day 6 and 30). Addition of PEG400 did not induce an appreciable burst effect in the release of leuprolide during the first 24 h (Figure 2). Changing the ratio of L-PLA compared to D-PLA generally affected the release profile of leuprolide, losing its first-order pattern. Adding L-PLA at a 3:2 ratio to D-PLA in a reaction with leuprolide (4% w/w), a first-order release pattern from the obtained microparticles was preserved. The observed release was slower than from complex particles containing D- and L-PLA in 1:1 ratio, as 35% of the leuprolide content was released over a period of 40 days (at 0.13 µg/day/mg complex, between day 7 and 35; Figure 3). Different timing of the addition of L-PLA to the reaction mixture of D-PLA and leuprolide did not affect the pattern of the leuprolide release. When L-PLA was added to a reaction of low molecular weight D-PLA (3.5-10 kDa) and leuprolide in acetonitrile, complexes were obtained from which leuprolide was released more slowly for a longer period of time. Of the total leuprolide content, 40% was released from the stereocomplex over a period of three months (at 0.23 µg/day/mg complex, between day 10 and 60; Figure 4). Particles obtained from the complexation of high and low molecular weight PLA and leuprolide by freeze-spraying, released 40% and 30% of their leuprolide content over a period of two weeks (at 0.37 µg/day and 0.31 µg/day/mg complex, between day 6 and 21), respectively (Figure 5). The release patterns were similar to stereocomplex microparticles obtained by spontaneous precipitation from an acetonitrile solution at 60 °C. In Vivo Evaluation of Leuprolide-PLA Stereocomplex. In vivo release of leuprolide was studied from two different
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Figure 7. Release rates of leuprolide. Summary of release rates from D-PLA/L-PLA stereocomplexes, described in Figures 2-5. Black bars: high molecular weight PLA. White bars: low molecular weight PLA.
formulations by monitoring testosterone blood levels. Leuprolide, a potent LHRH agonist, initially causes a boost in testosterone synthesis. Via negative feedback control, the testosterone systhesis is blocked. Low testosterone blood levels were recorded for a period of at least five weeks while the testosterone concentration had not yet returned to the control level. The results of the in vivo release of leuprolide from the PLA stereocomplexes were comparable to those published by Ogawa et al.6 (Figure 6). Summary The formation, characterization and controlled release of heterostereocomplexes of D-PLA and leuprolide have recently been reported. In this work, the effects of adding L-PLA to the stereocomplex reaction of D-PLA and leuprolide on the release pattern of leuprolide are reported. For most formulations, a small burst release was observed during the first 24 h, and an almost linear release profile of leuprolide was obtained in the release period between day 6 and day 25.
Slager and Domb
This pattern was lost when L-PLA was added to the complexation reaction in ratios (L-PLA:D-PLA) other than 1:1 or 3:2. Leuprolide release over a period of three months from complexes of low molecular weight D-PLA (
