Nonpeptide Growth Hormone Secretagogues Research Articles

Detection of the growth hormone secretagogue MK-0677 in equine hair following oral administration.

MK-0677 (ibutamoren) is an orally active non-peptide growth hormone secretagogue that binds to the ghrelin receptor stimulating the secretion of endogenous growth hormone. It is one of the most prevalent performance-enhancing compounds currently available online and is potentially subject to abuse both in human and equine sports. The aim of the current study was to investigate whether it could be detected in equine hair following oral administration of MK-0677 mesylate to a Thoroughbred racehorse. MK-0677 and its O-dealkylated metabolite were extracted using an existing method for prohibited substances in equine hair and analysed by liquid chromatography tandem mass spectrometry. This enabled the detection of MK-0677 in all hair samples collected, up to 209 days in mane and 358 days in tail. A follow-up methodology with an extensive wash procedure was carried out for selected hair samples, which unambiguously verified the presence of MK-0677. Wash criteria to differentiate between internal incorporation (via bloodstream) and external deposition (via sweat and sebum) was also assessed and indicated internal incorporation for the samples collected at later time points (≥52 days) and a combination of internal incorporation and external deposition for hair samples collected at the earlier time point (2days).

Characterization of growth hormone secretagogue small molecule ibutamoren (MK-0677) and its possible metabolites in thoroughbred horses for doping control.

It is important to remember that performance-enhancing agents such as non-peptide growth hormone secretagogues present a significant risk of abuse. Ibutamoren (MK-0677) is a potent, long-acting, selective non-peptide growth hormone secretagogue that can be taken orally. This study examines ibutamoren and its metabolites in thoroughbred horses after oral administration. Liquid chromatography/high-resolution mass spectrometry was used to determine the probable structures of the detected metabolites. In this study, 22 metabolites of ibutamoren were identified (17 phase I and 5 phase II). Oxidation of ibutamoren leads to hydroxylated metabolites (mono and di). The study also detected dissociated side chains (benzyl group and 2-amino-2-methylpropanamide) and hydrogenated metabolites. The glucuronic acid conjugated analogs of ibutamoren were detected during phase II of the study, but no sulfonic acid conjugated analogs were observed. The major metabolites can be detected up to 96 hours after a single dose, and ibutamoren can persist for up to 72 hours. These findings will aid in the detection of ibutamoren and the detection of its illegal use in competitive sports.

Equine metabolism of the growth hormone secretagogue MK-0677 in vitro and in urine and plasma following oral administration.

Ibutamoren mesylate, or MK-0677, is an orally active, nonpeptide growth hormone secretagogue that has been developed to stimulate excretion of endogenous growth hormone. It has been evaluated for the treatment of a range of clinical conditions but is not available therapeutically. Nonetheless, MK-0677 is widely available to purchase online, sold as 'supplement' products. The mode of action and relative ease of purchase make MK-0677 a potential threat with regard to sports doping. The aim of this study was to investigate the metabolism of MK0677 in the horse following in vitro incubation and oral administration to two Thoroughbred racehorses, in order to identify the most appropriate analytical targets for doping control laboratories. Liquid chromatography high resolution mass spectrometry was used for metabolite identification, and subsequently, liquid chromatography-tandem mass spectrometry was used to generate full metabolite profiles for post-administration urine and plasma samples. Fourteen phase I metabolites were identified in vitro; 13 of these were subsequently detected in urine and nine in plasma collected post-administration, alongside the parent compound in both matrices. In both urine and plasma, the longest duration of detection was observed for an O-dealkylated metabolite of MK-0677, and therefore, this would be the best target for the detection of MK-0677 administration. MK-0677 and the O-dealkylated metabolite were found to be excreted largely unconjugated in urine and plasma.

Characterization of equine liver microsome-generated metabolites of growth hormone secretagogue small molecule ibutamoren.

Interest in growth hormone secretagogues has intensified during the past several years based on capable, ever-widening investigational applications of recombinant growth hormone in animals and humans. Ibutamoren is a potent, long-acting, selective and orally active non-peptide growth hormone secretagogue, which has a great potential for abuse as a performance-enhancing agent in sports. To support drug metabolism and pharmacokinetic studies of chiral pharmaceuticals, it is necessary to combine the resolving power of high-performance liquid chromatography with the sensitivity of mass spectrometric techniques. This paper describes the metabolic conversion of ibutamoren using equine liver microsomes and metabolite characterization using a QExactive high-resolution mass spectrometer. A total of 32 metabolites for ibutamoren (20 phase I and 12 phase II) were detected. The important findings of the current research are as follows: (1) the growth hormone secretagogue ibutamoren was prone to oxidation, resulting in corresponding hydroxylated metabolites; (2) in ibutamoren, the dissociation of the phenyl ring and 2-amino-2-methylpropanamide side chain was also observed; (3) the glucuronic acid conjugates of mono-, di- and trihydroxylated analogues were detected; and (4) no sulfonic acid conjugated metabolites were observed in this study of ibutamoren. The reported data help in the speedy detection of the growth hormone secretagogue ibutamoren and reveal its illegal use in competitive sports.

Detection of bioactive peptides including gonadotrophin-releasing factors (GnRHs) in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS).

The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non-peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS). A simple mixed-mode cation exchange solid-phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analyzed using UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA, for MS2 ). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purposes. The validation data, including method specificity, method sensitivity, extraction recovery, method precision, and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin-releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin, and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).

Determination of Andarine (S-4), a Selective Androgen Receptor Modulator, and Ibutamoren (MK-677), a Nonpeptide Growth Hormone Secretagogue, in Urine by Ultra-High Performance Liquid Chromatography with Tandem Mass-Spectrometric Detection

A procedure for the determination of a selective androgen receptor modulator andarine (S-4) and a nonpeptide growth hormone secretagogue ibutamoren (MK-677) in urine has been developed including sample preparation by the “dilute-and-shoot” procedure, separation of analytes by ultra-high performance liquid chromatography in the gradient elution mode, and mass-spectrometric detection with heated electrospray ionization. The limits of detection are in the range of 0.5–2.5 ng/mL, the calibration curves are linear in the range of 2.5–250 ng/mL for andarine and 5–250 ng/mL for ibutamoren. The proposed procedure was used for the analysis of urine samples obtained from volunteers after a single administration of these drugs containing 15 mg of active substances.

Application of solid-phase extraction for the quantification of several abused drugs in sports in human urine using UHPLC-MS/MS method

A procedure for the determination of several selective androgen receptor modulators in human urine, namely, Andarine (S-4, GTx-007), Ostarine (S-22, GTx-024, MK-2866) and Ligandrol (LGD-4033, VK-5211), as well as a phytosteroid Laxogenin and Ibutamoren (MK-677), related to non-peptide growth hormone secretagogues, has been developed. Most of these are prohibited by the World Anti-Doping Agency due to their anabolic effects. With the aim of the analytes purification and concentration, a procedure for solid-phase extraction with the use of Varian Bond Elut C8 (100 mg, 1 mL), Biotage Isolute C18 (EC) (100 mg, 1 mL) and Waters Oasis HLB (30 mg, 1 mL) cartridges was applied. To achieve good resolution of the analytes, reversed-phase ultra-high-performance liquid chromatography coupled with the triple quadrupole mass spectrometer as a detector in positive and negative ion detection modes was used. The limits of detection were in the range of 0.1–0.5 ng/mL, limits of quantification were between 0.25–0.5 ng/mL. Calibration curves were examined to be linear in the wide range of concentrations. Matrix effects were also evaluated and were found not to influence the results of the analytes quantification. The proposed method was applied for the analysis of real samples after single 15 mg oral administration of the analytes. All the analytes were positively detected in the human urine samples in 12h, thus, proving the applicability of the procedure for the analytes quantification in the urine. Key words : UHPLC-MS/MS, SARM, Laxogenin, Ibutamoren, solid-phase extraction, doping control DOI: http://dx.doi.org/10.15826/analitika.2018.22.3.008 (Russian) E.V. Dmitrieva, A.Z. Temerdashev, A.A. Azaryan, E.M. Gashimova Kuban State University, Stavropolskaya st., 149, Krasnodar, 350040, Russian Federation

Novel Series of Highly Potent Non-peptide Growth Hormone Secretagogues with Im- proved Bioavailability

The discovery and the SAR of acylproline derivatives as highly potent growth hormone secretagogues (GHSs) with good oral bioavailability are described. One representative compound, N-[3-(2,2-dimethylpropylamino)-2-hydroxypropyl]-2(R)-[l-(2,2-dimethylpropionyl)pyrrolidine-2(S)-carbonylamino]-3-naphthalen-2-ylpropionamide (4e), showed potent GHS activity (ED 50 =1 nM) and good oral bioavailability (BA=33.2%). Moreover, the optically pure N-[3-(2,2-dimethylpropylamino)-2(S)-hydroxypropyl]-2(R)-[1-(2,2-dimethylpropionyl)pyrrolidine-2(S)-carbonylamino]-3-naphthalen-2-ylpropionamide ((2S)-4e) showed a good metabolic stability against in vitro clearance (human liver microsome) with potent GHS activity.

Signal transduction mechanism of the seabream growth hormone secretagogue receptor

We have recently cloned the full-length cDNAs of the two growth hormone secretagogue receptor (GHSR) subtypes from a teleost species, the black seabream ( Acanthopagrus schlegeli) [Mol. Cell. Endocrinol. 214 (2004) 81], namely sbGHSR-1a and sbGHSR-1b. Functional expression of these two receptor constructs in human embryonic kidney 293 (HEK293) cells indicated that stimulation of sbGHSR-1a by growth hormone secretagogues (GHS) could evoke increases in intracellular Ca 2+ concentration ([Ca 2+] i), whereas sbGHSR-1b appeared to play an inhibitory role on the signal transduction activity of sbGHSR-1a. In the present study, we have further investigated the signal transduction mechanism of sbGHSR-1a. The peptide GHS GHRP-6 and the non-peptide GHS L163,540 were able to trigger a receptor specific and phospholipase C (PLC)-dependent elevation of [Ca 2+] i in HEK293 cells stably expressing sbGHSR-1a. This GHS-induced calcium mobilization was also dependent on protein kinase C activated L-type calcium channel opening. It was found that sbGHSR-1a could function in an agonist-independent manner as it exhibited a high basal activity of inositol phosphate production in the absence of GHS, indicating that the fish receptor is constitutively active. In addition, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) were found to be activated upon stimulation of sbGHSR-1a by GHRP-6. This observation provides direct evidence in the coupling of sbGHSR-1a to ERK1/2 activation. Neither G s nor G i proteins are coupled to the receptor, as GHS did not induce cAMP production nor inhibit forskolin-stimulated cAMP accumulation in the sbGHSR-1a bearing cells. Furthermore, the ability of the GHSR antagonist d -Lys(3)-GHRP-6 to inhibit basal PLC and basal ERK1/2 activity suggests that this compound is an inverse agonist. In summary, the sbGHSR-1a appears to couple through the G q/11-mediated pathway to activate PLC, resulting in increased IP 3 production and Ca 2+ mobilization from both intracellular and extracellular stores. Moreover, sbGHSR-1a may trigger multiple signal transduction cascades to exert its physiological functions.

Stimulation of growth hormone secretion from seabream pituitary cells in primary culture by growth hormone secretagogues is independent of growth hormone transcription

The action of a number of growth hormone secretagogues (GHS) on growth hormone (GH) secretion and gene expression was studied in a primary culture of pituitary cells isolated from the black seabream Acanthopagrus schlegeli. The peptide GHS employed included growth hormone-releasing peptide (GHRP)-2, ipamorelin, and human ghrelin. The nonpeptide GHS employed included the benzolactam GHS L692,585 and the spiropiperidine GHS L163,540. Secreted GH was measured in the culture medium by an enzyme-linked immunosorbent assay (ELISA) method using a specific antibody against seabream GH. The GH mRNA content in the incubated cells was assessed by reverse transcription polymerase chain reaction (RT-PCR) using a pair of gene-specific primers designed from the cloned black seabream GH cDNA sequence. A dose-dependent stimulation of GH release was demonstrated by all the GHS tested, except human ghrelin, with EC 50 values in the nanomolar range. Simultaneous measurement of GH mRNA levels in the incubated seabream pituitary cells indicated that the GHS-stimulated increase in GH secretion was not paralleled by corresponding changes in GH gene expression. In contrast to the situation previously reported in the rat, no change in GH gene expression was noticed in the seabream pituitary cells even though the time of stimulation by GHS was increased up to 48 h, confirming that the GHS-stimulated GH secretion in seabream is independent of GH gene transcription.

Ghrelin and growth hormone (GH) secretagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3',5'-monophosphate production in cells expressing transfected GHRH and GH secretagogue receptors.

GHRH stimulates GH secretion from somatotroph cells of the anterior pituitary via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cAMP production. The actions of GHRH to release GH can be augmented by the synthetic GH secretagogues (GHS), which bind to a distinct G protein-coupled receptor to activate phospholipase C and increase production of the second messengers calcium and diacylglycerol. The stomach peptide ghrelin represents an endogenous ligand for the GHS receptor, which does not activate the cAMP signaling pathway. This study investigates the effects of GHS and ghrelin on GHRH-induced cAMP production in a homogenous population of cells expressing the cloned GHRH and GHS receptors. Each epitope-tagged receptor was shown to be appropriately expressed and to functionally couple to its respective second messenger pathway in this heterologous cell system. Although activation of the GHS receptor alone had no effect on cAMP production, coactivation of the GHS and GHRH receptors produced a cAMP response approximately twice that observed after activation of the GHRH receptor alone. This potentiated response is dose dependent with respect to both GHRH and GHS, is dependent on the expression of both receptors, and was observed with a variety of peptide and nonpeptide GHS compounds as well as with ghrelin-(1-5). Pharmacological inhibition of signaling molecules associated with GHS receptor activation, including G protein betagamma-subunits, phospholipase C, and protein kinase C, had no effect on GHS potentiation of GHRH-induced cAMP production. Importantly, the potentiation appears to be selective for the GHRH receptor. Treatment of cells with the pharmacological agent forskolin elevated cAMP levels, but these levels were not further increased by GHS receptor activation. Similarly, activation of two receptors homologous to the GHRH receptor, the vasoactive intestinal peptide and secretin receptors, increased cAMP levels, but these levels were not further increased by GHS receptor activation. Based on these findings, we speculate that direct interactions between the GHRH and GHS receptors may explain the observed effects on signal transduction.

HUMAN GROWTH HORMONE: RESEARCH AND CLINICAL PRACTICE

Overview of Human Growth Hormone: Research and Clinical Practice, Roy G. Smith. Part I Research. GHRP Historical Perspective: Basic and Clinical, C. Y. Bowers. The Design of Peptidomimetic Growth Hormone Secretagogues, Arthur A. Patchett and Matthew J. Wyvratt. Molecular Characterization of Growth Hormone Secretagogue Receptors, Andrew D. Howard, Scott D. Feighner, Roy G. Smith, and Lex H. T. Van der Ploeg. Central Actions of Peptide and Nonpeptide Growth Hormone Secretagogues, Suzanne L. Dickson and Gareth Leng. Pharmacology of Growth Hormone-Releasing Hormone and Its Peptide Analogs, David H. Coy. Somostatin Receptor Subtypes and Regulation of GH Secretion, Gurkeerat Singh, George Liapakis, and Terry Reisine. Mathematical Modeling of the GH Release Axis, David Brown, Elinor A. Stephens, Gareth Leng, and Roy G. Smith. Activation of the Human Growth Hormone Receptor: Structure and Function of the Ligand-Receptor Complex, Kenneth H. Pearce, and James A. Wells. The Central Nervous System as a Direct Target for Growth Hormone Action, Pamela A. Bennett and Iain C. A. F. Robinson. Part II Clinical Practice. Molecular Defects of the Growth Hormone Axis, Linda Anne DiMeglio, Paul L. Hofman, and Ora Hirsch Pescovitz. Growth Hormone: Relevance to Pediatrics, Barry B. Bercu and Howard J. Heinze. Growth Hormone Deficiency in Adults: The Rationale for Growth Hormone Replacement, Paul V. Carroll, Emanuel R. Christ, and Peter H. Soenksen. Growth Hormone and Osteoporosis, Howard B. A. Baum and Anne Klibanski. Growth Hormone and Syndrome X, Gudmundur Johannsson, Per Marin, Jan-Ove Johansson, and Bengt-Ake Bengtsson. Interactions Between Growth Hormone Secretion and Sleep, Eve Van Cauter and Georges Copinschi. Growth Hormone in AIDS, Kathleen Mulligan and Morris Schambelan. Clinical and Physiological Studies With Growth Hormone-Releasing Hormone, George R. Merriam and Fernando Cassorla. Clinical Use of Growth Hormone Secretagogues, Mark A. Bach and Glenn J. Gormley. Index.

Actions of Leptin on Growth Hormone Secretagogue‐Responsive Neurones in the Rat Hypothalamic Arcuate Nucleus Recorded In Vitro

AbstractIn the arcuate nucleus, the growth hormone (GH) secretagogue (GHS)‐responsive cells include a subpopulation of the neuropeptide Y (NPY) neurones. It is not known whether these include the orexigenic NPY population that are inhibited by the satiety hormone, leptin. Thus we investigated whether (i) the arcuate nucleus cells electrically excited by GHS are inhibited by leptin and (ii) chronic central leptin infusion alters GHS‐induced Fos expression. Of 36 cells recorded from a trimmed hypothalamic slice containing arcuate nucleus, 13 cells were excited by the nonpeptide GHS, CP‐459,599. The predominant response of these cells to leptin was inhibitory: six inhibited, three excited and four unresponsive. Similar responses were observed in a population of arcuate cells recorded from a preparation in which synaptic transmission was blocked, suggesting that leptin acts directly on a subpopulation of GHS‐responsive neurones. Intracerebroventricular infusion of leptin for 1 week did not alter the number of cells expressing Fos following GHS administration. Thus, while leptin does not appear to influence the central actions of GHS to induce immediate early gene expression, it does act directly on a subpopulation of cells excited by GHS, eliciting mostly inhibitory but also some excitatory responses. It will be interesting to discover the consequences of leptin's inhibitory effects on the hypothalamic circuits excited by GHS, particularly since leptin paradoxically has a stimulatory effect on GH secretion, presumed to reflect a suppression of central NPY pathways.

Actions of leptin on growth hormone secretagogue-responsive neurones in the rat hypothalamic arcuate nucleus recorded in vitro.

In the arcuate nucleus, the growth hormone (GH) secretagogue (GHS)-responsive cells include a subpopulation of the neuropeptide Y (NPY) neurones. It is not known whether these include the orexigenic NPY population that are inhibited by the satiety hormone, leptin. Thus we investigated whether (i) the arcuate nucleus cells electrically excited by GHS are inhibited by leptin and (ii) chronic central leptin infusion alters GHS-induced Fos expression. Of 36 cells recorded from a trimmed hypothalamic slice containing arcuate nucleus, 13 cells were excited by the nonpeptide GHS, CP-459,599. The predominant response of these cells to leptin was inhibitory: six inhibited, three excited and four unresponsive. Similar responses were observed in a population of arcuate cells recorded from a preparation in which synaptic transmission was blocked, suggesting that leptin acts directly on a subpopulation of GHS-responsive neurones. Intracerebroventricular infusion of leptin for 1 week did not alter the number of cells expressing Fos following GHS administration. Thus, while leptin does not appear to influence the central actions of GHS to induce immediate early gene expression, it does act directly on a subpopulation of cells excited by GHS, eliciting mostly inhibitory but also some excitatory responses. It will be interesting to discover the consequences of leptin's inhibitory effects on the hypothalamic circuits excited by GHS, particularly since leptin paradoxically has a stimulatory effect on GH secretion, presumed to reflect a suppression of central NPY pathways.

Central actions of the nonpeptide growth hormone secretagogue GHS-25.

Growth hormone secretagogues (GHSs) increase the activity of hypothalamic arcuate nucleus neurons thought to be involved in controlling the release of growth hormone (GH). The GHS receptor is also found in hypothalamic regions not associated with the release of GH, suggesting that GHSs may influence other hypothalamic systems. This study utilized double-labeling immunocytochemical techniques to examine the hypothalamic actions of a novel nonpeptide GHS, GHS-25. In common with other GHSs, GHS-25 induced significant amounts of Fos immunoreactivity in the arcuate nucleus of conscious male rats. However, unlike other GHSs, GHS-25 also induced Fos immunoreactivity in the supraoptic nucleus. Double labeling revealed that approx 66% of supraoptic nucleus cells that were Fos positive after the administration of GHS-25 were also immunoreactive for oxytocin. Thus, in addition to its actions on the GH axis, GHS-25 may influence the release of neurohypophyseal hormone.

Growth hormone secretagogue activation of the arcuate nucleus and brainstem occurs via a non-noradrenergic pathway.

Noradrenergic systems are integrally involved in the release of growth hormone (GH) from the anterior pituitary gland and in regulating the activity of hypothalamic growth hormone-releasing hormone (GHRH) neurones. GH secretagogues act at both the pituitary and the hypothalamus to facilitate the release of GH. In male rats, using the induction of Fos protein as an indicator of neuronal activation, we examined whether neurones in the brainstem, the main noradrenergic input to the hypothalamus, were activated by systemic administration of peptide and non-peptide GH secretagogues. In addition, we examined the effects of chronic central noradrenaline depletion upon GH secretagogue-induced activation of the arcuate nucleus. Systemic injection of the GH secretagogues, GHRP-6 and MK-0677 induced Fos protein expression in a population of area postrema cells, but less than 10% of these cells were noradrenergic. Depletion of hypothalamic noradrenaline by the specific neurotoxin, 5-ADMP, did not alter GH secretagogue-induced activation of Fos protein in the arcuate nucleus compared to vehicle-treated controls. We conclude that the central actions of GH secretagogues involve the activation of non-noradrenergic cells in the area postrema and that GH secretagogue-induced activation of the arcuate nucleus occurs independently of noradrenergic tone.

Attenuation of the growth hormone secretagogue induction of Fos protein in the rat arcuate nucleus by central somatostatin action.

We set out to determine whether the central action of growth hormone (GH) secretagogues to induce Fos protein expression in the arcuate nucleus is influenced by central somatostatin action. Conscious male rats were injected i.v. with 100 micrograms sandostatin (octreotide, a long-acting somatostatin analogue) or saline, 10 min before an i.v. injection of either 50 micrograms GH-releasing peptide (GHRP-6), 50 micrograms MK-0677 (a non-peptide GH secretagogue) or saline. In a separate study, conscious male rats were injected i.c.v. with either 2 micrograms sandostatin or artificial cerebrospinal fluid (aCSF) vehicle 20 min before an i.v. injection of 50 micrograms GHRP-6. In all studies, rats were anaesthetized 90 min following GH secretagogue injection, perfused with fixative and the brains processed for the immunocytochemical detection of Fos protein. The number of Fos-positive nuclei detected in the arcuate nucleus of the i.v. sandostatin/i.v. GHRP-6 treated rats (28 +/- 5 nuclei/section) and the i.v. sandostatin/i.v. MK-0677-injected rats (8 +/- 2 nuclei/section) was significantly less than the i.v. saline/i.v. GHRP-6-treated group (56 +/- 5 nuclei/section) and the i.v. saline/ i.v. MK-0677-treated group (20 +/- 2 nuclei/section) respectively. Intracerebroventricular sandostatin injection attenuated the GHRP-6-induced Fos response, from 53 +/- 6 nuclei/section in the i.c.v. aCSF/i.v. GHRP-6 group, to 39 +/- 5 nuclei/section in the i.c.v. sandostatin/i.v. GHRP-6 group. Thus, the central action of GH secretagogues to induce Fos protein expression in the arcuate nucleus appears to be subject to central inhibitory control by somatostatin.

Synthesis and biological activities of camphor-based non-peptide growth hormone secretagogues

The synthesis and growth hormone (GH) releasing activities of a novel series of camphor-based non-peptide GH secretagogues is presented. Use of the (R)-nipecotic acid amino side-chain and N-terminal derivatization of it with an (R)-(2-hydroxy)propyl group provided a potent secretagogue 18 (EC 50 = 90 nM). An o-tolyl piperazine was identified as a good replacement for the spiroindanyl piperidine.

Design and biological activities of L-163,191 (MK-0677): a potent, orally active growth hormone secretagogue.

A potent, orally active growth hormone (GH) secretagogue L-163,191 belonging to a recently synthesized structural class has been characterized. L-163,191 releases GH from rat pituitary cells in culture with EC50 = 1.3 +/- 0.09 nM and is mechanistically indistinguishable from the GH-releasing peptide GHRP-6 and the prototypical nonpeptide GH secretagogue L-692,429 but clearly distinguishable from the natural GH secretagogue, GH-releasing hormone. L-163,191 elevates GH in dogs after oral doses as low as 0.125 mg/kg and was shown to be specific in its release of GH without significant effect on plasma levels of aldosterone, luteinizing hormone, thyroxine, and prolactin after oral administration of 1 mg/kg. Only modest increases in cortisol were observed. Based on these properties, L-163,191 has been selected for clinical studies.

Central Actions of Peptide and Non-Peptide Growth Hormone Secretagogues in the Rat

Evidence for a central site of action of growth-hormone-releasing peptide (GHRP-6) was sought by (1) counting the number of Fos-positive nuclei within the brain following intracerebroventricular or intravenous injection of peptide and non-peptide GH secretagogues and (2) characterizing the electrophysiological responses of neuroendocrine arcuate neurones (recorded in vivo) following intravenous injection of GHRP-6. Conscious male rates were chronically implanted with intracerebroventricular or intravenous catheters. Dense nuclear Fos staining was induced throughout the ventral arcuate nucleus of rats injected intracerebroventricularly with low doses of GHRP-6 but not in rats injected with the endogenous GH-releasing hormone GHRH or in vehicle-treated controls. The non-peptidyl GH secretagogues L-692,585 and L-692,429 also induced Fos expression in the arcuate nucleus, and the pattern of distribution was similar to that described for GHRP-6. No increase in Fos expression was observed in rats given a systemic injection of a high dose of GHRH. In pentobarbitone-anaesthetized male rats, the effects of intravenous injection of GHRP-6 on the electrical activity of arcuate neurones was predominantly excitatory for putative neuroendocrine cells and inhibitory for the remaining unidentified cells. These results suggest that (1) GHRP-6 and non-peptidyl GH secretagogues have a central site of action involving the activation of a subpopulation of arcuate neurones and (2) this action is not mimicked by the central or peripheral effects of GHRH.