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Prolonged Stimulation of Growth Hormone (GH) and Insulin-Like Growth Factor I Secretion by CJC-1295, a Long-Acting Analog of GH-Releasing Hormone, in Healthy Adults
Sam L. Teichman 1
Ann Neale 1
Betty Lawrence 1
Catherine Gagnon 1
Jean-Paul Castaigne 1
Lawrence A. Frohman 2
1 WinPharm Associates (S.L.T., A.N.), Alamo, California 94507; ConjuChem, Inc. (B.L., C.G., J.-P.C.), Montréal, Québéc, Canada;
2 and Section of Endocrinology, Department of Medicine, University of Illinois (L.A.F.), Chicago, Illinois 60612
Received July 11, 2005.
Accepted December 6, 2005.
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Address all correspondence and requests for reprints to: Dr. Lawrence A. Frohman, Section of Endocrinology, Department of Medicine, University of Illinois, 1819 West Polk Street (M/C 640), Chicago, Illinois 60612. E-mail:
[email protected].
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This work was supported by ConjuChem, Inc. (Montréal, Canada).
Copyright © 2006 by The Endocrine Society
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Abbreviations:
AUC
Area under the curve
Cmax
peak plasma concentration
DAC-GRF
drug affinity complex-GH-releasing factor
MPA
maleimidoproprionic acid
Tmax
time to peak plasma concentration
Context: Therapeutic use of GHRH to enhance GH secretion is limited by its short duration of action.
Objective: The objective of this study was to examine the pharmacokinetic profile, pharmacodynamic effects, and safety of CJC-1295, a long-acting GHRH analog.
Design: The study design was two randomized, placebo-controlled, double-blind, ascending dose trials with durations of 28 and 49 d.
Setting: The study was performed at two investigational sites.
Participants: Healthy subjects, ages 21–61 yr, were studied.
Interventions: CJC-1295 or placebo was administered sc in one of four ascending single doses in the first study and in two or three weekly or biweekly doses in the second study.
Main Outcome Measures: The main outcome measures were peak concentrations and area under the curve of GH and IGF-I; standard pharmacokinetic parameters were used for CJC-1295.
Results: After a single injection of CJC-1295, there were dose-dependent increases in mean plasma GH concentrations by 2- to 10-fold for 6 d or more and in mean plasma IGF-I concentrations by 1.5- to 3-fold for 9–11 d. The estimated half-life of CJC-1295 was 5.8–8.1 d. After multiple CJC-1295 doses, mean IGF-I levels remained above baseline for up to 28 d. No serious adverse reactions were reported.
Conclusions: Subcutaneous administration of CJC-1295 resulted in sustained, dose-dependent increases in GH and IGF-I levels in healthy adults and was safe and relatively well tolerated, particularly at doses of 30 or 60 μg/kg. There was evidence of a cumulative effect after multiple doses. These data support the potential utility of CJC-1295 as a therapeutic agent. ( J Clin Endocrinol Metab 91: 799–805, 2006)
THE USE OF GH for the treatment of children with impaired linear growth has been accepted as an important therapeutic modality for more than 50 yr [1] . An unlimited supply of the hormone, made possible by the availability of recombinant GH in the 1980s, permitted expansion of the target population to include GH-deficient adults. Most adults receiving GH today have primary pituitary disease with impaired GH secretory capacity. However, most children being treated with GH have no evidence of pituitary disease and are believed to have an impaired hypothalamic signaling mechanism due to a GHRH neurosecretory dysfunction. GH has also been used for therapy of disorders in children and adults in which pituitary function is either intact or only slightly impaired, such as chronic renal failure and Turner syndrome (in children) and HIV-related wasting and lipodystrophy and burn therapy (in adults).
In patients with intact pituitary function, there has been interest in the use of GHRH rather than GH in the hope of producing a more physiological pattern of tissue exposure to GH than occurs by a single daily injection of the hormone. In fact, several studies in both children and adults have suggested that comparable or near-comparable results can be achieved with GHRH therapy [2] [3] [4] .
A major limitation in the use of GHRH for therapy, however, is its short half-life. Native GHRH, a 44-amino acid peptide, has a half-life of 7 min [5] , which is even shorter than that of GH (12 min) [6] , necessitating daily or even more frequent injections. Polyethylene glycol-conjugated GHRH has been studied in an effort to overcome this limitation [7] .
A synthetically modified form of GHRH has been linked to a reactive chemical that enables binding to endogenous serum albumin after sc administration. The chemical structure of this compound, drug affinity complex-GH-releasing factor (DAC-GRF; CJC-1295, ConjuChem, Inc., Montréal, Canada) is shown in Fig. 1 . The core therapeutic moiety is GHRH-(1–29)NH2 , which contains the full biological activity of GHRH-(1–44)NH2 modified by substitution of four amino acids that serve to render the compound more resistant to proteolytic cleavage (herein called GRF). GRF is linked by the amino acid, lysine, to a reactive chemical [maleimidoproprionic acid (MPA)] that binds to unpaired thiol (sulfhydryl)
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First Published Online December 13, 2005
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Figure 1. Chemical structure of the CJC-1295 (DAC-GRF). The core therapeutic moiety is a tetrasubstituted GHRH-(1–29)NH2 . The substituted amino acids are shown in italics. The linker is lysine, and the reactive chemical is maleimidoproprionic acid that binds covalently to the single unpaired cysteine (cysteine 34) in serum albumin.
groups. The predominant free thiol group available for binding after parenteral administration is the single unpaired cysteine (cysteine 34) in serum albumin. At least 90% of CJC-1295 binds covalently to albumin in this fashion, with trace amounts found bound to fibrinogen and IgG. No other chemical species have been found bound to DAC-GRF after administration (data on file, ConjuChem, Inc.). This binding extends the half-life of the active pharmacophore, resulting in a markedly prolonged duration of action in several animal species [8] . Moreover, studies in both dogs and pigs indicate that physiological GH secretion is maintained, and IGF-I levels are enhanced for several days after a single administration.
We assessed the safety, tolerability, pharmacokinetic profile, and effect of CJC-1295 on circulating concentrations of GH and IGF-I in two randomized, placebo-controlled, double-blind, dose-escalating studies in healthy adult subjects.
Subjects and Methods
The subjects consisted of healthy men and women, ages 21–61 yr, with a body mass index of 30 kg/m2 or less and IGF-I levels in the normal range for age and gender. Appropriately constituted independent ethics committees reviewed and approved each of the studies, and written informed consent of all subjects was obtained before participation.
Study design
Study 1 was an ascending, randomized, double-blind, placebo-controlled single-dose trial performed at SFBC International, Inc. (Miami, FL). Study 2 was an ascending, randomized, double-blind, placebocontrolled multiple-dose trial performed at Kendle International BV (Utrecht, The Netherlands).
In study 1, four sequential, dose-escalation groups were evaluated. These dose levels were 30 μg/kg (n = 6, five active and one placebo), 60 μg/kg (n = 6, five active and one placebo), 125 μg/kg (n = 6, five active and one placebo), and 250 μg/kg (n = 6, five active and one placebo). An additional cohort of 18 subjects (15 active and three placebo) subsequently received 125 μg/kg.
Serum GH was measured on d 0 at 60, 30, and 15 min before study drug dosing; at 15, 30, and 60 min and 2, 3, 4, 6, 8, 10, 12, and 24 h after dosing; and then every 8 h on d 2–3, then daily on d 4, 5, 6, 7, 9, 11, 14, 21, and 28. Serum IGF-I and CJC-1295 were measured on d 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 14, 21, and 28.
In study 2, 24 subjects were enrolled in one of four sequential dosing cohorts. Group 1 (n = 6, five active and one placebo) received two injections of 30 μg/kg (d 0 and 14), group 2 (n = 6, five active and one placebo) received two injections of 60 μg/kg (d 0 and 14), group 3 (n = 6, five active and one placebo) received three injections of 30 μg/kg (d 0, 7, and 14), and group 4 (n = 6, five active and one placebo) received three injections of 20 μg/kg (d 0, 7, and 14). Sample collection was similar to that in study 1, with the addition of more frequent pre- and postinjection sampling on d 7 (groups 3 and 4 only) and 14 as well as a final sample collection on d 49 in all subjects.
Serial clinical evaluations (vital signs, adverse events, and physical examination) and laboratory safety assessments (serum chemistry, hematology, and urinalysis) were performed ending on d 28 in study 1 and on d 49 in study 2.
Laboratory methods
GH.
Serum GH was measured by a double antibody RIA (Esoterix Laboratory Services, Inc., Calabasas Hills, CA). The assay sensitivity was 0.3 ng/ml, and the coefficient of variation was 10%.
IGF-I.
Serum IGF-I was measured by a double antibody RIA by Esoterix Laboratory Services, Inc., after ethanol extraction and with the addition of IGF-2 as a blocking agent. The assay sensitivity was 10 ng/ml, and the coefficient of variation was 5.4%. Normal ranges for the assay are age and gender adjusted.
Other hormones.
Serum cortisol, prolactin, TSH, and LH concentrations were measured in patients receiving 60 μg/kg CJC-1295 in study 1 by Esoterix Laboratory Services, Inc.
CJC-1295.
Plasma CJC-1295 concentrations were measured by RIA at PPD Development, LP (Richmond, VA), using a rabbit anti-(tetra-substituted)GRF-(1–29) coupled to keyhole limpet hemocyanin and radioiodinated GRF-(1–29). The antibody exhibited 100% cross-reactivity with albumin-bound DAC of the tetrasubstituted GRF-(1–29). There was no cross-reactivity with native GRF-(1–29), native GRF-(3–29), or the DAC of the fragment 12–29 of the tetrasubstituted GRF. There was 25% cross-reactivity with free (nonalbumin-bound) tetrasubstituted GRF-(1–29). The lower limit of detection was 0.2 nmol/liter, and the mean intra-and interassay coefficients of variation were 5.5% and 8.2%, respectively.
Pharmacokinetic analysis.
Pharmacokinetic parameters [peak plasma concentrations (Cmax ), time to peak plasma concentrations (Tmax ), and area under the curve (AUC)] of CJC-1295, GH, and IGF-I were calculated from the concentration vs. time values for each patient using a compartment model in the single-dose study (WinNonlin Professional version 4.1, Pharsight Corp., Mountain View CA) and a noncompartmental model in the multiple-dose study (WinNonlin Professional version 4.0.1).
Antibody formation.
A validated immunoradiometric assay was used to determine the presence of antibodies to CJC-1295. The anti-CJC-1295 antibody was raised in rabbits by immunization with a CJC-1295 analog [the tetrasubstituted GRF-(1–29)] to which a cysteine residue was added at position 30 to permit direct conjugation to keyhole limpet hemocyanin to make the molecule more immunogenic. This antibody was also used in the assay for plasma CJC-1295 concentrations. In this immunoradiometric assay, tubes are coated with CJC-1295 bound to inactivated MPA. Test samples or affinity-purified rabbit anti-CJC-1295 antibody controls in human serum were added. After incubation, tubes were washed and [125 I]protein LA (Sigma-Aldrich, St. Louis, MO) was added. After incubation, tubes were washed again, radioactivity was determined in a γ-counter, and the specific binding of the samples was calculated.
Statistical analysis
Mean and variance estimates were calculated for all pharmacokinetic parameters by dose group. Cmax and AUC to the last sampling time (AUCt ) were log transformed before analysis, and AUC values were calculated using the linear trapezoidal rule. Differences in GH and IGF-I levels and AUC between groups were compared by ANOVA and/or one-tailed t test; P < 0.05 was considered significant. All statistical analyses were performed using SAS version 8.02 (SAS Institute, Cary, NC).
Because all enrolled subjects received at least one dose of the study drug, all available data are included in analyses of safety, pharmacokinetic,
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and pharmacodynamic parameters. No effort to estimate missing data was made, with the exception of AUC calculations.
Subjects receiving placebo in all dosing groups in each study were pooled for comparison with groups treated with active drug.