Sustained suppression of pituitary‐gonadal axis with an injectable, in situ forming implant of leuprolide acetate

Sustained Suppression of Pituitary-Gonadal Axis with an Injectable, In Situ Forming Implant of Leuprolide Acetate HARISH B. RAVIVARAPU, KATIE L. MOYER, RICHARD L. DUNN Atrix Laboratories, Inc., 2579 Midpoint Drive, Fort Collins, Colorado 80525

Received 7 May 1999; revised 18 January 2000; acccepted 20 January 2000

The objective of these studies was to develop a leuprolide acetate depot based on an in situ forming drug delivery system (Atrigel®) to suppress the pituitarygonadal axis and in turn the serum testosterone to chemical castration levels for a period of at least 3 months. Formulations with biodegradable lactide/glycolide copolymers that varied in molecular weight, lactide/glycolide ratio, and hydrophilicity were evaluated in rats for their efficacy by measuring serum testosterone levels. The effect of polymer irradiation was also investigated. Molecular weight of the polymers was characterized by gel-permeation chromatography, and retrieved implants at the termination of animal studies were assayed for residual drug content by high-performance liquid chromatography. These initial rat studies showed that a formulation containing a 75/25 lactide/glycolide copolymer dissolved in N-methyl-2-pyrrolidone with 3% w/w leuprolide acetate suppressed serum testosterone for a period of 3 months or longer. This formulation with its advantages of biodegradability, biocompatibility, ease of injection, and no need for removal after use should be beneficial in treating patients with hormonal-dependent prostate and mammary cancers, endometriosis, and precocious puberty. In addition, this formulation with its simple manufacturing process is expected to provide an economic benefit to the user compared with products currently available on the market. © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association

ABSTRACT:

J Pharm Sci 89: 732–741, 2000

Keywords: leuprolide acetate, Atrigel, polyester polymers, rat, serum testosterone, chemical castration

INTRODUCTION Leuprolide acetate (leuprorelin, D-Leu6- (desGly10-NH2)-LH-RH ethylamide) is a potent luteinizing hormone-releasing hormone (LHRH)agonist analog that is useful in the palliative treatment of hormonal-related prostate and mammary cancers, endometriosis, and precocious puberty.1–5 With continued use leuprolide acetate causes pituitary desensitization and down-regulation to affect the pituitary gonadal axis, leading to suppressed circulating levels of luteinizing and Coprrespondence to: H. B. Ravivarapu. (E-mail: hraviv@ atrixlab.com) Journal of Pharmaceutical Sciences, Vol. 89, 732–741 (2000) © 2000 Wiley-Liss, Inc. and the American Pharmaceutical Association

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sex hormones. In patients with advanced prostate cancer, achieving circulating testosterone levels of less than or equal to 0.5 ng/mL (“chemical castration levels”) is a desired pharmacologic indicator of therapeutic action6. Originally, leuprolide acetate was launched in the united States as a daily subcutaneous (SC) injection of the analog solution. The inconvenience of chronic repetitive injections was later eliminated by the development of a 1-month sustained-release depot product based on poly(DL-lactide-co-glycolide) microspheres (Lupron® Depot).6,7 At present, 1-, 3-, and 4-month formulations are widely available. These formulations, in addition to improving patient convenience and compliance, reduced the needed drug dose by one fourth to one eighth com-

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pared with the conventional SC dose.8 This was possible because of the constant blockade of LHRH receptors with the sustained levels of the drug present at the target. The objective of these studies was to develop a 3-month depot system of leuprolide acetate based on an in situ forming drug delivery system (Atrigel®)9,10 that would have the same benefits as the Lupron® Depot microsphere product, namely, biodegradability, biocompatibility, and ease of injection with a lack of need for removal of the delivery system. However, preparing microspheres usually is a complex and expensive multistep process. The simpler oil/water emulsionsolvent evaporation may not be useful in efficient encapsulation of highly water-soluble drugs such as the studied peptide and is modified to water/ oil/water multiple emulsion or oil/oil emulsion approaches.11 In comparison, a simple mix and fill preparation of the Atrigel® formulation would be easier to manufacture and therefore more economical to the patient. The Atrigel® system combines the benefits of microsphere and implant delivery systems. In this system, a water-insoluble and biodegradable polymer is dissolved in a biocompatible water-soluble solvent. When this system is injected into the body as a liquid, the organic solvent dissipates into the surrounding tissue as the water permeates in. This leads to precipitation or coagulation of polymer to form an in situ implant. Active drug agents can be added to Atrigel® system to provide for sustained release of the compound. Once the drug containing implant is formed in situ, the drug is encapsulated within the polymer matrix. On complete dissipation of the solvent, drug release is controlled by the polymer and drug properties as in the case of any other biodegradable implant. Polymer properties such as molecular weight, hydrophobicity, and comonomer ratio can be modified to obtain the desired drug release profile. Other factors that are not evaluated in this study but have major effects on the drug release include polymer concentration, solvent used, drug load, and state of the drug-solution or dispersion. In this article we describe our efforts in developing an Atrigel®-leuprolide acetate product that was shown to effectively suppress serum testosterone levels of rats over a period of 3 months after dosage. Because serum levels of leuprolide are not always predictable of efficacy, these were not measured in the described formulation screening studies. Instead, testosterone levels in-

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dicating true efficacy were determined for a number of possible candidate formulations.1 The effect of polymer irradiation on efficacy of the candidate formulations was also evaluated.

MATERIALS AND METHODS Materials Leuprolide acetate was purchased from Bachem California, Inc. (Torrance, CA). N-methyl-2pyrrolidone (NMP, Pharmasolve™) was obtained from International Specialty Products (Wayne, NJ). Poly (DL-lactide-co-glycolide) with a comonomer ratio of 50:50 (50/50 PLG) and an inherent viscosity (IV) of 0.38 (Resomer RG503), carboxyl end-group 50/50 PLG (50/50 PLGH) (Resomer RG504H) with an IV of 0.47, and carboxyl end-group poly(D,L-lactide) with an IV of 0.20 (PLAH, R202H) polymers were supplied by Boehringer Ingelheim (Wallingford, CT). 50/50 PLGH (IV, 0.75) and 75/25 PLG (IV, 0.20 and 0.54) were purchased from Birmingham Polymers, Inc. (Birmingham, AL). All the IV values reported were provided by the manufacturer and are in dL/g units. All other reagents used were of highperformance liquid chromatography (HPLC) grade. Methods

Preparation of Formulations Appropriate amounts of individual polymer and NMP were weighed into glass vials. After initial mixing of the contents, vials were placed on a continuous shaker (Labline® orbit shaker, Melrose Park, IL) overnight at room temperature to completely dissolve the polymer. A portion of each of these preparations in glass scintillation vials was sent to a contract irradiation facility (Isomedix, Morton Grove, IL) for ␥-irradiation at a dose of 20 to 25 kGy. Only polymer solutions and not the drug were irradiated. Because the scission effect of ␥-irradiation on polymer chains is welldocumented, we did not characterize the irradiated system for any degradants. To prevent degradation of the polymer by the drug on long-term storage, the appropriate amount of leuprolide acetate based on the intended drug load was added to the polymer/solvent preparations 30 to 45 minutes before animal injections. Formulations were kept on a continuous shaker with occasional mixing. All formulations were prepared with a 3% JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

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w/w leuprolide acetate load and at the time of injection, formulations were all clear solutions. These were filled into 1-mL polypropylene syringes (Becton Dickinson, B. D., Franklin Lines, NJ) and the required weight of each formulation was injected into animals. Compositions of evaluated individual formulations are tabulated in Table 1 along with other related information.

Polymer Molecular Weight Known weights of polymer solutions without drug, both irradiated and nonirradiated, were dissolved in chloroform to yield a polymer concentration of approximately 0.5% w/v. Filtered samples were analyzed by gel permeation chromatography (GPC) to determine the weight average polymer molecular weight. Narrow molecular weight polystyrenes in the range of 580 to 370,000 daltons (Polymer Laboratories, Amherst, MA) were used as standards. The chromatography conditions were as follows: Polymer Laboratories MIXED-D (5 ␮m, 30 cm × 7.5 mm) column maintained at 40°C, Hewlett Packard (Santa Clara, CA) 1050 series isocratic pump, autosampler, 1047A refractive index detector, and 50 ␮L injection volume. Chloroform with 1% triethylamine at a flow rate of 1 mL/min was used as the mobile phase. Polymer Laboratories CALIBER software was used for GPC calculations.

In Vivo Evaluation: Rat studies. Male Sprague-Dawley (Harlan Sprague Dawley, Inc., Chicago, IL) rats with a baseline weight range of 275.7 to 395.2 g were used in this study. Animals were housed in polycarbonate cages with a 12-h on/off lighting cycle. Laboratory rodent chow and tap water were provided ad li-

bitum. The animals were maintained according to AAALAC requirements and were in accordance with the Guide for the Care and Use of Laboratory Animals (DHEW Pub. No. [NIH] 78-23). Randomized rats were identified by ear notch and cage cards and were acclimated for at least a week before the studies. Each treatment group had five rats. On the study date, rats were weighed, anesthetized with isoflurane, and each given a single SC injection of specific formulation in the dorsothoracic region using 20- to 22-gauge needles. On the basis of the literature reports, the targeted dose for rats was set at 100 ␮g/kg/day.12 Syringes were weighed before and after dosage administration to determine the formulation amount received by animals. Dose information for each group of rats is provided in Table 1. Four days before injection and on days 3, 7, 15, 21, 35, 49, 63, 70, 80, and 92 after administration of the test articles, approximately 0.7 mL of blood was collected into a clot tube from the lateral tail vein by serial bleeding from each animal. On day 105, rats in all groups except for 1, 2, 3, and 2R were anesthetized, bled by cardiac puncture, and killed with carbon dioxide. The remaining rats were terminated on day 150, after additional bleeding on 121 and 136 days. Any remaining polymer/drug implants at the injection site were retrieved for the analysis of residual drug content. All the rats were periodically observed for overt toxicity and weight changes. At termination, the injection site was evaluated macroscopically for any tissue reactions. Serum testosterone assay. Serum was separated from the blood by centrifugation at 3,500 rpm for 10 min and frozen at −20°C until analysis by solid-phase radioimmunoassay (RIA). Standard

Table 1. Atrigel-Leuprolide Acetate Formulations Injected into Rats (n ⳱ 5) (Irradiated Versions (Suffixed with R in Text) of Formulations 1-6 were also Evaluated) Average Dose (␮g/kg/day) No.

Composition

1 2 3 4

PLAH (IV 0.2) : NMP 45:55 75/25 PLG (IV 0.2) : NMP 45:55 75/25 PLG (IV 0.54) : NMP 34:66 50/50 PLGH (IV 0.47) : 50/50 PLGH (IV 0.75) : NMP 15:15:70 50/50 PLGH (IV 0.47) : 50/50 PLG (IV 0.38) : NMP 17:17:66 50/50 PLGH (IV 0.75) : NMP 28:72

5 6

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Drug Load (% w/w)

Nonradiated

Irradiated

3 3 3 3

108.6 130.4 129.4 115.6

111.1 120.5 114.9 104.4

3

114.3

99.4

3

113.7

99.8

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commercial RIA kits were used and the mean ± S. D. of testosterone levels (ng/mL) was reported. Samples, standards, and controls were analyzed in duplicate. Predose administration serum testosterone values were averaged and used as the baseline value. Retrieved implant analysis by HPLC. The excised implants from rats were dissolved in the sample diluent solution, which was a 1:1 mixture of dimethyl sulfoxide and methanol with 1% w/v polyethylenimine, by placing the contents on a continuous shaker for 24 h at room temperature. Samples were then vortex-mixed for 5 s and filtered through 0.45 ␮m PTFE syringe filters into HPLC vials. Leuprolide acetate was separated on a Vydac (Hesperia, CA) Protein and Peptide C-18 column (4.6 × 250 mm, 5 ␮m particle size) using a modified literature method. 13 The chromatographic equipment used was a Shimadzu LC-10A that consisted of autosampler, pump, variable UV/Vis detector, computer, and data acquisition/ analysis software. The mobile phase was 23:77 acetonitrile and 87 mM ammonium acetate buffer solution that was pH adjusted to 6.5 with 6 M HCl. The flow rate was set at 1 mL/min and the detection wavelength was 280 nm. Sample run time was 30 min, with leuprolide eluting at 15 min. The limit of detection was 1 ␮g/mL, and the leuprolide standard curve was linear over the range of 1 to 1,200 ␮g/mL.

RESULTS AND DISCUSSION The objective of this study was to develop an efficacious leuprolide acetate product on the basis of the in situ forming implant drug delivery system. As a preliminary screening study, nonirradiated and irradiated formulations 1 through 6 (Table 1, irradiated formulations represented with suffix R) were evaluated in rats for their efficacy in suppressing serum testosterone levels to approximately 0.5 ng/mL (human castration) over a period of 3 months after dosage administration. In vivo drug release from the formulations (by analyzing retrieved implants at specific time points) or serum leuprolide levels were not determined in this study. One reason is the large number of animals required to terminate five animals at each time point for all the groups and retrieve implants and collect serum. The additional cost of study animals and leuprolide analysis was not deemed necessary at this initial stage

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of product development. Moreover, it is known that once the pituitary-gonadal axis is suppressed, a minimum amount of leuprolide is needed to maintain the suppression, and no good correlation exists between the serum leuprolide levels and pharmacologic efficacy.14 Thus, even though different drug levels from various formulations could potentially be observed, it would not help in selecting an optimum formulation without efficacy data. A number of polymers that varied in their monomer composition (50/50 PLG to PLA), molecular weight (10.3–121.8 kilo daltons [kd]), and hydrophilicities (acid end group vs nonacid end groups) were used in preparing these formulations. Preliminary animal studies (data not shown) had demonstrated that a formulation of irradiated 50/50 PLGH (Mw 38 kd) at 34% w/w with NMP and 3% leuprolide acetate was efficient in suppressing testosterone levels for a period of approximately 1 month. At the end of the study, only a small amount of polymer with leuprolide was retrieved as implants. It was reasoned that the use of a more hydrophobic or higher molecular weight polymer would retain the drug for a longer time because of its slower degradation rate, while still releasing sufficient drug during the initial period to be efficacious. Therefore, formulations were prepared with more hydrophobic copolymers containing a higher lactide monomer content and different molecular weights (formulations 1–3). It was also hypothesized that the addition of a more hydrophobic or higher molecular weight polymer to the 1-month system would provide the longer release of drug needed for the 90-day product, therefore, formulations with 1:1 combinations of the 50/50 PLGH (38 kd) with a 50/50 PLGH (Mw 121.9 kd) (formulation 4) and with a noncarboxyl end-capped 50/50 PLG (40 kd) (formulation 5) were also prepared. One formulation was evaluated with simply the higher molecular weight 50/ 50 PLGH (121.9 kd) polymer (formulation 6). The leuprolide acetate loading in all formulations was at 3% w/w. All polymer solutions of the formulations, irradiated or nonirradiated, were analyzed by GPC to characterize polymer molecular weight and the results are shown in Table 2. As can be seen, polymers with a wide range of molecular weight were included in the study. In the case of polymer combination formulations 4 and 5, the molecular weight reported is a composite of both the polymers. There was a molecular weight loss in the range of 3.7 to 51.2% when the systems were irJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

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Table 2. Molecular Weight (Weight Average) Characterization of Polymers

Formulation No.

Nonradiated

Irradiated

% Drop in Molecular Weight

1 2 3 4 5 6

11974 17862 76648 82833 45316 121829

10361 17194 50389 46943 33896 59425

13.5 3.7 34.3 43.3 25.2 51.2

Molecular Weight

radiated at an approximate dose of 21 kGy. The loss was more prominent in the case of higher molecular weight polymers. Concentrations of the polymers used in the formulations were based on in vitro injectability experiments. The criterion was to inject the final formulations through a 20 or higher gauge needle with relative ease and accordingly the polymer concentration was adjusted depending on the polymer composition and molecular weight. At the set polymer/solvent composition, all the formulations were easily injected into animals through 20- to 22-gauge and 1-inch length needles. Formulations 1 through 6 and 1R through 6R were clear, homogeneous solutions at the time of injection. The intended dose for the rat studies was 100 ␮g/kg/day of leuprolide acetate or for 90 days, 9 mg/kg. Literature reports suggest this dose to be effective in suppressing rat testosterone levels for at least 3 months and comparable to a dose of 25.6 ␮g/kg/day in dogs and 22.5 mg in humans.12 The actual dose given to rats is shown in Table 1 as an average value, which ranged from 85.4 to 130.4 ␮g/kg/day. Many of the values presented were greater than the intended 100 ␮g/kg/day dose. Because of the large number of animals involved (n = 60), for practical reasons, an average rat weight of 285 g was assumed and an attempt was made to give all the rats a fixed amount of formulation. As a result of this limitation and a wide variation in rat weights, the actual doses given to rats were not always the same as the targeted doses. All the rats gained weight steadily during the study period. Macroscopic evaluations of the injection sites at the termination of the study showed no untoward local reactions. There was no vasodilation, erythema, or edema. Minimal capsule formation was noted in formulation groups 1, 1R, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

and 3R. Implants could only be retrieved from groups 1R and 3R (day 106) and 1 and 3 (day 150). Recovered implants were located SC and most were firm. Figures 1 through 6 show the serum testosterone profiles of rats after administering formulations 1 through 6. Data from formulations that show the comparative effect of irradiation are presented together for direct comparison. Baseline testosterone values were determined as an average of predose values from all the study rats to provide a larger n value (n = 60). During the formation of polymeric implant in vivo, a certain portion of leuprolide acetate dissolved in the polymer solution was expected to be taken away by the dissipating NMP. The factors that can affect this initial release include drug solubility in solvent, solvent properties that determine its rate of outflow, formation of a polymer “skin” around the implant, and properties of the implant itself. Because of this expected initial release and leuprolide acetate being a LHRH agonist, an immediate increase in testosterone levels was expected followed by suppressed levels because of down-regulation of the pituitary. However, the peak levels were not determined in this preliminary screening study because the primary focus was the long-term efficacy of the formulations. Thus, the first data point in these studies was at 3 days, by which time the levels appeared to be already in decline. Figure 1 shows the profile obtained with the more hydrophobic PLAH formulation (1 and 1R). No significant effect of irradiation on the efficacy was noted. With both formulations, testosterone levels declined from as early as the third day of the study; however, they did not approach castration levels until 35 days. Suppression once achieved was maintained until day 105. At the day of termination (day 105 or 150), a significant amount of residual polymer was found in vivo. However, the mean values of residual leuprolide acetate were low: 1.46 (day 150) and 4.63% (day 105) of the initial amount for formulations 1 and 1R, respectively. This would suggest that even though enough polymer was present, the remaining drug and its release at that point was not enough to saturate the receptors. The long delay in approaching castration levels with this formulation is probably related to the slower degradation of the more hydrophobic PLAH polymer and the subsequent lower release of peptide. Formulation 2 in either irradiated or nonirra-

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Figure 1. Rat serum testosterone profile with 45% PLAH (IV 0.2) + 55% NMP with 3% leuprolide acetate formulation (1, 1R).

diated form appeared to be the best of the formulations evaluated (Fig. 2). By 14 days the testosterone levels were less than or close to castration levels, and near castration levels were main-

tained up to day 105 of the study. After 105 days, testosterone levels were elevated. At day 150, no retrievable implants could be detected in vivo. Because the polymer used was of low molecular

Figure 2. Rat serum testosterone profile with 45% 75/25 PLG (IV 0.2) + 55% NMP with 3% leuprolide acetate formulation (2, 2R). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

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weight (Mw 17.8 kd), it appeared to have been degraded some time between 105 and 150 days. Figure 3 shows the testosterone levels with formulations 3 and 3R. Compared with formulation 2, the polymer used in 3 and 3R had a higher molecular weight (Table 2). Suppression to castration was obtained by day 14, which, however, was not maintained. Testosterone levels remained elevated from day 21 to 63 for the irradiated form and day 77 with the nonirradiated form. This difference was expected because the irradiated form with a lower starting molecular weight (Table 2) loses mass faster and starts releasing drug early. Very little residual leuprolide acetate was detected at the termination, and it is not known when most of the drug was released. Testosterone levels obtained with formulations 4, 4R, 5, and 5R are shown in Figures 4 and 5.. These results do not support our hypothesis that addition of slower degrading polymers would help in retaining and releasing drug for more time compared with the 50/50 PLGH (38 kd) polymer formulation alone. The profiles resembled those obtained with the 50/50 PLGH (38 kd) formulation alone (data not shown). The concept of blending microspheres with different polymer molecular weights was shown in the literature to be effective in modifying the drug release.15 However, in contrast to microspheres, the Atrigel® implant

is a single unit and actually provides a solution of the combined polymers. Theoretically, as the 50/ 50 PLGH (38 kd) polymer degrades and produces an acidic environment from the degradants (lactic and glycolic acids), degradation of polymers that are in immediate contact can be catalyzed. This appears to be the case with formulations 4 and 5 and is supported to an extent by the finding that no retrievable implants were found at the termination in vivo. Formulation 5 had a slightly greater total polymer concentration and contained the 50/50 PLG (40 kd) compared with the more hydrophilic 50/50 PLGH (121.9 kd) of formulation 6. Accordingly, the implant from this formulation seems to have lasted longer and was efficient for a longer time. Finally, formulations 6 and 6R that contained 50/50 PLGH (121.9 kd) at 28% w/w showed efficient suppression by day 14 (Fig. 6). However, testosterone levels rose rapidly after day 35 and remained high. This polymer compared with the 50/50 PLGH (38 kd) had a significantly higher molecular weight (Table 2) and was expected to be effective for longer time. Because of the lack of any other studies that can explain this absence of prolonged efficacy, it can only be concluded that the polymer concentration at 28% was not sufficient to hold the drug for a very long time.

Figure 3. Rat serum testosterone profile with 34% 75/25 PLG (IV 0.54) + 66% NMP with 3% leuprolide acetate formulation (3, 3R). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

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Figure 4. Rat serum testosterone profile with 15% 50/50 PLGH (IV 0.47) + 15% 50/50 PLGH (0.75) + 70% NMP with 3% leuprolide acetate formulation (4, 4R).

CONCLUSIONS In summary, these studies have led to a promising in situ implant forming drug delivery formu-

lation, 75/25 PLG (18 kd):NMP 45:55 with 3% w/w leuprolide acetate, that is effective in suppressing rat testosterone levels for a period of at least 3 months. No significant effect of the irradiation of

Figure 5. Rat serum testosterone profile with 17% 50/50 PLGH (IV 0.47) + 17% 50/50 PLG (IV 0.38) + 66% NMP with 3% leuprolide acetate (5, 5R). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 89, NO. 6, JUNE 2000

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Figure 6. Rat serum testosterone profile with 28% 50/50 PLGH (IV 0.75) + 72% NMP with 3% leuprolide acetate formulation (6, 6R).

polymer on formulation efficacy was observed. On the basis of these studies, this formulation appears feasible to be developed into a clinical product.

ACKNOWLEDGMENTS The authors would like to sincerely acknowledge and thank the other Atrix Laboratories, Inc. personnel, Catherine Balliu, Steve Volker, Michele Gehring, Brad Thomson, and Dennis Wilson, for their assistance in the animal studies and the analysis of the test formulations.

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