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Pharmacokinetics of Recombinant Methionyl Human Leptin after Subcutaneous Administration: Variation of Concentration-Dependent Parameters According to Assay

Division of Endocrinology, Diabetes, and Metabolism (J.L.C., C.O., P.R., C.S.M.), Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; and Amgen, Inc. (S.L.W.), Thousand Oaks, California 91320

Address all correspondence and requests for reprints to: Christos S. Mantzoros, M.D., D.Sc., Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, ST 816, Boston, Massachusetts 02215. E-mail: cmantzor{at}bidmc.harvard.edu.

	Abstract

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Context: Recombinant human leptin (r-metHuLeptin) has demonstrated efficacy in improving hormonal and metabolic parameters in leptin-deficient states, but pharmacokinetic parameters after sc administration have not yet been published. In addition, the effect of potential variability across different leptin assays on concentration-dependent pharmacokinetic parameters remains unknown.

Objective: The objective of the study was to characterize pharmacokinetic parameters after sc r-metHuLeptin administration using three commercially available leptin assays (Linco, Diagnostic Systems Laboratories, and Alpco).

Design, Setting, Patients, and Intervention: We analyzed pharmacokinetic profiles in five lean and five obese men after sc administration of physiological (0.01 mg/kg) and pharmacological (0.3 mg/kg) doses of r-metHuLeptin.

Main Outcome Measures: Leptin pharmacokinetic parameters were measured.

Results: Measurement of leptin produced typical pharmacokinetic profiles in all assays with time to maximal concentration and half-life of approximately 3 h. Diagnostic Systems Laboratories consistently measured leptin higher than Linco, with Alpco measuring intermediate between or similar to Linco. There was high correlation among assays (R² ranging from 0.89 to 0.98, all P < 0.01). Concentration-dependent parameters such as maximal concentration, area under the curve, and clearance were significantly different among assays, whereas concentration-independent parameters such as time to maximal concentration and half-life were generally not different.

Conclusions: We report novel data on leptin pharmacokinetic parameters after sc administration, which will be relevant for the future therapeutic use of r-metHuLeptin. Although commercially available assays demonstrated high correlation, they can provide substantially different measures of leptin levels. This demonstrates the importance of standardizing leptin assays for diagnosing patients with relative leptin deficiency, determining appropriate doses of r-metHuLeptin for administration, and monitoring response to therapy.

	Introduction

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LEPTIN PLAYS AN important role in the regulation of appetite, energy homeostasis, and neuroendocrine and immune function. As a therapeutic agent, recombinant human leptin (r-metHuLeptin) has demonstrated beneficial effects in several disease states associated with low leptin levels, including congenital leptin deficiency (1) and lipoatrophy (2), hypothalamic amenorrhea (3, 4), and HIV-associated lipoatrophy (5).

The future clinical use of r-metHuLeptin depends critically on clarification of pharmacokinetic parameters to guide accurate dosing, but pharmacokinetic parameters based on sc administration have not yet been published, nor is it known whether these parameters are affected by potential differences among different leptin assays. We performed a pharmacokinetic study with administration of sc r-metHuLeptin in healthy lean and obese subjects and measured leptin levels using three commercially available assays.

	Subjects and Methods

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Human subjects

Five lean men with body mass index (BMI) less than 25 kg/m² and five obese men with BMI greater than 30 kg/m² were studied during two separate 1-d admissions in the fed state. Subjects were healthy and not on any medications. r-metHuLeptin was administered by sc injection at 0.01 mg/kg during one study (0800 h) and at 0.3 mg/kg during the second study (6.4 ± 1.0 wk later). Serum leptin levels were measured at time 0 before r-metHuLeptin administration and at +30 min and +1, + 2, +3, +4, +5, +6, +8, +10, +12, +18, and +24 h [except Diagnostic Systems Laboratories (Webster, TX) at +24 h]. This study was approved by the Beth Israel Deaconess Medical Center (BIDMC) Institutional Review Board. Written informed consent was obtained from subjects. Clinical quality r-metHuLeptin was supplied by Amgen, Inc. (Thousand Oaks, CA) and administered under an investigator-initiated (C.S.M.) Investigational New Drug application submitted to the Food and Drug Administration.

Measurements

Leptin levels (nanograms per milliliter) were measured in the same samples by immunoassays from Linco Research [St. Louis, MO; now Millipore (Billerica, MA)], Diagnostic Systems Laboratories, and Alpco (Salem, NH). The Linco RIA (catalog no. HL-81K) has a sensitivity 0.5 ng/ml, limit of linearity of 100 ng/ml, reported intraassay and interassay coefficients of variation (CVs) of 8.3 and 6.2% (mean level 4.9 ng/ml) and 3.4 and 3.6% (mean level 25.6 ng/ml), in-house intraassay CV approximately 7% (mean level 22 ng/ml), and interassay CV approximately 18–20% (mean levels 3 and 28 ng/ml). The Alpco RIA (catalog no. 22-LEP-R42) has a sensitivity of 0.1 ng/ml, limit of linearity 64 ng/ml, reported intraassay and interassay CVs less than 5% and less than 7.6%, in-house intraassay CV approximately 7% (mean level 2 ng/ml), and interassay CV approximately 11% (mean level 7 ng/ml). The Diagnostic Systems Laboratories immunoradiometric assay (catalog no. 23100) has a sensitivity 0.1 ng/ml, limit of linearity 100 ng/ml, reported intraassay and interassay CVs 3.7 and 6.6% (mean level 2.8 ng/ml) and 2.6 and 3.7% (mean level 74 ng/ml), in-house intraassay CV approximately 3% (mean levels 9 and 16 ng/ml), and interassay CV approximately 20% (mean level 3 ng/ml). Samples for the same subject were run within the same assay (in duplicate) to decrease interassay variability.

Noncompartmental pharmacokinetic analysis

Pharmacokinetic analyses were performed on baseline-subtracted leptin concentrations, using leptin values obtained immediately before dosing as baseline. After baseline subtraction, peak serum concentration (C_max), T_max (defined as the time that C_max was reached), terminal-phase elimination half-life (t_1/2), and area under the serum concentration vs. time curve from zero to infinity (AUC_0-) were calculated (WinNonlin, version 5.0.1; Pharsight, Mountain View, CA), as previously described (6). The apparent clearance (CL/F) of r-metHuLeptin is the ratio of actual dose administered to AUC_0-, where F (an unknown factor herein) is the bioavailability factor after sc administration and represents the fraction of drug absorbed into the systemic circulation relative to that available after direct systemic administration because routes of administration other than systemic are associated with a fraction lost through factors such as degradation, hepatic metabolism, etc. The infusion rate of exogenous r-metHuLeptin required to raise serum leptin levels can be calculated as R_syn=R_inf = CL*(L) where R_syn and (L) represent the endogenous production rate and serum leptin concentration, respectively (6). Because only sc r-metHuLeptin was administered in the current study, the equation is further modified as R_inf,s.c = (CL/F)*(L).

Statistical analysis

Data are presented as mean ± SD. Nonparametric Wilcoxon signed ranks or rank sum tests were used to compare age and weight between lean and obese subjects and pharmacokinetic parameters measured using the three assays. Simple regression was performed on serum leptin values measured by each assay against those measured by the other assays, with best fit according to linear or exponential equations and expression of goodness-of-fit as R² and testing of the slope for significance.

	Results

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Lean and obese men had similar age (lean: 22.2 ± 2.0 vs. obese: 23.4 ± 3.4, P = 0.92) but significantly different weights (lean: 66.4 ± 6.4 vs. obese: 104.4 ± 10.7 kg, P = 0.008). r-metHuLeptin was generally well tolerated, without systemic side effects in this study, and only one mild injection site reaction after 0.3 mg/kg r-metHuLeptin, which resolved without treatment.

Pharmacokinetic profiles

Figure 1 depicts average leptin pharmacokinetic profiles. Serum leptin levels determined by Diagnostic Systems Laboratories were consistently higher than corresponding Linco values. Alpco measured leptin at levels intermediate between Linco and Diagnostic Systems Laboratories assays (lean men) or similar to Linco (obese men). Leptin returned to levels similar to baseline values by 24 h after 0.01 mg/kg r-metHuLeptin in lean [2.22 ± 0.41 ng/ml (Linco), 11.48 ± 1.58 ng/ml (Alpco)] and obese men [14.10 ± 6.11 ng/ml (Linco), 17.87 ± 4.07 ng/ml (Alpco)] but higher than baseline after 0.3 mg/kg r-metHuLeptin in lean [4.11 ± 1.73 ng/ml (Linco), 13.81 ± 1.84 ng/ml (Alpco)] and obese men [27.60 ± 10.22 ng/ml (Linco), 41.31 ± 0.50 ng/ml (Alpco)].

View larger version (30K): [in this window] [in a new window]   FIG. 1. Pharmacokinetic profiles (mean ± SD) for serum leptin levels measured using Linco (), Diagnostic Systems Laboratories (), and Alpco (•) assays in lean men (n = 5) (A) and obese men (n = 5) (B) after 0.01 and 0.3 mg/kg r-metHuLeptin administration. Arrow depicts r-metHuLeptin administration.

The regression curve for Linco and Diagnostic Systems Laboratories in lean men demonstrated a linear relationship (R² = 0.91) but was best described by an exponential curve (R² = 0.92) for obese men. The regression curves were exponential for Alpco and Diagnostic Systems Laboratories (R² = 0.98 and 0.96 in lean and obese men, respectively) and for Linco vs. Alpco (R² = 89 and 0.90 in lean and obese men, respectively).

Bland-Altman plots of the average of two measurements against the corresponding difference between measurements for each pair of assay methods are presented in Fig. 2 and generally show greater divergence at higher leptin levels when all data were considered. When only baseline leptin values prior to r-metHuLeptin were evaluated, the difference between Alpco and Linco assays was largely independent from average leptin measurements, whereas the difference from Diagnostic Systems Laboratories increased with increasing leptin measurements (ng/ml).

View larger version (20K): [in this window] [in a new window]   FIG. 2. Bland-Altman plots depicting the average of leptin measurements (all data and baseline values alone) by Linco and Diagnostic Systems Laboratories (A), Alpco and Diagnostic Systems Laboratories (B), and Linco and Alpco (C) assays against the corresponding difference in leptin measurements between the two assays in lean (open circles) and obese (closed circles) men after 0.01 and 0.3 mg/kg dose r-metHuLeptin administration. Data are actual (not baseline corrected) leptin values for all time points at which leptin was measured. Solid horizontal line indicates the mean difference, and dashed horizontal line indicates the mean difference + 1.96 x the SD.

Pharmacokinetic parameters (Table 1) that depend on absolute serum leptin levels, such as L₀, C_max, AUC_0-, and CL/F were quite different between assays. In contrast, concentration-independent parameters such as T_max and t_1/2 were generally not significantly different because these are determined primarily by leptin biology. We also calculated the dose of exogenously administered r-metHuLeptin required to raise serum leptin levels by 1 ng/ml based on 0.01 mg/kg and 0.3 mg/kg doses (Table 1). Within a specific assay, the calculated dose was generally similar, indicating dose-independent linearity of pharmacokinetic behavior across broad ranges of leptin levels and consistency within an assay but varied by severalfold, depending on the assay.

Finally, we evaluated differences between assays using an independent source of recombinant human leptin (National Hormone and Peptide Program, Torrance, CA; http://www.humc.edu/hormones). For expected leptin levels of 3.13, 6.25, 12.5, 25, 50, and 100 ng/ml (serial dilutions), Diagnostic Systems Laboratories measured leptin at 7.8, 15.0, 33.7, 85.5, 161.4, and 312.5 ng/ml, respectively, whereas Linco measured the same samples at 3.9, 4.8, 9.7, 20.6, 46.8, and 104.5 ng/ml, respectively, and Alpco measured the samples at 11.1, 12.0, 14.9, 26.1, 59.4, and 163.6, respectively. In general, the values obtained using the Diagnostic Systems Laboratories assay measured higher than expected values but parallel to the expected values, whereas Linco measured closer but slightly lower than expected, and Alpco measured slightly higher than expected with loss of linearity less than 12.5 ng/ml and above 50 ng/ml.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Our prior study using iv r-metHuLeptin administration elucidated effects of adiposity, age, and gender on pharmacokinetic parameters (6). Leptin levels, but not pharmacokinetic data, after sc r-metHuLeptin administration have been reported in leptin-deficient children (1, 7), but these data are limited to a few subjects with an extremely rare condition and may not be generalizable to those without congenital leptin deficiency. The data presented herein provide novel information on leptin pharmacokinetic parameters based on sc dosing in healthy lean and obese subjects, using the route of administration that would most likely be used when r-metHuLeptin is available for clinical use. We confirm findings of our previous iv pharmacokinetic study showing higher leptin levels and lower clearance with obesity (6). Previously we did not find differences in absorption or elimination of r-metHuLeptin based on gender (6), but similar data in women will be important to establish.

In this study, Diagnostic Systems Laboratories measured leptin consistently higher than Linco, with Alpco measuring similar to Linco or intermediate between Linco and Diagnostic Systems Laboratories assays. The literature also reports leptin levels that may differ by 10-fold in normal-weight women, depending on the assay used (8, 9). The standard use of serial dilutions as necessary to measure samples in the linear range of the standard curve makes the likelihood of a hook effect due to interference of the assay antibody from very high leptin levels unlikely. Correlations between assays were generally tight at physiological leptin levels and less close at higher pharmacological levels. Concentration-dependent pharmacokinetic parameters, such as baseline leptin level, C_max, AUC, and clearance, were significantly affected by the assay used. Compared with our prior pharmacokinetic study (6), which used an in-house Amgen ELISA, Linco measured baseline leptin levels closer to the Amgen assay than Diagnostic Systems Laboratories or Alpco in men with comparable BMI. Clearance based on the Amgen assay was calculated at an intermediate level [70–80 (ml/h)/kg] (6). These findings reflect differences in leptin measurement, rather than inherent differences in leptin biology because concentration-independent parameters (e.g. T_max and t_1/2) did not demonstrate major assay-specific differences.

Similar to the issue of assay standardization for hormones such as GH (10), gonadotropins (11, 12), thyroglobulin (13), and 25-hydroxy vitamin D (14), a reliable, standardized leptin assay will be critical for the future use of r-metHuLeptin from both diagnostic and monitoring standpoints. We and others (2, 3, 5) have previously used a leptin level of 3 ng/ml (men) or 4 ng/ml (women), based on the Linco assay, for treating subjects with lipodystrophy (2, 5) or hypothalamic amenorrhea (3). Currently, different assay-specific criteria for diagnosing leptin deficiency would be required, and r-metHuLeptin dose adjustments based on leptin levels would be valid only if the same assay were used in the same patient over time.

A number of factors could explain differences between assays, including variation in calibration standards (11, 12) or differential recognition of different epitopes on the leptin molecule; free vs. total leptin [because leptin circulates in part bound to a soluble leptin receptor (15)]; endogenous vs. exogenous leptin (perhaps suggested by differences in calculated replacement doses between Alpco and Diagnostic Systems Laboratories despite relatively similar baseline levels in lean men); and/or biologically active leptin fragments (leptin_22–56, leptin_57–92, leptin_116–130) (16). Our experience demonstrated high interassay CVs in some cases, which may reflect lot-to-lot variability. One prior study found a highly linear correlation between Linco and Diagnostic Systems Laboratories leptin assays at leptin levels between 1 and 100 ng/ml but in a nearly 1:1 relationship (17), in contrast to our findings of a linear correlation but with severalfold difference in levels. Further investigation into the mechanisms underlying the differences among leptin assays, including determining the precise leptin epitopes recognized by the assay antibody, will be important in identifying an accurate assay that reliably measures the true leptin level.

	Acknowledgments

 
We gratefully thank the General Clinical Research Center nurses at BIDMC for assistance with collecting the samples for this research, the General Clinical Research Center nutritionists for assistance with the isocaloric diet, and Jennifer Blakeman for assistance with the assays.

	Footnotes

 
This work was supported by National Institutes of Health (NIH) Grant MO1-RR01032; NIH Grant R01-58785; an Amgen grant; a BIDMC discretionary grant (to C.S.M.); and NIH Grant K23 RR018860 (to J.L.C.).

Disclosure Summary: J.L.C., C.O., and P.R. have nothing to declare. S.L.W. was previously employed by and has equity interests in Amgen, Inc. C.S.M. has received grant support from Amgen, Inc. and Amylin, Inc. as well as consulting honoraria from Amgen, Inc.; Amylin, Inc.; and DSL.

First Published Online April 3, 2007

Abbreviations: AUC_0-, Area under the serum concentration vs. time curve from zero to infinity; BMI, body mass index; CL/F, apparent clearance; C_max, peak serum concentration; CV, coefficient of variation; t_1/2, terminal-phase elimination half-life; T_max, time that C_max was reached.

Received December 26, 2006.

Accepted March 23, 2007.

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