Abstract
GHRP-6 is a growth hormone secretagogue that also enhances tissue viability in different organs. In the present work, we studied the pharmacokinetics of this short therapeutic hexapeptide (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2, MW = 872.44 Da) in nine male healthy volunteers after a single intravenous bolus administration of 100, 200 and 400 μg/kg of body weight. GHRP-6 was quantified in human plasma by a specific LC–MS method, previously developed and validated following FDA guidelines, using 13C3Ala-GHRP-6 as internal standard (Gil et al., 2012, J. Pharm. Biomed. Anal. 60, 19–25). The Lower Limit of Quantification (5 ng/mL) was reached in all subjects at 12 h post-administration, which was sufficient for modeling a pharmacokinetic profile including over 85% of the Area under the Curve (AUC). Disposition of GHRP-6 best fitted a bi-exponential function with R2 higher than 0.99, according to a mathematic modeling and confirmed by an Akaike index (AIC) lower than that of the corresponding one-compartment model for all subjects. Averaging all three dose levels, the distribution and elimination half-life of GHRP-6 were 7.6 ± 1.9 min and 2.5 ± 1.1 h, respectively. These values are coherent with existing data for other drugs whose disposition also fits this model. Dose dependence analysis revealed a noticeable trend for AUC to increase proportionally with administered dose. Atypical GHRP-6 concentration spikes were observed during the elimination phase in four out of the nine subjects studied.
Introduction
The Growth Hormone-Releasing Peptides (GHRPs) are a family of synthetic molecules structurally related to Met-enkephalin that stimulate the secretion of growth hormone (Pihoker et al., 1998). GHRP-6 (Growth Hormone-Releasing Peptide 6) is a six amino acid synthetic peptide (His-(D-Trp)-Ala-Trp-(D-Phe)-Lys-NH2, MW = 872.44 Da) first described in 1994 by Deghenghi et al. (1994). This molecule is a strong growth hormone secretagogue that acts via specific G-protein-coupled receptors at the hypothalamus and pituitary gland (Smith et al., 1999).
GHRP-6 exhibits a potent cytoprotective effect, extending tissue viability during acute ischemia/reperfusion episodes in different organs (e.g. small bowel, liver, kidneys) (Cibrián et al., 2006). This peptide has also shown antioxidant activity in preclinical experimental models, and may partially contribute to reduce myocardial ischaemic damage (Berlanga et al., 2007). Thus, pathologies associated with ischemia or some types of tissue damage may potentially benefit from treatment with this promising therapeutic candidate.
Peptides in biological samples are usually quantified by means of bioanalytical techniques. Methods based on Liquid Chromatography coupled to Mass Spectrometry (LC–MS or LC–MS/MS) are often used for this purpose, although their sensitivity and selectivity and therefore, the reliability of their results depend to a large extent on the specific analyte. In these methods, isotopically labeled internal standards are used for either relative or absolute quantification of the analyte, as previously described for large peptides like insulin (Darby et al., 2001) and Enfuvirtide (Chang et al., 2005). Similar methods have been implemented to quantify certain members of the GHRP family or their metabolites in urine (Okano et al., 2010, Thomas et al., 2011).
Although GHRP-6 is a potential therapeutic candidate, little is known about its disposition in vivo. To our knowledge, pharmacokinetic studies of GHRPs have been limited to the administration of GHRP-2 to pediatric patients with short stature (Pihoker et al., 1998), and of hexarelin (a GHRP-6 analog) to rats (Roumi et al., 2000), quantifying and monitoring the administered peptide in both cases by Radio Immuno Assay (RIA). Immunoassays based on radioactive labels are not, however, very accurate, since they are unable to distinguish between analyte and radiolabeled metabolites. This limitation is overcome by bioanalytical methods based on mass spectrometric determinations, which simultaneously identify and quantify the analyte (Carrascal et al., 1998).
The pharmacokinetics (PK) of GHRP-6 in humans has to be known in detail before undertaking large scale clinical trials to estimate its safety and efficacy. The aim of this work, therefore, was to characterize the PK of this peptide in healthy volunteers after a single intravenous administration at one of three different dose levels: 100, 200, or 400 μg of GHRP-6 kg−1 of body weight. This pharmacokinetic study was included in a dose scaling and safety phase I clinical trial. Plasma concentrations of GHRP-6 were determined by a previously developed and validated bioanalytical method based on LC–MS using the fully labeled 13C Alanine peptide (13C3Ala-GHRP-6) as internal standard (Gil et al., 2012).
Access through your organization
Check access to the full text by signing in through your organization.Access through your organization
Section snippets
Subjects
Nine male healthy volunteers were included in this study. Written informed consent to the protocol, previously approved by the National Center for Drug Control (CECMED, Cuba), was obtained from each volunteer before inclusion. Subject health was assessed by physical examinations and determinations of the main blood biochemistry parameters before administering GHRP-6. The age of the subjects included in this study ranged from 18 to 35 years. The subjects were distributed into groups of three
Results and discussion
GHRP-6 was well tolerated at the lowest dose level and, progressively, at higher dose levels throughout the dose scale-up procedure from 100 μg/kg to 400 μg/kg, enabling all nine subjects to complete the study without any serious or moderate adverse event. Plots of mean GHRP-6 concentration vs. time for the three dose levels studied are shown in Fig. 1. After 12 h post-administration, GHRP-6 concentrations fell below the LLOQ (5 ng/mL); these values were, therefore, excluded from the
Conclusions
The pharmacokinetic behavior of GHRP-6 was characterized by a bi-exponential profile with distribution and elimination phases of 7.6 ± 1.9 min and 2.5 ± 1.1 h, respectively. This behavior was similar for the three dose levels studied. Biliary excretion was hypothesized as one of the possible elimination routes for GHRP-6 in 4 out of 9 subjects. Dose dependence analysis revealed a noticeable trend for AUC towards a proportional increase with administered dose.
The total distribution volume determined
Acknowledgements
The authors thank the Center for Medical and Surgical Research (CIMEQ, Cuba) for clinical and laboratory assistance. We also thank Alejandro Martín from CIGB (Cuba) for critical review and English correction of the manuscript.
References (28)
et al.
System suitability in bioanalytical LC/MS/MS
J. Pharm. Biomed. Anal.
(2007) M.E. Burton
Applied pharmacokinetics & pharmacodynamics: principles of therapeutic drug monitoring M. Carrascal et al.
Quantitative electrospray LC–MS and LC–MS/MS in biomedicine
J. Pharm. Biomed. Anal.
(1998) D. Chang et al.
Bioanalytical method development and validation for a large peptide HIV fusion inhibitor (Enfuvirtide, T-20) and its metabolite in human plasma using LC–MS/MS
J. Pharm. Biomed. Anal.
(2005) C.B. Davis et al.
Disposition of growth hormone-releasing peptide (SK&F 110679) in rat and dog following intravenous or subcutaneous administration
Drug Metab. Dispos.
(1994) R. Deghenghi et al.
GH-releasing activity of hexarelin, a new growth hormone releasing peptide, in infant and adult rats
Life Sci.
(1994) J. Gil et al.
Development and validation of a bioanalytical LC–MS method for the quantification of GHRP-6 in human plasma
J. Pharm. Biomed. Anal.
(2012) K.A. Hoffmaster et al.
Multiple transport systems mediate the hepatic uptake and biliary excretion of the metabolically stable opioid peptide [D-penicillamine2,5] enkephalin
Drug Metab. Dispos.
(2005) M. Roumi et al.
Kinetics and disposition of hexarelin, a peptidic growth hormone secretagogue, in rats
Drug Metab. Dispos.
(2000) H.H. Szeto et al.
In vivo pharmacokinetics of selective μ-opioid peptide agonists
J. Pharmacol. Exp. Ther.
(2001)
1
These authors contributed equally to this work.View full text
Copyright © 2012 Elsevier B.V. All rights reserved.