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PHARMACOKINETICS AND PHARMACODYNAMICS |
From the Departments of Pharmacokinetics and Drug Metabolism (Dr. Wong, Dr. Lau, Dr. Baughman), Clinical Research (Dr. Menchaca), and Biostatistics (Dr. Fotheringham), Amgen, Inc., Thousand Oaks, California, and Clinical Research, Praecis Pharmaceuticals, Inc., Waltham, Massachusetts (Dr. Garnick).
Address for reprints: Shekman L. Wong, PhD, Department of Pharmacokinetics and Drug Metabolism, MS-1-1-A, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320.
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: Abarelix • pharmacokinetics • pharmacodynamics • GnRH • depot formulation
)-isopropyllysyl-L-prolyl-D-alanyl-amide) with high antagonistic activity against naturally occurring GnRH.4-6 As a GnRH antagonist, abarelix binds directly to and provides immediate and competitive blocking of the GnRH receptors on pituitary gonadotropes without causing release of gonadotropins.4-8 A previous study has demonstrated that abarelix, given as a 50-µg/kg/day subcutaneous infusion over 3 months, is sufficient enough to produce clinically significant effects, based on prostate gland volume reduction and the suppression of gonadotropins, without changes in pharmacokinetics.9 Therefore, the ideal regimen for abarelix would be as a subchronic to chronic therapeutic medication formulated as a depot injection. This study compares the pharmacokinetics of abarelix given as an intramuscular injectable solution or as a depot formulation (a final formulation used in phase III clinical trials) in elderly healthy male volunteers and evaluates the pharmacodynamics of abarelix given as a depot formulation. |
MATERIAL AND METHODS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Subjects
Mean age (± SD) of the subjects was 62 ± 9 years (range: 52-75 years inclusive), and mean body weight was 81 ± 12 kg (range: 61-111 kg). The study protocol was approved by the Ethics Committee of the study site. All subjects gave their written informed consent before participation in the study. Subjects were judged to be in good health based on the results of medical history, physical examination, clinical laboratory evaluations, and electrocardiogram (ECG) obtained within 14 days prior to the initial study drug administration. Special inclusion criteria for subjects included serum testosterone levels above 220 ng/dL and prostate-specific antigen levels within normal limits for the corresponding age. Subjects were excluded if they had a history of or currently have secondary cancer or had a history of known allergy to GnRH agonists or antagonists. Subjects were excluded if they had unstable medical conditions such as poorly controlled diabetes, cardiovascular disease, and/or respiratory disease. Subjects were also excluded if they were likely to receive corticosteroids or other agents known to modify serum androgen levels, had received any such drug in the past 90 days, or were currently receiving or had received finasteride or other 5 alpha-reductase inhibitors in the past 30 days.
Clinical Safety
The tolerance and safety assessments involved evaluation of vital signs, ECG, hematology and routine clinical laboratory examinations, and adverse events. These assessments took place prior to and at scheduled times after dosing.
Sample Collection
Venous blood samples (10 mL) for drug assay were collected prior to dosing (0 h); at 0.25, 0.5, 1, 2, 4, 6, 10, 15, and 24 hours; and at 1.25, 1.5, 2, 3, 4, 7, 10, 14, and 21 days after the 1.5-µg/kg administration of intramuscular injectable solution (period 1). During period 1, subjects remained at the study center through collection of the 96-hour sample on day 5. Serial sampling of blood during period 2 of the study was performed at 1, 2, 4, 6, 10, 15, and 24 hours and at 1.25, 1.5, 2, 3, 4, 7, 10, 14, 21, 28, 42, and 56 days after the 100-mg intramuscular abarelix depot dosing. During period 2, subjects were discharged from the study center after the collection of the 36-hour sample and returned to the center for blood sampling at scheduled times.
During period 1, urine was collected from days 1 to 8 (every 24 h) after dosing. For period 2, 24-hour urine collection was performed on days 1, 14, and 28 after dosing. The serum and urine samples were stored at approximately -20°C until analysis.
Assay for Abarelix in Serum
Serum concentrations of abarelix were determined via LC-MS/MS (PE Sciex, API III). In brief, 200-µL aliquots of human serum samples spiked with 5.00 ng of the internal standard PPI-258 (an analog of abarelix synthesized by Praecis Pharmaceuticals, Inc.) were treated with 0.8 mL of 0.2% acetic acid in acetonitrile to precipitate the protein. The mixtures were vortexed for 5 minutes and centrifuged at about 3000 rpm at approximately 4°C for about 10 minutes. The supernate was transferred and dried, reconstituted with 100 µL methanol/water (1:4 v/v), and injected onto an LC-MS/MS instrument using Turbo Ionspray. Chromatography was performed using a Zorbax RX-C8 (2.1 x 150 mm) column. The mobile phase was composed of 70:30 (v/v) of 0.1% acetic acid in methanol and 0.1% of acetic acid in water, with a flow rate of 0.2 mL/min. The linear range of the method was 0.200 to 100 ng/mL for abarelix. The limit of quantitation (LOQ) was 0.200 ng/mL, defined as the lowest quantifiable concentration of the standard curve. Overall precision for the calibration standards and quality control samples, as measured by %RSD, was within 8.96%; the overall accuracy, as measured by %REC for these calibration standards and quality control samples, ranged from 92.3% to 109%. The precision for the dilution integrity quality control samples was 1.30%, and the overall accuracy was 114%.
For urine samples, the analyses of abarelix were performed with the LC-MS/MS (PE Sciex, API III) method similar to the serum assay described above without extraction. In brief, 1.0 mL of water, 1.0 mL of 1 M acetic acid, and 50.0 µL of a 1.00-µg/mL internal standard solution were added to 500 µL human urine. The samples were then loaded onto Isolute C-18 columns and the analytes eluted with methanol. The eluant was evaporated and then reconstituted with 100 µLof1:4methanol/water. Then, 20 µL was injected onto the LC-MS/MS system. The linear range of the method was 1.0 to 200 ng/mL for abarelix. The LOQ was 1.0 ng/mL. Overall precision for the calibration standards and quality control samples, as measured by %RSD, was within 8.20%, and the overall accuracy, as measured by %REC for these calibration standards and quality control samples, ranged from 103% to 106%.
Hormonal Assay
Serum concentrations of total testosterone (T), dihydrotestosterone (DHT), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were determined using clinical diagnostic methods. The total T in subject sera was measured using a diagnostic method approved by the Food and Drug Administration (test kits from Diagnostic Products, Inc., Los Angeles). The LOQ of the method was 8 ng/dL, and the reportable range of the method was from 8 to 1600 ng/dL. DHT levels in subject sera were measured using commercially available test kits (Diagnostic Systems Laboratories, Inc., Webster, TX). The LOQ of the method was 25 pg/mL, and the reportable range of the method was from 25 to 2500 pg/mL. Levels of FSH and LH in subject sera were measured by an automated immunoassay performed on the AxSYM system manufactured by Abbott Diagnostics. The LOQ of the FSH method was 0.6 mIU/mL, and the reportable range of the method was from 0.6 to 150 mIU/mL. The LOQ of the LH method was 0.5 mIU/mL, and the reportable range of the method was from 0.5 to 250 mIU/mL. All intra-assay and interassay variability were within the specification of the manufacturers.
Detection of Human Antibodies to Abarelix
The testing for potential anti-abarelix antibodies in subject sera was measured using an enzyme-linked immunosorbent assay (ELISA). In brief, abarelix was immobilized on plastic 96-well microplates. The peptide-coated microplates were subsequently coated with gelatin to cover any remaining unoccupied peptide-binding sites. Serum specimens for study subjects were then incubated in the peptide/gelatin-coated plastic wells. If present in the specimen, antibodies would bind to the immobilized abarelix during this incubation period. After washing away nonspecifically bound immunoglobulins and other proteins in the specimen, an enzymatic detection system composed of an anti-human IgG/M conjugated to alkaline phosphatase and p-nitrophenyl phosphate was used to detect what would be the presence of antibodies bound to the immobilized abarelix to produce a yellow water-soluble product absorbing light at 405 nm.
Noncompartmental Pharmacokinetic Analysis
Pharmacokinetic parameters of abarelix, including Cmax (maximum serum concentration observed), tmax (time to Cmax), the area under the serum concentration-time curve (AUC), the apparent total serum clearance (CL/F), the elimination rate constant (
z), half-life (t1/2), and the apparent volume of distribution during the terminal elimination phase (Vz/F) were estimated for each subject using standard noncompartmental methods. The area under the first moment curve (AUMC) was calculated using the trapezoidal rule. The mean residence time (MRT) of abarelix after the administration of injectable solution or the depot formulation was calculated as AUMC/AUC. The relative bioavailability (Fr) was calculated as the ratio of the dose-normalized AUC0-
after depot administration to the dose-normalized AUC0- after injectable solution administration.
During period 1, the total amount of abarelix excreted unchanged in urine (Ae) was estimated as 
Xu0-t from time zero to time t. The corresponding renal clearance (CLr) was calculated as Ae/AUC0-t. The percentage of abarelix excreted through the urinary route is fe, defined as the ratio of 100%•Ae to the dose administered. During period 2, since urine was only collected for 36 hours during the first interval after the depot injection, and for two discrete intervals at a later time, the corresponding renal clearance (CLr) was calculated as Xu0-36/AUC0-36 and Xut1-t2/AUCt1-t2, respectively. Since CLr/CL is fe, total body clearance of abarelix can be estimated as CLr•fe; thus, bioavailability (F) of the formulations from this study can be estimated from the corresponding apparent clearance (CL/F).
All of the pharmacokinetic calculations were performed using WinNonlin (version 3.3, Pharsight Corporation, Mountain View, CA).
Pharmacodynamic Analysis
Pharmacokinetic/pharmacodynamic (PK/PD) relationships were investigated for the data obtained from the depot administration. The estimates of percentage inhibition of T, DHT, FSH, and LH biosynthesis were computed according to the following equation:
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where %I = percent inhibition, Bt = concentration of the biomarker measurement at time t postdosing, and B0 = baseline level of the biomarker measurement at time zero on day 1 of the corresponding period. When Bt was below the detection limit, the percentage inhibition was reported as 100%, and when Bt was greater than B0, the percentage inhibition was reported as zero.
Pharmacodynamic Modeling for Abarelix Depot Injection
Pharmacokinetic and pharmacodynamic concentration analyses between each biomarker (T, DHT, FSH, LH) versus abarelix were explored using SAAM II (version 1.1.2, SAAM Institute, University of Washington, Seattle, WA). For each subject, the measure (endogenous biomarker such as testosterone, DHT, FSH, and LH) of response to abarelix was modeled using an indirect-response model.10 The model uses an indirect mechanism in which the production rate of the response variables (testosterone, DHT, FSH, or LH) was inhibited by the serum levels of abarelix. In general, the endogenous hormones were assumed to be at steady state before abarelix administration, and their levels can be described by the following differential equations:
 | (1) |
In equation (1), Rsyn, [B], and (B) represent the endogenous production rate, the amount of the biomarker in the body, and the serum concentration of the biomarker, respectively. CLout defines the clearance for the biomarker. When abarelix was injected, it was assumed that abarelix inhibited the production of the biomarkers without changing the clearance of the biomarker, and therefore the equation can be modified to
 | (2) |
In equation (2), C is the abarelix serum concentration at the time of the inhibition measurement, IC50 is the abarelix serum concentration at which 50% of the maximum inhibition is observed, and 
defines the slope and sigmoidicity of the effect-concentration curve.
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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One subject received 10 µg/kg abarelix injectable solution instead of 15 µg/kg. Another subject did not receive the period 2 study medication. According to the statistical plan of the protocol, these 2 subjects were to be excluded from the analyses.
Pharmacokinetics of Abarelix
The mean ± SD pharmacokinetic parameters of abarelix are listed in Table I. The mean total dose of the abarelix injectable solution (period 1 portion) administered was 1.24 mg. For most study subjects, abarelix peaked 1 hour after abarelix injectable solution administration (Figure 1A). For subjects who received the abarelix depot injection, individual peak concentrations occurred between 1 hour and 10 days during the period 2 portion (Figure 1B). Mean Cmax values for the abarelix injectable solution and the abarelix depot dosing were 57.8 and 43.4 ng/mL, respectively. The mean observed elimination t1/2 values of abarelix following the abarelix injectable solution administration was 0.22 days (5.3 h), while the t1/2 values of abarelix following the abarelix depot administration was 13.2 days. The mean observed CL/F values of abarelix following the injectable solution and the depot administrations were 105 and 208 L/day, respectively. The corresponding mean observed weight-normalized CL/F values of abarelix were 1.3 and 2.5 L/day/kg, respectively. The mean observed Vz/F values of abarelix following the injectable solution administration and depot administration were 34 and 4040 L, respectively. The corresponding mean observed weight-normalized Vz/F values of abarelix were 0.42 and 49.1 L/kg, respectively. The mean observed mean residence time (MRT) values of abarelix following administration of the injectable solution and depot were 0.22 and 17.8 days, respectively. The mean relative bioavailability of the depot formulation compared to the injectable solution was 0.52. The mean renal clearance (CLr) of abarelix was approximately 13.3 L/day after injectable solution administration, with the total abarelix excreted unchanged in urine representing approximately 12.8% of the total dose. The mean renal clearance (CLr) of abarelix was approximately 14.4 L/day after depot administration; the total abarelix excreted unchanged in urine could not be estimated due to incomplete urine collection.
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Pharmacodynamic Effects of Abarelix
The pharmacological effects of abarelix on the inhibition of biomarkers were immediate. Within 1 hour of administration of the 15-µg/kg injectable solution during period 1, T, DHT, FSH, and LH concentrations in serum started to decrease, with the peak inhibition occurring at 6 to 15 hours after dosing. The maximum mean inhibition by abarelix on T, DHT, LH, and FSH was 76.5%, 65.2%, 76.5%, and 33.6%, respectively. However, depression of the concentrations of T, DHT, FSH, and LH was not prolonged, and the concentrations began to recover to normal levels within several days (Figure 2).
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Likewise, T, DHT, FSH, and LH concentrations in serum also began to decrease within several hours after administration of the abarelix depot during period 2. However, the peak inhibition of these biomarkers occurred 2 to 14 days after dosing (Figure 2), and the duration of inhibition was longer. The maximum mean inhibition by abarelix on T, DHT, FSH, and LH was 93.6%, 88.5%, 71.2%, and 94.6%, respectively.
Pharmacodynamic Modeling for Abarelix Depot Formulation
The parameters defining the pharmacodynamic models were estimated for each subject individually as described in equation (2), assuming the production rates of the response variables (T, DHT, FSH, or LH) were inhibited by the serum levels of abarelix. Figure 3 shows the observed and model-fitted curves of all biomarkers, including T, DHT, FSH, and LH. Table II shows the mean (± SD) model-fitted IC50, the endogenous steady-state clearance (CLout), and production rate (Rsyn), respectively, for each biomarker.
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the major concerns related to depot formulation is the safety issue consequent to formulation-related dose dumping-effects. Based on the results of this study, the first 24-hour AUC of the depot formulation represented approximately only 4.6% of the overall AUC, suggesting that the initial release that occurred shortly after depot injection did not contribute to a major portion of the entire exposure. The mean Cmax values for the abarelix injectable solution and the abarelix depot administration at 57.8 and 43.4 ng/mL, respectively, were comparable. The results suggest that the initial burst of abarelix depot formulation is not likely to be higher than levels achieved from 1.24 mg (or the equivalent of 15 µg/kg).
Similar to another newly developed GnRH antagonist,11 abarelix had a relatively short elimination t1/2 value (0.22 days or 5.3 h) following abarelix injectable solution administration. However, the t1/2 value of abarelix following abarelix depot administration was 13.2 days, representing the absorption t1/2 value of the depot formulation. These results show that the depot formulation successfully provided a slow release of the active drug from the injection site, thus prolonging the duration of the pharmacological effects. The mean observed MRT values of abarelix following administration of injectable solution and depot were 0.22 and 17.8 days, respectively, confirming that the abarelix depot injection was absorption rate limited, with an abarelix MRT in the depot injection area of about 17.5 days (MRTIM depot - MRTIM injectable). Only 12.8% of administered abarelix was excreted unchanged in urine, which suggests that renal excretion is not a major elimination pathway for the drug. Based on the results from this study, it is estimated that the total body clearance (CLr/fe; 13.3 L/day by 0.128; data from Table I) for abarelix would be approximately 104 L/day, and the absolute bioavailability for the abarelix injectable solution and depot formulation would be 99% and 50%, respectively.
The results in this study demonstrate that abarelix is a potent antagonist for GnRH (Table II), with IC50 of 2.1 ng/mL on testosterone production. It was demonstrated in this study that abarelix binds directly to and provides immediate (within hours) competitive blocking of the GnRH receptors on pituitary gonadotropes without causing release of gonadotropins, following injectable solution administration. The suppression of T, DHT, FSH, and LH production was not prolonged, and the concentrations of these gonadotropins began to recover to normal levels within several days. Based on the pharmacodynamic results (Figure 2), the novel abarelix depot formulation used in this study significantly improved the duration of abarelix delivery and pharmacological activities compared to the injectable formulation, without causing any safety issues.
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FOOTNOTES |
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Dr. Menchaca died in the terrorist attack on the U.S. Pentagon, September 11, 2001. Presented in part at the 2001 Annual American Association of Pharmaceutical Scientist Meeting, Denver, Colorado, October 21-25, 2001.
Submitted for publication November 23, 2002; Revised version accepted February 8, 2004.
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REFERENCES |
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8. Hall JE, Whitcomb RW, River JE, et al: Differential regulation of luteinizing hormone, follicle stimulating hormone, and the free alpha-subunit secretion from the gonadotrope by gonadotropin-releasing hormone (GnRH): evidence from the use of two GnRH antagonists. J Clin Endcrinol Metab 1992;75: 1220-1225.[Abstract]
9. Wong SL, Lau DT-W, Baughman SA, Menchaca D, Garnick MB: Pharmacokinetics and pharmacodynamics of abarelix, a-gonadotropin releasing hormone (GnRH) antagonist after subcutaneous, continuous infusion in patients with prostate cancer. Clin Pharmacol Ther 2003;73: 304-311.[Medline] [Order article via Infotrieve]
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