GHRP-2

Ipamorelin, the first selective growth hormone secretagogue.

The development and pharmacology of a new potent growth hormone (GH) secretagogue, ipamorelin, is described. Ipamorelin is a pentapeptide (Aib-His-D-2-Nal-D-Phe-Lys-NH2), which displays high GH releasing potency and efficacy in vitro and in vivo. As an outcome of a major chemistry programme, ipamorelin was identified within a series of compounds lacking the central dipeptide Ala-Trp of growth hormone-releasing peptide (GHRP)-1. In vitro, ipamorelin released GH from primary rat pituitary cells with a potency and efficacy similar to GHRP-6 (ECs) = 1.3+/-0.4nmol/l and Emax = 85+/-5% vs 2.2+/-0.3nmol/l and 100%). A pharmacological profiling using GHRP and growth hormone-releasing hormone (GHRH) antagonists clearly demonstrated that ipamorelin, like GHRP-6, stimulates GH release via a GHRP-like receptor. In pentobarbital anaesthetised rats, ipamorelin released GH with a potency and efficacy comparable to GHRP-6 (ED50 = 80+/-42nmol/kg and Emax = 1545+/-250ng GH/ml vs 115+/-36nmol/kg and 1167+/-120ng GH/ml). In conscious swine, ipamorelin released GH with an ED50 = 2.3+/-0.03 nmol/kg and an Emax = 65+/-0.2 ng GH/ml plasma. Again, this was very similar to GHRP-6 (ED50 = 3.9+/-1.4 nmol/kg and Emax = 74+/-7ng GH/ml plasma). GHRP-2 displayed higher potency but lower efficacy (ED50 = 0.6 nmol/kg and Emax = 56+/-6 ng GH/ml plasma). The specificity for GH release was studied in swine. None of the GH secretagogues tested affected FSH, LH, PRL or TSH plasma levels. Administration of both GHRP-6 and GHRP-2 resulted in increased plasma levels of ACTH and cortisol. Very surprisingly, ipamorelin did not release ACTH or cortisol in levels significantly different from those observed following GHRH stimulation. This lack of effect on ACTH and cortisol plasma levels was evident even at doses more than 200-fold higher than the ED50 for GH release. In conclusion, ipamorelin is the first GHRP-receptor agonist with a selectivity for GH release similar to that displayed by GHRH. The specificity of ipamorelin makes this compound a very interesting candidate for future clinical development.

Pralmorelin: GHRP 2, GPA 748, growth hormone-releasing peptide 2, KP-102 D, KP-102 LN, KP-102D, KP-102LN.

Pralmorelin [GPA 748, GHRP 2, growth hormone-releasing peptide 2, KP-102 D, KP 102 LN] is an orally active, synthetic growth hormone-releasing peptide from a series of compounds that were developed by Polygen in Germany and Tulane University in the US. Researchers at Tulane University led by Dr Cyril Bowers synthesised a series of small highly active peptides ranging in size from 3-5 amino acids or partial peptides that were suitable for a variety of administration formats (subcutaneous, buccal, oral, depot). These peptides mimic the actions of ghrelin, a 28 amino acid octanoyl peptide that regulates the release of growth hormone (GH), and may play an important role in bone and muscle growth, food intake and possibly improve recovery from injury. The use of pralmorelin as a diagnostic agent for GH deficiency is based on its ability to markedly increase plasma levels of GH in healthy subjects irrespectively of gender, obesity or age. However, in patients with GH deficiency, the effect of pralmorelin on GH levels is significantly lower compared with healthy controls. Analysis of the receiver-operating characteristics curve provided the cut-off threshold value for the GH peak of 15.0 micro g/L for the identification of patients with GH deficiency from those of healthy controls. Kaken acquired worldwide manufacturing and marketing rights to pralmorelin, and then sublicensed it to Wyeth (formerly American Home Products) for the US and Canada. Kaken retains rights to pralmorelin in Japan. On 11 March 2002 American Home Products changed its name and the names of its subsidiaries Wyeth-Ayerst and Wyeth Lederle to Wyeth. Kaken also granted exclusive sublicense options in Africa, Australia, Europe, Latin America and New Zealand to unspecified partners. Pralmorelin as KP-102 D [KP-102D] is currently awaiting approval in Japan as a diagnostic agent for hypothalamo-pituitary function. It is planned to be launched in Japan for this indication in 2004. Pralmorelin is also undergoing phase II clinical trials with Kaken in Japan for short stature (pituitary dwarfism) as KP-102 LN [KP-102LN]. Its launch for the treatment of short stature is planned for 2009 (Kaken, Annual Report 2003). The agent was undergoing phase II trials in the US for the treatment of GH deficiency with Wyeth; however, it appears that its development was discontinued. Tulane University was granted a US Patent (6,468,974 issued in October 2002), as well as patent protection in Europe and other countries for a series of synthesised GH-releasing peptides.

Growth hormone-releasing peptides.

Growth hormone-releasing peptides (GHRPs) are synthetic, non-natural peptides endowed with potent stimulatory effects on somatotrope secretion in animals and humans. They have no structural homology with GHRH and act via specific receptors present either at the pituitary or the hypothalamic level both in animals and in humans. The GHRP receptor has recently been cloned and, interestingly, it does not show sequence homology with other G-protein-coupled receptors known so far. This evidence strongly suggests the existence of a natural GHRP-like ligand which, however, has not yet been found. The mechanisms underlying the GHRP effect are still unclear. At present, several data favor the hypothesis that GHRPs could act by counteracting somatostatinergic activity both at the pituitary and the hypothalamic level and/or, at least partially, via a GHRH-mediated mechanism. However, the possibility that GHRPs act via an unknown hypothalamic factor (U factor) is still open. GHRP-6 was the first hexapeptide to be extensively studied in humans. More recently, a heptapeptide, GHRP-1, and two other hexapeptides, GHRP-2 and Hexarelin, have been synthesized and are now available for human studies. Moreover, non-peptidyl GHRP mimetics have been developed which act via GHRP receptors and their effects have been clearly demonstrated in animals and in humans in vivo. Among non-peptidyl GHRPs, MK-0677 seems the most interesting molecule. The GH-releasing activity of GHRPs is marked and dose-related after intravenous, subcutaneous, intranasal and even oral administration. The effect of GHRPs is reproducible and undergoes partial desensitization, more during continuous infusion, less during intermittent administration: in fact, prolonged administration of GHRPs increases IGF-1 levels both in animals and in humans. The GH-releasing effect of GHRPs does not depend on sex but undergoes age-related variations. It increases from birth to puberty, persists at a similar level in adulthood and decreases thereafter. By the sixth decade of life, the activity of GHRPs is reduced but it is still marked and higher than that of GHRH. The GH-releasing activity of GHRPs is synergistic with that of GHRH, is not affected by opioid receptor antagonists, such as naloxone, and is only blunted by inhibitory influences, including neurotransmitters, glucose, free fatty acids, gluco corticoids, recombinant human GH and even exogenous somatostatin, which are known to almost abolish the effect of GHRH. GHRPs maintain their GH-releasing effect in somatotrope hypersecretory states such as in acromegaly, anorexia nervosa and hyperthyroidism. On the other hand, their good GH-releasing activity has been shown in some but not in other somatotrope hyposecretory states. In fact, reduced GH responses after GHRP administration have been reported in idiopathic GH deficiency as well as in idiopathic short stature, in obesity and in hypothyroidism, while in patients with pituitary stalk disconnection or Cushing's syndrome the somatotrope responsiveness to GHRPs is almost absent. In short children an increase in height velocity has also been reported during chronic GHRP treatment. Thus, based on their marked GH-releasing effect even after oral administration, GHRPs offer their own clinical usefulness for treatment of some GH hyposecretory states.

Beyond the androgen receptor: the role of growth hormone secretagogues in the modern management of body composition in hypogonadal males.

Male hypogonadism is an increasingly prevalent clinical condition that affects patients' quality of life and overall health. Obesity and metabolic syndrome can both cause and result from hypogonadism. Although testosterone remains the gold standard for hypogonadism management, its benefits are not always conserved across different populations, especially with regards to changes in body composition. Partially in response to this, growth hormone secretagogues (GHS) have emerged as a potential novel adjunctive therapy for some of the symptoms of hypogonadism, although current data on their clinical efficacy largely remain lacking. The present review examines the existing literature on the use of GHS and explores their potential complementary role in the management of hypogonadal and eugonadal males with metabolic syndrome or subclinical hypogonadism (SH). The GHS that will be discussed include sermorelin, growth hormone-releasing peptides (GHRP)-2, GHRP-6, ibutamoren, and ipamorelin. All are potent GH and IGF-1 stimulators that can significantly improve body composition while ameliorating specific hypogonadal symptoms including fat gain and muscular atrophy. However, a paucity of data examining the clinical effects of these compounds currently limits our understanding of GHS' role in the treatment of men with hypogonadism, but does open opportunities for future investigation.

Determination of growth hormone secretagogue pralmorelin (GHRP-2) and its metabolite in human urine by liquid chromatography/electrospray ionization tandem mass spectrometry.

GHRP-2 (pralmorelin, D-Ala-D-(beta-naphthyl)-Ala-Ala-Trp-D-Phe-Lys-NH(2)), which belongs to a class of growth hormone secretagogue (GHS), is intravenously used to diagnose growth hormone (GH) deficiency. Because it may be misused in expectation of a growth-promoting effect by athletes, the illicit use of GHS by athletes has been prohibited by the World Anti-Doping Agency (WADA). Therefore, the mass spectrometric identification of urinary GHRP-2 and its metabolite D-Ala-D-(beta-naphthyl)-Ala-Ala-OH (AA-3) was studied using liquid chromatography/electrospray ionization tandem mass spectrometry for doping control purposes. The method consists of solid-phase extraction using stable-isotope-labeled GHRP-2 as an internal standard and subsequent ultra-performance liquid chromatography/tandem mass spectrometry, and the two target peptides were determined at urinary concentrations of 0.5-10 ng/mL. The recoveries ranged from 84 to 101%, and the assay precisions were calculated as 1.6-3.8% (intra-day) and 1.9-4.3% (inter-day). Intravenous administration of GHRP-2 in ten male volunteers was studied to demonstrate the applicability of the method. In all ten cases, unchanged GHRP-2 and its specific metabolite AA-3 were detected in urine.

Adult growth hormone deficiency: current concepts.

The clinical syndrome of adult growth hormone deficiency (AGHD) was widely recognized in the 1980s. In this review, we first describe the clinical features and diagnosis of AGHD and then state the effects of growth hormone (GH) therapy for these patients. The main characteristics of AGHD are abnormal body composition, dyslipidemia, insulin resistance, and an impaired quality of life (QoL) due to decreased psychological well-being. For diagnosing AGHD, the international consensus guidelines have suggested that an insulin tolerance test (ITT) is the gold standard, but in Japan, the growth hormone releasing peptide-2 (GHRP-2) test is available and is recommended as a convenient and safe GH stimulating test. The cut-off for diagnosing severe AGHD is a peak GH concentration of 9 g/L during the GHRP-2 test. Since 2006, GH therapy has been approved for Japanese patients with severe AGHD. For adults, GH replacement therapy should be initiated at a low dose (3 g/kg body weight/day), followed by individualized dose titration while monitoring patients' clinical status and serum insulin-like growth factor-I (IGF-I) concentrations. A variety of favorable effects of GH replacement have been indicated; however, it has not yet been established fully whether there is a direct effect of GH treatment on reducing mortality.

Growth hormone-releasing peptides and their analogs.

Growth hormone-releasing peptides (GHRPs) are a series of hepta (GHRP-1)- and hexapeptides (GHRP-2, GHRP-6, Hexarelin) that have been shown to be effective releasers of GH in animals and humans. More recently, a series of nonpeptidyl GH secretagogues (L-692,429, L-692,585, MK-0677) were discovered using GHRP-6 as a template. Some cyclic peptides as well as penta-, tetra-, and pseudotripeptides have also been described. This review summarizes recent developments in our understanding of the GHRPs, as well as the current nonpeptide pharmacologic analogs. GHRPs and their analogs have no structural homology with GHRH and act via specific receptors present at either the pituitary or the hypothalamic level. The GHRP receptor has recently been cloned and it does not show sequence homology with other G-protein-coupled receptors known so far. This evidence strongly suggests the existence of a natural GHRP-like ligand which, however, has not yet been found. Although the exact mechanism of action of GHRPs has not been fully established, there is probably a dual site of action on both the pituitary and the hypothalamus, possibly involving regulatory factors in addition to GHRH and somatostatin. Moreover, the possibility that GHRPs act via an unknown hypothalamic factor (U factor) is still open. The marked GH-releasing activity of GHRPs is reproducible and dose-related after intravenous, subcutaneous, intranasal, and even oral administration. The GH-releasing effect of GHRPs is the same in both sexes, but undergoes age-related variations. It increases from birth to puberty and decreases in aging. The GH-releasing activity of GHRPs is synergistic with that of GHRH and not affected by opioid receptor antagonists, while it is only blunted by inhibitory influences that are known to nearly abolish the effect of GHRH, such as neurotransmitters, glucose, free fatty acids, glucocorticoids, rhGH, and even exogenous somatostatin. GHRPs maintain their GH-releasing effect in somatotrope hypersecretory states, such as acromegaly, anorexia nervosa, and hyperthyroidism. On the other hand, GHRPs and their analogs have been reported to be effective in idiopathic short stature, in some situations of GH deficiency, in obesity, and in hypothyroidism, while in patients with pituitary stalk disconnection and in Cushing's syndrome the somatotrope responsiveness to GHRPs is almost absent. A potential role in the treatment of short stature, aging, catabolic states, and dilated cardiomyopathy has been envisaged.

GH releasing peptides--structure and kinetics.

A new class of small synthetic peptides has been developed which specifically release GH. They consist of 6-7 amino acids and release GH in animals as well as humans. So far 3 of these peptides have been administered to humans, i.e., GHRP-6, GHRP-1 and GHRP-2. As in rats, these 3 peptides have been found to be increasingly more effective in releasing GH in humans. All 3 GHRPs release GH more efficaciously than GHRH 1-44 NH2 in humans. Particularly note-worthy is that GHRP-6, GHRP-1 AND GHRP-2 all release GH after oral administration. Near maximal amounts of GH can be released after GHRP-1 and GHRP-2 oral administration. In the present studies, the GH responses and serum irGHRP levels after i.v., s.c. and oral administration have been determined in normal younger men and/or women. By each route of administration GH was very effectively released. Additionally, GH release was induced by oral GHRP-6 in children with various degrees of GH deficiency. Noteworthy is the synergistic release of GH induced by the combined i.v. bolus administration of 1 microgram/kg of GHRP-1 + GHRH 1-44NH2. Thus, these results demonstrate GHRPs' potential importance at the theoretical as well as pharmaceutical level.

Growth hormone-releasing peptides: clinical and basic aspects.

Growth hormone (GH)-releasing peptides (GHRPs), a family of synthetic oligopeptides which stimulate GH release, were identified more than a decade ago. The effects of these peptides on GH release have been described in vivo and in vitro, in both animals and humans, using various doses and administration routes. It is generally accepted that GHRPs stimulate the release of GH by acting at the level of the pituitary through a receptor different to that for the endogenous GH-releasing hormone (GHRH). In addition, it has been reported that there are specific binding sites for these peptides in the hypothalamus and that systemic administration of GHRPs increases the expression of the immediate early gene c-fos in a subpopulation of hypothalamic neurons. However, the identity of these hypothalamic neurons and the mechanism of action of GHRPs at both the hypothalamic and pituitary levels remain unknown. One interesting aspect of GHRPs is that they are orally active and this phenomenon has been demonstrated in both animals and humans. Furthermore, these drugs stimulate GH secretion in humans dose-dependently with the magnitude and duration of this response being comparable to that seen with an intravenous peptide bolus. We have studied the oral activity of GHRP-2 on GH release in normal children. In addition, we have analyzed the response to GHRP-2 of obese adolescents, as well as the effects of an intravenous bolus of GHRH alone and GHRH plus GHRP-2. Orally administered GHRP-2 stimulates GH secretion in normal children and, although it seems that this drug is more potent in girls, there were no statistical differences between the groups. Characteristically, GH levels started to increase by 15 min, peaked at 60 min and returned to basal concentrations by 180 min. The effect of GHRP-2 was synergistic with GHRH 1-29 NH2. In addition, obese subjects appeared to have a greater response to this peptide than did normal controls. To study the effects of GHRPs on hypothalamic GHRH and somatostatin neurons, female dwarf rats (dw/dw) were treated continuously with GHRP-6 (1 mg/kg per 24 h) for 14 days. In situ hybridization for GHRH and SS was performed. We found that GHRP-6 stimulated GHRH mRNA levels in the posterior arcuate nucleus (ARC), with no significant effect in the anterior ARC or ventromedial hypothalamic neurons. SS mRNA levels in the posterior periventricular nucleus (PeN) were decreased after GHRP-6 treatment, while no effect was seen in the anterior PeN, ARC, or lateral paraventricular nucleus. These results suggest that GHRP-6 treatment modulates hypothalamic neurons controlling GH secretion; however, whether this effect is direct or mediated through another factor remains to be elucidated.

Robust growth hormone responses to GH-releasing peptide 2 in adolescents.

GH-releasing peptide-2 (GHRP2) can be used for provocative growth hormone testing (GHT). Since it acts as a powerful stimulus for GH secretion, cut-off peak GH level in GHRP2 loading test (GHRP2T) is higher than in other GHT. Nevertheless, data on response at adolescents are limited. This report aimed to investigate peak GH levels in GHRP2T in adolescents. Clinical data of adolescents after onset of puberty who underwent GHRP2T at our institution from May 2010 to March 2023 were collected retrospectively. Subjects were classified into three groups according to underlying diseases. A total of 23 patients were included: 12 in organic or genetic GHD (o/gGHD) group, three in idiopathic GHD (iGHD) group, and eight in short stature (SS) group. The median GH peak levels were 3.4 ng/mL in o/gGHD group, 88.9 ng/mL in iGHD group, and 90.1 ng/mL in SS group, indicating a robust response of GH peak levels in iGHD and SS groups. Two patients exceeded the cut-off for GHRP2T but below for other GHT, indicating the current cut-off for GHRP2T may miss some GHD patients. The GH response to GHRP2T in adolescents except the o/gGHD group may be robustly responsive. For the correct diagnosis of GHD, the cut-off peak GH levels in GHRP2T in adolescents may require revisiting.

The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3',5'-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs.

The mechanism of action of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on GH release was investigated in ovine and rat pituitary cells in vitro. In partially purified sheep somatotrophs, GHRP-2 and GH-releasing factor (GRF) increased intracellular cyclic AMP (cAMP) concentrations and caused GH release in a dose-dependent manner; GHRP-6 did not increase cAMP levels. An additive effect of maximal doses of GRF and GHRP-2 was observed in both cAMP and GH levels whereas combined GHRP-6 and GHRP-2 at maximal doses produced an additive effect on GH release only. Pretreatment of the cells with MDL 12,330A, an adenylyl cyclase inhibitor, prevented cAMP accumulation and the subsequent release of GH that was caused by either GHRP-2 or GRF. The cAMP antagonist, Rp-cAMP also blocked GH release in response to GHRP-2 and GRF. The cAMP antagonist did not prevent the effect of GHRP-6 on GH secretion whereas MDL 12,330A partially reduced the effect. An antagonist for the GRF receptor, [Ac-Tyr1,D-Arg2]-GRF 1-29, significantly diminished the effect of GHRP-2 and GRF on cAMP accumulation and GH release, but did not affect GH release induced by GHRP-6. Somatostatin prevented cAMP accumulation and GH release responses to GHRP-2, GRF and GHRP-6. Ca2+ channel blockade did not affect the cAMP increase in response to GHRP-2 or GRF but totally prevented GH release in response to GHRP-2, GRF and GHRP-6. These results indicated that GHRP-2 acts on ovine pituitary somatotrophs to increase cAMP concentration in a manner similar to that of GRF; this occurs even during the blockade of Ca2+ influx. GHRP-6 caused GH release without an increase in intracellular cAMP levels. GH release in response to all three secretagogues was reduced by somatostatin and was dependent upon the influx of extracellular Ca2+. The additive effect of GHRP-2 and GRF or GHRP-6 suggested that the three peptides may act on different receptors. In rat pituitary cell cultures, GHRP-6 had no effect on cAMP levels, but potentiated the effect of GRF on cAMP accumulation. The synergistic effect of GRF and GHRP-6 on cAMP accumulation did not occur in sheep somatotrophs. Whereas GHRP-2 caused cAMP accumulation in sheep somatotrophs, it did not do so in rat pituitary cells. These data indicate species differences in the response of pituitary somatotrophs to the GHRPs and this is probably due to different subtypes of GHRP receptor in rat or sheep.

Clinical Usefulness of the Growth Hormone-Releasing Peptide-2 Test for Hypothalamic-Pituitary Disorder.

Growth hormone deficiency (GHD) develops early in patients with hypothalamic-pituitary disorder and is frequently accompanied by other anterior pituitary hormone deficiencies, including secondary adrenal insufficiency (AI). A growth hormone-releasing peptide-2 (GHRP2) test, which is widely used for the diagnosis of patients with GHD, is thought to induce release of not only growth hormone (GH) but also ACTH. However, its clinical usefulness in hypothalamic-pituitary disorder is unclear. We aimed to determine the clinical utility of the GHRP2 test in patients with hypothalamic-pituitary disorders, particularly for AI concomitant with GHD. The GHRP2 test, a cosyntropin stimulation test, corticotropin-releasing hormone (CRH) tests, and/or insulin tolerance tests (ITTs) were performed on 36 patients with hypothalamic-pituitary disorder. Twenty-two (61%) had severe GHD, and 3 (8%) had moderate GHD by GHRP2. There was no difference in baseline ACTH and cortisol between non-GHD, moderate GHD, and severe GHD participants. However, a cosyntropin stimulation test and subsequent CRH tests and/or ITTs revealed that 17 (47%) had secondary AI and 16/17 (94%) cases of secondary AI were concomitant with severe GHD. ROC curve analysis demonstrated that the ACTH response in the GHRP2 test was useful for screening pituitary-AI, with a cutoff value of 1.55-fold (83% sensitivity and 88% specificity). Notably, the combination of ACTH response and the peak cortisol level in the GHRP2 test using each cutoff value (1.55-fold and 10 µg/dL, respectively) showed high specificity (100%) with high accuracy (0.94) for diagnosis of pituitary-AI. We recommend measuring ACTH as well as GH during the GHRP2 test to avoid overlooking or delaying diagnosis of secondary AI that frequently accompanies GHD.

Postoperative growth hormone dynamics in clinically nonfunctioning pituitary adenoma.

Growth hormone deficiency (GHD) is an endocrine disorder characterized by insufficient production of growth hormone (GH). Non-functioning pituitary adenoma (NFPA) is one of common causes of GHD. Although most patients with NFPA have transsphenoidal surgery, the time-dependent changes in GH after operation have yet to be investigated. In this study, we analyzed patients with NFPAs that underwent transsphenoidal surgery. Postoperatively, GH secretion was evaluated in response to GH-releasing peptide-2 (GHRP2) infusion. We also investigated how several factors affected GH dynamics. Of 119 patients analyzed, 94 (79.0%) had peak GH levels less than 9.0 ng/mL and were diagnosed with severe GHD (sGHD) immediately after surgery. Of those patients, 27 (28.7%) recovered from sGHD within 1-2 years after surgery. Univariate analyses confirmed that sGHD recovery improved significantly in patients that were younger, had only undergone a single primary surgery, had not had anterior hormone deficiency except GH, and had cystic adenoma or normal insulin-like growth factor-1 (IGF1) standard deviation score (SD-S) levels immediately after surgery. Multivariate analyses confirmed that younger age and absence of hormone replacement therapy significantly predicted sGHD recovery within 1-2 years after surgery. Taken together, our results indicated that postoperative sGHD should be assessed by GHRP2 infusion, regardless of IGF1 SD-S levels. Furthermore, recovery from sGHD occurs more frequently at 1-2 years after surgery especially in younger patients and/or those with GH deficiency alone. These patients, therefore, should be reassessed for GHD by appropriate tests including GHRP2 test at 1-2 years after surgery.

The effect of GH-releasing peptide-2 (GHRP-2 or KP 102) on GH secretion from primary cultured ovine pituitary cells can be abolished by a specific GH-releasing factor (GRF) receptor antagonist.

A newly synthesised GH-releasing peptide, KP 102 (also named GHRP-2), was studied in an in vitro perifusion system of primary cultured ovine anterior pituitary cells. Application of KP 102 to the perifusion medium caused a dose-dependent increase in GH secretion. Dose-response relationships indicated that KP 102 had similar potency to GRF and was 10-fold more potent than earlier generations of GH-releasing peptide (GHRP-6 and GHRP-1) tested in same system. The response to a second application of KP 102 given within 1 h of initial application was significantly lower than the response to the first application. When KP 102 (or GRF) was applied first and then GRF (or KP 102) given 1 h later, the second response was not attenuated. When GRF and KP 102 were coadministered, an additive effect on release of GH was obtained. The effect of maximal dose of KP 102 (100 nM) on GH release was totally abolished by [Ac-Tyr1,D-Arg2] GRF 1-29 (1 microM) which is believed to be a specific antagonist for the GRF receptor. Blockade of Ca2+ channels by Cd2+ (2 mM) diminished the basal GH secretion and abolished the increase in GH release in response to KP 102 (100 nM). These data suggest that the action of KP 102 is blocked by a GRF receptor antagonist and therefore acts through a different receptor to that employed by earlier generations of GH-releasing peptides. GH release in response to KP 102 involves an increase in Ca2+ influx and there is no cross-desensitization between KP 102 and GRF responses.

Evaluation of Hypothalamic-Pituitary-Adrenal Axis by the GHRP2 Test: Comparison With the Insulin Tolerance Test.

GH-releasing peptide 2 (GHRP2) stimulates the hypothalamic-pituitary-adrenal axis (HPA) through the GH secretagogue receptor (GHSR) in the hypothalamus, in which ghrelin is a natural ligand. Therefore, the GHRP2 test (GHRP2T) could be used instead of the insulin tolerance test (ITT). Can the GHRP2T replace the ITT for evaluation of HPA? The present retrospective study analyzed the clinical features and laboratory data from 254 patients admitted for evaluation of hypopituitarism who underwent both GHRP2T and ITT. We analyzed the association between the maximum cortisol level (Fmax) during both tests. Adrenocortical insufficiency was diagnosed by ITT. The suitability of GHRP2T was examined using the receiver operating characteristic curve. A strong correlation was found between Fmax measured using both tests (r = 0.777, P < 0.0001). However, the sensitivity (64%) and specificity (79%) showed that the GHRP2T was not suitable for clinical use. Various factors influenced the correlation, probably through their effects on ghrelin and/or GHSR, including functional adenoma (P < 0.05) and sex (P < 0.05). No substantial correlation was found between Fmax measured using both tests in patients with prolactinoma (n = 30). The exclusion of patients with functional adenoma revealed no factors that affected the association in male patients; however, age and menstruation significantly influenced it in female patients (P < 0.05). Analysis of the data from male subjects without functional adenoma (n = 104) showed high sensitivity (95%) and specificity (85%) for the GHRP2T. ITT can be substituted with GHRP2T for assessment of HPA in male patients free of functional adenoma.

Detection of black market follistatin 344.

Follistatin, a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA 2019 List of Prohibited Substances and Methods". While currently no approved pharmaceutical formulations of follistatin are available, follistatin can be bought on the black market. Most of the products are labeled "follistatin 344" (FS344), a few "follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only nine of the 17 tested products actually contained follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Surprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western blotting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-follistatin antibody can be used. For immunoprecipitation (IP), a polyclonal anti-follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practically all currently available follistatin standards were investigated. The detection limit of the method for black market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 &#x3bc;L, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous follistatin is possible.

Gateways to clinical trials.

Gateways to Clinical Trials is a guide to the most recent clinical trials in current literature and congresses. The data in the following tables can be retrieved from the Clinical Studies knowledge area of Prous Science Integrity, the drug discovery and development portal, http://integrity.prous.com. This issue focuses on the following selection of drugs: Abacavir sulfate, abarelix, abciximab, acarbose, alefacept, alteplase, amisulpride, amoxicillin trihydrate, apomorphine hydrochloride, aprepitant, argatroban monohydrate, aspirin, atenolol; Betamethasone dipropionate, betamethasone valerate, bicalutamide, bleomycin sulfate; Calcium carbonate, candesartan cilexetil, celecoxib, cetirizine hydrochloride, cisplatin, clarithromycin, clavulanate potassium, clomethiazole edisilate, clopidogrel hydrogensulfate, cyclophosphamide, chorionic gonadotropin (human); Dalteparin sodium, desloratadine, dexamethasone, doxorubicin, DPC-083; Efalizumab, efavirenz, enoxaparin sodium, eprosartan mesilate, etanercept, etoposide, ezetimibe; Faropenem daloxate, fenofibrate, fluocinolone acetonide, flutamide, fluvastatin sodium, follitropin beta, fondaparinux sodium; Gabapentin, glibenclamide, goserelin, granisetron hydrochloride; Haloperidol, hydrochlorothiazide; Imiquimod, interferon beta-1a, irbesartan, iseganan hydrochloride; L-758298, lamivudine, lanoteplase, leflunomide, leuprorelin acetate, loratadine, losartan potassium; Melagatran, metformin hydrochloride, methotrexate, metronidazole, micafungin sodium, mitoxantrone hydrochloride; Nelfinavir mesilate, neutral insulin injection, nizatidine; Olopatadine hydrochloride, omeprazole, ondansetron hydrochloride; Pamidronate sodium, paracetamol, paroxetine hydrochloride, perindopril, pimecrolimus, pioglitazone hydrochloride, piroxicam, pleconaril, pralmorelin, pravastatin sodium, prednisolone, prednisone, propofol; Raloxifene hydrochloride, ranpirnase, remifentanil hydrochloride, risedronate sodium, risperidone, rofecoxib, ropinirole hydrochloride, rosuvastatin calcium; Sevoflurane, sildenafil citrate, simvastatin, somatropin; Tacrolimus, tamoxifen citrate, telmisartan, temozolomide, thiopental sodium, tinzaparin sodium, tirofiban hydrochloride, treosulfan, triamcinolone acetonide; Urokinase; Valsartan, vardenafil, vincristine; Warfarin sodium; Ximelagatran; Zidovudine.

Analysis of new growth promoting black market products.

Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22&#x202f;kDa, the GHRPs are short (3-6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22&#x202f;kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124&#x202f;Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195&#x202f;Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration.

Growth hormone-releasing peptide-2 (GHRP-2) does not act via the human growth hormone-releasing factor receptor in GC cells.

Effect of growth hormone-releasing peptide-2 (GHRP-2) on ovine somatotrophs is abolished by a growth hormone-releasing factor (GRF) receptor antagonist, which raises the possibility that GHRP-2 may act on GRF receptors. In the present study, we used rat pituitary GC cells with or without stable transfection of cDNA coding for the human GRF receptor (GC/R+ or GC/R-) to determine whether or not GHRP-2 acts via the GRF receptor. Northern blot analysis indicated that GRF receptor mRNA was undetectable in GC/R-cells, whereas a high level of expression occurred in GC/R+ cells that were transfected by GRF receptor cDNA. In GC/R- cells, incubation with up to 10(-7)M of either hGRF or GHRP-2 did not alter the intracellular cAMP, [Ca2+]i, or GH secretion. In GC/R+ cells, hGRF (10(-11)-10(-7)M) increased cAMP levels in a concentration-dependent manner up to 20-fold. This increase in cAMP levels was blocked by a GRF receptor antagonist, [Ac-Tyr1, D-Arg2]-GRF 1-29, but not by a Ca2+ channel blocker, NiCl2 (0.5 mM). GH secretion and [Ca2+]i were, however, not increased by hGRF. Incubation of the transfected cells with 10(-1)-10(-8)MGH RP-2 did not modify intracellular cAMP levels. This result suggests that GHRP-2 does not act through the GRF receptor.

Determination of growth hormone releasing peptides metabolites in human urine after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin.

Growth hormone releasing peptides (GHRPs) stimulate secretion of endogenous growth hormone and are listed on the World Anti-Doping Agency (WADA) Prohibited List. To develop an effective method for GHRPs anti-doping control we have investigated metabolites of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin in urine after nasal administration. Each compound was administrated to one volunteer. Samples were collected for 2 days after administration, processed by solid-phase extraction on weak cation exchange cartridges and analyzed by means of nano-liquid chromatography - high resolution mass spectrometry. Six metabolites of GHRP-1 were identified. GHRP-1 in the parent form was not detected. GHRP-1 (2-4) free acid was detected in urine up to 27 h. GHRP-2, GHRP-2 free acid and GHRP-2 (1-3) free acid were detected in urine up to 47 h after administration. GHRP-6 was mostly excreted unchanged and detected in urine 23 h after administration, its metabolites were detectable for 12 h only. Hexarelin and Ipamorelin metabolized intensively and were excreted as a set of parent compounds with metabolites. Hexarelin (1-3) free acid and Ipamorelin (1-4) free acid were detected in urine samples after complete withdrawal of parent substances. GHRPs and their most prominent metabolites were included into routine ultra-pressure liquid chromatography-tandem mass spectrometry procedure. The method was fully validated, calibration curves of targeted analytes were obtained and excretion curves of GHRPs and their metabolites were plotted. Our results confirm that the detection window after GHRPs administration depends on individual metabolism, drug preparation form and the way of administration.

Physiologically based pharmacokinetic model for pralmorelin hydrochloride in rats.

Pralmorelin hydrochloride (pralmorelin), consisting of six amino acid residues, is a growth hormone-releasing peptide. The aim of this study is to analyze the pharmacokinetics of pralmorelin after intravenous bolus administration to rats, and to develop a physiologically based pharmacokinetic (PB-PK) model to describe and predict the concentrations of pralmorelin in blood and tissues. Pralmorelin (3 mg/kg) was administered intravenously to 24 Sprague-Dawley rats. Groups of three rats were sacrificed by decapitation at each designated time point (up to 4 h), and plasma and tissues (brain, lung, heart, liver, kidney, small intestine, muscle, adipose, and skin) were collected. Bile was also pooled until decapitation. The concentration of pralmorelin in samples was determined by liquid chromatography-tandem mass spectrometry. Plasma concentrations of pralmorelin declined rapidly in a biexponential manner. Biliary excretion of pralmorelin was so rapid that 80% of the dose was recovered unchanged in the bile within 1 h after administration. The distribution parameters in each tissue were obtained by using a hybrid model and an integration plot. They revealed that the distribution of pralmorelin into liver was blood flow-limited, and its distribution was permeability-limited in all other tissues. The PB-PK model developed in this study well described the time courses of pralmorelin concentration in the blood and tissues of rats.

Estradiol regulates GH-releasing peptide's interactions with GH-releasing hormone and somatostatin in postmenopausal women.

Estrogen stimulates pulsatile secretion of GH, via mechanisms that are largely unknown. An untested hypothesis is that estradiol (E&#x2082;) drives GH secretion by amplifying interactions among GH-releasing hormone (GHRH), somatostatin (SS), and GH-releasing peptide (GHRP). The design comprised double-blind randomized prospective administration of transdermal E&#x2082; vs placebo to healthy postmenopausal women (n=24) followed by pulsatile GHRH or SS infusions for 13 h overnight with or without continuous GHRP2 stimulation. End points were mean concentrations, deconvolved secretion, and approximate entropy (ApEn; a regularity measure) of GH. By generalized ANOVA models, it was observed that E&#x2082; vs placebo supplementation: i) augmented mean (13-h) GH concentrations (P=0.023), GHRH-induced pulsatile GH secretion over the first 3 h (P=0.0085) and pulsatile GH secretion over the next 10 h (P=0.054); ii) increased GHRP-modulated (P=0.022) and SS-modulated (P<0.001) GH ApEn; and iii) did not amplify GHRH/GHRP synergy during pulsatile GH secretion. By linear regression, E&#x2082; concentrations were found to be positively correlated with GH secretion during GHRP2 infusion (P=0.022), whereas BMI was found to be negatively correlated with GH secretion during GHRH (P=0.006) and combined GHRH/GHRP (P=0.015) stimulation. E&#x2082; and BMI jointly determined triple (combined l-arginine, GHRH, and GHRP2) stimulation of GH secretion after saline (R&#xb2;=0.44 and P=0.003) and pulsatile GHRH (R&#xb2;=0.39 and P=0.013) infusions. In summary, in postmenopausal women, E&#x2082; supplementation augments the amount (mass) and alters the pattern (regularity) of GH secretion via interactions among GHRH, SS, GHRP, and BMI. These outcomes introduce a more complex model of E&#x2082; supplementation in coordinating GH secretion in aging women.

Metabolism of growth hormone releasing peptides.

New, potentially performance enhancing compounds have frequently been introduced to licit and illicit markets and rapidly distributed via worldwide operating Internet platforms. Developing fast analytical strategies to follow these new trends is one the most challenging issues for modern doping control analysis. Even if reference compounds for the active drugs are readily obtained, their unknown metabolism complicates effective testing strategies. Recently, a new class of small C-terminally amidated peptides comprising four to seven amino acid residues received considerable attention of sports drug testing authorities due to their ability to stimulate growth hormone release from the pituitary. The most promising candidates are the growth hormone releasing peptide (GHRP)-1, -2, -4, -5, -6, hexarelin, alexamorelin, and ipamorelin. With the exemption of GHRP-2, the entity of these peptides represents nonapproved pharmaceuticals; however, via Internet providers, all compounds are readily available. To date, only limited information on the metabolism of these substances is available and merely one metabolite for GHRP-2 is established. Therefore, a comprehensive in vivo (po and iv administration in rats) and in vitro (with human serum and recombinant amidase) study was performed in order to generate information on urinary metabolites potentially useful for routine doping controls. The urine samples from the in vivo experiments were purified by mixed-mode cation-exchange solid-phase extraction and analyzed by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution/high-accuracy mass spectrometry. Combining the high resolution power of a benchtop Orbitrap mass analyzer for the first metabolite screening and the speed of a quadrupole/time-of-flight (Q-TOF) instrument for identification, urinary metabolites were screened by means of a sensitive full scan analysis and subsequently confirmed by high-accuracy product ion scan experiments. Two deuterium-labeled internal standards (triply deuterated GHRP-4 and GHRP-2 metabolite) were used to optimize the extraction and analysis procedure. Overall, 28 metabolites (at least three for each GHRP) were identified from the in vivo samples and main metabolites were confirmed by the human in vitro model. All identified metabolites were formed due to exopeptidase- (amino- or carboxy-), amidase-, or endopeptidase activity.

GHRP-2, GHRH and SRIF interrelationships during chronic administration of GHRP-2 to humans.

Studies with chronic GHRP-2 or GHRH administration were performed to demonstrate and better understand the interrelationships between GHRP-2, GHRH and SRIF. Normal younger and older men and women received chronic GHRP-2, GHRH or GHRP-2 + GHRH for 7-30 days. It was demonstrated that chronic administration of either GHRP-2 or GHRH could convert an additive GHRP-2 + GHRH GH response to a synergistic one. In addition, the type of synergistic response induced by chronic GHRP-2 versus GHRH was different. Whether the GH response becomes desensitized during chronic administration depends in part on the dosage and frequency of administration. The potential to learn more about the in vivo actions of GHRP relative to the regulation of GH secretion is underscored by studying the GH responses to GHRP-2, GHRH and GHRP-2 + GHRH.

Association between overweight and growth hormone secretion in patients with non-functioning pituitary tumors.

Growth hormone (GH) deficiency (GHD) is often complicated by non-functioning pituitary tumors (NFPTs); however, its prevalence remains unclear because preoperative screening for GHD with provocative tests is not recommended. Accordingly, we attempted to clarify the characteristics of GHD in unoperated patients with NFPT. We retrospectively reviewed adult patients with non-functioning pituitary adenoma (NFPA) and Rathke's cyst who underwent preoperative GH-releasing peptide-2 (GHRP-2) tests from January 2013 to December 2016. We investigated the association between peak GH response to GHRP-2 and background characteristics. Among 104 patients (85 NFPA and 19 Rathke's cysts), 45 (43%) presented severe GHD, as diagnosed using GHRP-2 tests. Body mass index (&#x3b2; = -0.210, P = 0.007), free thyroxine (&#x3b2; = 0.440, P < 0.001), and tumor height (&#x3b2; = -0.254, P < 0.001) were significant variables for determining the peak GH response to GHRP-2 in multiple regression analyses. Overweight (odds ratio, 3.86; 95% confidence interval, 1.02-14.66) was significantly associated with severe GHD after adjustment for age, sex, creatinine, free thyroxine, tumor height and clinical diagnosis. The regression slopes between tumor height and peak GH response to GHRP-2 significantly differed between overweight patients and non-overweight individuals, as determined by analysis of covariance (P = 0.040). In the 48 patients who underwent postoperative GHRP-2 tests, severe postoperative GHD was significantly more common in overweight patients than non-overweight individuals (100% vs. 48%, P < 0.001). We observed a negative synergistic effect between overweight and tumor size on GH secretion in patients with NFPTs, indicating that GH provocation tests for diagnosing underestimated GHD could be considered in overweight unoperated patients with large NFPTs.

Detection of GHRP-2 and GHRP-6 in urine samples from athletes.

Pharmacotherapy in Cachexia: A Review of Endocrine Abnormalities and Steroid Pharmacotherapy.

Cachexia is a state of increased metabolism associated with high morbidity and mortality. Dysregulation of cytokines and hormone activity causes reduced protein synthesis and excessive protein breakdown. various treatments are available, depending on the primary disease and the patient's state. Besides pharmacological treatment, crucial is nutritional support as well as increasing physical activity. The main purpose of pharmacological treatment is to diminish inflammation, improve appetite and decrease muscle wasting. Therefore a lot of medications aim at proinflammatory cytokines such as Interferon-&#x3b1; or Tumor Necrosis Factor-&#x3b2;, but because of the complicated mechanism of cachexia, the range of targets is very wide. in cachexia treatment, use of corticosteroids is common, which improve appetite, diminish inflammation, inhibit prostaglandin metabolism, Interleukin-1 activity. They can also decrease protein synthesis and increase protein degradation, which can be prevented by resveratrol. Estrogen analogs, progesterone analogs, testosterone analogs, Selective Androgen Receptor Modulators (SARM), Angiotensin-Converting-Enzyme Inhibitors (ACEI), Nonsteroidal anti-inflammatory drugs (NSAIDs), thalidomide, melatonin, Growth Hormone Releasing Peptide-2 (GHRP-2) may play important role in wasting syndrome treatment as well. However, for the usage of some of them, evidence-based recommendations are not available. This review highlights current therapeutic options for cachexia with a specific focus on steroid therapy.

Growth hormone-releasing peptides and the cardiovascular system.

Growth Hormone (GH)-releasing peptides (GHRPs) and their non peptidyl analogues are synthetic molecules which exhibit strong, dosedependent and reproducible GH-releasing activity but also significant PRL- and ACTH/cortisol-releasing effects. An influence of these compounds on food intake and sleep pattern has been also shown. The neuroendocrine activities of GHRPs are mediated by specific receptors subtypes that have been identified in the pituitary gland, hypothalamus and various extra-hypothalamic brain regions with (125)I-Tyr-Ala-hexarelin, an octapeptide of the GHRP family. In addition, GHRP receptors were also present in different peripheral tissues such as heart, adrenal, ovary, testis, lung and skeletal muscle, with a density significantly higher than that found in the hypothalamo-pituitary -system. A remarkable specific (125)I-Tyr-Ala-hexarelin binding was observed in the human cardiovascular system where the highest binding levels were detected in ventricles, followed by atria, aorta, coronaries, carotid, endocardium and vena cava. The binding of the radioligand to cardiac membranes was inhibited by unlabeled Tyr Ala hexare lin and hexarelin as well as by GHRP-6, GHRP-1 and GHRP-2 but not by MK-677, a non peptidyl GHRP analog. In other experiments on H9c2 myocytes, a fetal cardiomyocytes-derived cell line, specific GHRP binding was found and hexarelin showed an anti-apoptotic activity. On the other hand, in vivo studies in animals and in humans showed that GHRPs possess direct cardiotropic actions. In fact, hexarelin protects from ischemia-induced myocardial damage in aged and GH deficient rats while hexarelin shows a positive inotropic effect in normal subjects as well as in patients with GH deficiency. In conclusion, GHRPs possess extra--neuroendocrine biological activity and, particularly, show direct GH-independent cardiotropic effects.

Identification of a novel growth hormone releasing peptide (a glycine analogue of GHRP-2) in a seized injection vial.

Growth hormone releasing peptides (GHRPs) are synthetic peptides with the ability to stimulate human growth hormone (hGH) secretion. Several GHRPs have been developed as drug candidates; however, only one of them, GHRP-2 (Pralmorelin), has received a clinical approval. Nevertheless, they are distributed on the black market and misused by cheating athletes, due to their performance-enhancing effects. Hence, GHRPs have been included in the World-Anti-Doping-Agency's Prohibited List as forbidden substances in sport. Predominantly, analytical methods for detection and unequivocal identification of doping substances are based on mass spectrometry. Therefore, in the present work, a qualitative analysis by liquid chromatography coupled to high-resolution tandem mass spectrometry with a quadrupole time-of-flight analyzer was performed to identify a new heptapeptide (MW&#xa0;=&#xa0;874.02&#xa0;Da) - a glycine analogue of GHRP-2. Structure determination using de novo sequencing is described here in detail. The results of this study may indicate a new approach to circumvent a detection of doping practices.

Growth hormone releasing peptide-2 (GHRP-2), like ghrelin, increases food intake in healthy men.

GHRP-2 is a synthetic agonist of ghrelin, the newly-discovered gut peptide which binds to the growth hormone (GH) secretagogue receptor. Ghrelin has two major effects, stimulating both GH secretion and appetite/meal initiation. GHRP-2 has been extensively studied for its utility as a growth hormone secretagogue (GHS). Animal studies have shown its effect on food intake. However, whether GHRP-2 can also stimulate appetite in humans when administered acutely is not known. We subcutaneously infused 7 lean, healthy males with GHRP-2 (1 microg/kg/h) or saline for 270 minutes and then measured their intake of an ad libitum, buffet-style meal. Similar to what has been reported for ghrelin administration, our subjects ate 35.9 +/- 10.9% more when infused with GHRP-2 vs. saline, with every subject increasing their intake even when calculated per kg body weight (136.0 +/- 13.0 kJ/kg [32.5 +/- 3.1 kcal/kg] vs. 101.3 +/- 10.5 kJ/kg [24.2 +/- 2.5 kcal/kg], p = 0.008). The macronutrient composition of consumed food was not different between conditions. As expected, serum GH levels rose significantly during GHRP-2 infusion (AUC 5550 +/- 1090 microg/L/240 min vs. 412 +/- 161 microg/L/240 min, p = 0.003). These data are the first to demonstrate that GHRP-2, like ghrelin, increases food intake, suggesting that GHRP-2 is a valuable tool for investigating ghrelin effects on eating behavior in humans.

Refractory hypoglycaemia in a localised gastrointestinal stromal tumour: Case report.

GIST and NICTH are mesenchymal in origin however there are very few reports of GIST associated with NICTH which is a para neoplastic syndrome, generally diagnosed when a tumour induced hypoglycaemia is noted. A 46&#xa0;years old female with prime complain of awareness of a mass in the upper abdomen was admitted for evaluation and further management. Detailed investigation revealed the mass to be gastrointestinal stromal tumour. On the day of admission patient was found to be hypoglycaemic which didn't resolve even after 10% glucose infusion. A growth hormone releasing peptide-2 (GHRP-2) assay was carried out which showed an excessive reaction of basal growth hormone however corticotropin releasing hormone (CRH) tests were within normal limits. She was suspected to be Non Islet cell tumour hypoglycaemia (NICTH) and hypoglycaemia resolved upon administering dexamethasone. Later she underwent chemotherapy and surgical resection after which her blood sugar levels were within normal limits. Expression of big IGF-II on the surface of GIST be it metastatic or nonmetastatic can cause refractory hypoglycaemia and can be fatal if left untreated. Clinicians should be aware of refractory hypoglycaemia in patients with large GIST's as glucocorticoid therapy may prove to be extremely useful and lifesaving even before considering any forms of definitive management of the tumour.

Glycine-modified growth hormone secretagogues identified in seized doping material.

A number of unknown pharmaceutical preparations seized by Danish customs authorities were submitted for liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis. Comparison with reference standards unequivocally identified the content of the powders as analogs of the growth hormone secretagogues GHRP-2 (Pralmorelin), GHRP-6, Ipamorelin, and modified growth hormone releasing factor (modified GRF 1-29), which can be used as performance-enhancing substances in sports. In all cases, the detected modification involved the addition of an extra glycine amino acid at the N-terminus, and analytical methods targeting growth hormone secretagogues should hence be updated accordingly.

Treatment effects of intranasal growth hormone releasing peptide-2 in children with short stature.

Growth hormone-releasing peptide (GHRP)-2 is a synthetic six amino acid peptide that is a potent GH secretagogue. Although it shares no structural homology with GH-releasing hormone, in clinical studies its actions on the pituitary release of GH are similar. It is effective when administered orally and intranasally. For children with GH deficiency, such noninvasive treatments are most desirable and in need of development. Fifteen children with short stature participated in this study. All of the children had a height < 2 S.D. below mean for age, poor height velocity, delayed bone age, and low serum concentrations of IGF-1. These children had been tested with standard GH secretagogues, e.g. arginine, insulin, and L-dopa. Fifty percent of the children were GH deficient, the remainder had idiopathic short stature. The children received testing with GHRH and GHRP-2 as an acute i.v. bolus of 1 microgram/kg; all children in this study demonstrated a GH response > 20 micrograms/l. Each child in this study also demonstrated a GH response > 10 micrograms/l in response to intranasal GHRP-2, in the dose range of 5-20 micrograms/kg. The children were administered intranasal GHRP-2, 5-15 micrograms/kg, twice a day for 3 months, then three times a day. Fifteen children participated in the study for 6 months; six of the children have participated for 18-24 months. Height velocity, serum IGF-1, IGF-binding protein 3 (IGFBP-3) and GH-binding protein (GHBP) concentrations, and GH responses to GHRP-2 by i.v. bolus and intranasal spray were examined during treatment. Height velocity increased from 3.7 +/- 0.2 cm/year to 6.1 +/- 0.3 cm/year at 6 months, 6.0 +/- 0.4 cm/year at 18-24 months. There were no significant changes in IGF-1 or IGF-PB3 concentrations, or in acute GH responses to i.v. or intranasal GHRP-2. GHBP concentrations rose significantly, from 439 +/- 63 pmol/l to 688 +/- 48 pmol/l. In this study, intranasal GHRP-2 administration was well tolerated, and produced a modest but significant increase in growth velocity.

Structure-activity relationship for pept&#xed;dic growth hormone secretagogues.

Growth hormone releasing peptides (GHRPs) could be widely used by cheating athletes because they produce growth hormone (GH) secretion, so may generate an ergogenic effect in the body. Knowledge of the essential amino acids needed in GHRP structure for interaction with the target biological receptor GHSR1a, the absorption through different administration routes, and the maintenance of pharmacological activity of potential biotransformation products may help in the fight against their abuse in sport. Several GHRPs and truncated analogues with the common core Ala-Trp-(D-Phe)-Lys have been studied with a radio-competitive assay for the GHSR1a receptor against the radioactive natural ligand ghrelin. Relevant chemical modifications influencing the activity for positions 1, 2, 3, and 7 based on the structure aa-aa-aa-Ala-Trp-(D-Phe)-Lys have been obtained. To test in vivo the applicability of the activities observed, the receptor assay activity in samples from excretion studies performed after nasal administration of GHRP-1, GHRP-2, GHRP-6, Hexarelin, and Ipamorelin was confirmed. Overall results obtained allow to infer structure-activity information for those GHRPs and to detect GHSR1a binding (intact GHRPs plus active metabolites) in excreted urines. Copyright &#xa9; 2016 John Wiley & Sons, Ltd.

Pharmacological characteristics of KP-102 (GHRP-2), a potent growth hormone-releasing peptide.

KP-102 (D-alanyl-3-(2-naphthyl)-D-alanyl-L-alanyl-L-tryptophyl-D-phenylalanyl-L-lysinamide dihydrochloride, growth hormone-releasing peptide-2, GHRP-2, pralmorelin, CAS 158861-67-7), is a potent synthetic growth hormone (GH) secretagogue. In the present study, the pharmacological characteristics of the GH-releasing property of KP-102 were investigated by means of in vivo and in vitro experiments. In conscious rats, the GH-releasing activity of KP-102 was more potent than that of exogenously injected GH-releasing hormone (GHRH). Under pentobarbital anesthesia in which endogenous somatostatin secretion is known to be decreased, KP-102 and GHRH, both showed an almost equivalent GH-releasing potency, which was also similar to that of KP-102 in conscious rats. Besides, KP-102 showed GH-releasing activity in conscious dogs as well, while GHRH failed to increase serum GH levels in conscious dogs. These findings suggest that the GH-releasing activity of KP-102 was less sensitive to GH suppression by endogenous somatostatin as compared with that of GHRH. The GH-releasing activity of KP-102 was completely absent in hypophysectomized rats, but present in median eminence-lesioned rats in which secreted GH amounts were significantly less than those normal rats, indicating necessity of the median eminence (endogenous GHRH) to exert the full activity of KP-102 in GH stimulation. KP-102 directly stimulated GH secretion from cultured rat anterior pituitary cells, although the GH-releasing potency of KP-102 was significantly weaker than that of GHRH in vitro. In conscious rats, KP-102 stimulated the secretion of both adrenocorticotrophic hormone (ACTH) and corticosterone, but not of prolactin. Three weeks administration of KP-102 showed growth-accelerating effect, a slight increase of body weight and wet weight of some organs in both normal and monosodium glutamate (MSG)-treated rats. These results suggest that KP-102 showed specific GH-releasing activity apart from slight ACTH secretion, and that the GH-releasing activity was stable in comparison with that of exogenously injected GHRH.

Is GHRH receptor essential to GHRP-2-induced GH secretion in primary cultured rat pituitary cells?

It is still controversial in rat whether the stimulation of GH secretion by GH-releasing peptides (GHRP) requires both GHRP receptor (GHRP-R) and GH-releasing hormone receptor (GHRH-R). To clarify this issue, we have postulated that inhibition of GHS-R or GHRH-R gene transcription should block GHRP-2-induced GH secretion. Rat pituitary cells were incubated for 3 days in the presence or absence of antisense 18-mer phosphorothiate oligonucleotides (ONs) complementary to the codon region of GHS-R or GHRH-R mRNAs. A significant decrease in GHRH-R and GHS-R mRNA levels was found in corresponding antisense-treated cells compared with the control cells treated with sense ON. Treatment with antisense GHS-R ON reduced (but not abolished) GHRP-2-induced GH secretion although GHRH-induced GH secretion was not altered. GHRH-stimulated GH secretion was totally abolished by the treatment with antisense GHRH-R ON, whereas GHRP-2 induced GH secretion was not affected. Treatment of cells with both GHS-R and GHRH-2 ONs however completely inhibited GHRH and GHRP-2-stimulated GH secretion. These results suggest that GHRH-R is vital for GHRH-induced GH secretion but only partially involved in GHRP-2-stimulated GH secretion under the condition of down-regulation of GHS-R gene transcription.

The role of protein kinase C in GH secretion induced by GH-releasing factor and GH-releasing peptides in cultured ovine somatotrophs.

The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P < 0.001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP-protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP-PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP-PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP-PKA pathway in ovine somatotrophs during the action of GRF or GHRP.

Growth hormone response to GH-releasing peptide-2 in children.

The insulin tolerance test (ITT) has been considered the most reliable test in the diagnosis of growth hormone deficiency (GHD), but it is contraindicated in some patients. Recently, the use of GH-Releasing Peptide-2 (GHRP-2) has been validated and reported as a safe and reliable test for the diagnosis of adult severe GHD. We evaluated the GH response to GHRP-2 in 56 children with growth disorders to assess its efficacy. A dose of 2 microg/kg of GHRP-2 was administered intravenously and serum GH concentrations were determined. The Spearman correlation coefficient for GH peak values indicated a favorable correlation with the ITT (P<0.0001). Peak GH concentrations were significantly (p<0.0001) lower in children with (median: 3.39 microg/l (ng/ml)) than without (25.10 microg/l (ng/ml)) GHD. In the analysis of sensitivity-specificity curves, the serum concentration at the point where sensitivity crosses specificity was 15 microg/l (ng/ml). The GHRP-2 test was safe and required only one hour or less, and was capable of diagnosing GHD in children.

The combined administration of GH-releasing peptide-2 (GHRP-2), TRH and GnRH to men with prolonged critical illness evokes superior endocrine and metabolic effects compared to treatment with GHRP-2 alone.

Central hyposomatotrophism, hypothyroidism and hypogonadism are present concomitantly in men with prolonged critical illness. This study evaluated the impact of combined treatment with GH-releasing peptide-2 (GHRP-2), TRH and GnRH for 5 days compared with GHRP-2 + TRH and with GHRP-2 alone. Thirty-three men with prolonged critical illness participated at baseline compared to 50 age- and body mass index (BMI)-matched controls. Patients were randomly assigned to 5 days of placebo (n = 7), GHRP-2 (1 microg/kg/h; n = 9), GHRP-2 + TRH infusion (1 + 1 microg/kg/h; n = 9) or pulsatile GnRH (0.1 microg/kg every 90 min) together with GHRP-2 + TRH infusion (n = 8). GH, TSH and LH secretion were quantified by deconvolution analysis of serum concentration time series obtained by sampling every 20 min from 2100 to 0600 h at baseline and on nights 1 and 5 of treatment. Serum concentrations of IGF-I, IGFBPs, thyroid hormones, gonadal and adrenal steroids, proinflammatory cytokines and selected metabolic and inflammation markers were measured daily. Patients revealed suppressed pulsatile GH, TSH and LH secretion in the face of low serum concentrations of IGF-I, IGFBP-3 and the acid-labile subunit (ALS) (P < 0.0001 each), thyroid hormones (P < 0.0001) and total and estimated free testosterone (P < 0.0001) levels, whereas free oestradiol (E2) estimates were normal. Serum dehydroepiandrosterone sulphate (DHEAS) levels were also suppressed whereas morning cortisol was normal. Serum levels of type I procollagen (PICP) and bone alkaline phosphatase (sALP) were elevated whereas osteocalcin (OC) was low (P = 0.03). Ureagenesis (P < 0.0001) and breakdown of bone tissue (P < 0.0001) were increased. Baseline serum TNF-alpha, IL-6 and C-reactive protein level and white blood cell (WBC) count were elevated; serum lactate was normal. Only low T4 and high IGFBP-1 levels independently predicted mortality. GHRP-2 infusion reactivated GH secretion and normalized serum IGF-I, IGFBP-3 and ALS. GHRP-2 + TRH infusion reactivated both the GH axis and the thyroid axis, with normal levels of T4 and T3 reached within 1 day. Only GHRP-2 + TRH infusion combined with GnRH pulses reactivated the GH and TSH axis and at the same time increased pulsatile LH secretion compared to placebo. Only GnRH pulses together with GHRP-2 + TRH infusion increased testosterone significantly from day 2 (peak increase of + 312%) through day 5 and serum E2 with > 80% from day 1 through day 3 (all P = 0.05). Ureagenesis was reduced by GHRP-2 + TRH + GnRH (P = 0.01) and by GHRP-2 + TRH (P = 0.009) but not by GHRP-2 alone. Serum OC levels were increased only by GHRP-2 + TRH + GnRH (P = 0.03), with a trend for GHRP-2 + TRH (P = 0.09), but not by GHRP-2 alone. On day 5, serum lactate levels and WBC count were increased by GHRP-2 infused alone and in combination with TRH but not by GHRP-2 + TRH + GnRH. Coadministration of GHRP-2, TRH and GnRH reactivated the GH, TSH and LH axes in prolonged critically ill men and evoked beneficial metabolic effects which were absent with GHRP-2 infusion alone and only partially present with GHRP-2 + TRH. These data underline the importance of correcting the multiple hormonal deficits in patients with prolonged critical illness to counteract the hypercatabolic state.

Growth hormone secretagogues: out of competition.

Growth hormone secretagogues (GHS) constitute a new GH deficiency treatment increasing exponentially in number and improved potency and bioavailability over the last decade. The growth hormone releasing activity makes these compounds attractive for the artificial improvement of the human sports skills, now that recombinant human growth hormone (rhGH) administration is effectively detected. The GHS family is extremely diverse both in number and chemical heterogeneity and keeps growing continuously. In this paper, a general screening test is proposed. To develop a universal method, the single common property of growth hormone secretagogues has been targeted: their capacity to bind to the GHS receptor 1a (GHS-R1a). Pretreated urine samples have been tested in a competition assay where eventually the GHS presence detached a radiolabelled ligand from the receptor in a dose-dependent manner. Blank urine samples were processed to determine potential age, gender and exercise effects, and to define a threshold beyond which a specimen is considered positive. Samples from a growth hormone releasing peptide 2 (GHRP-2) excretion study corroborated the screening assay applicability with a detection window of approximately 4.5 h, and results were confirmed by comparison with a dedicated LC-MS quantification of the intact compound.