GHRP-2 Research & Studies

Dried blood spots (DBSs) sampling is gaining support by the antidoping community because of simplicity and cost-effective characteristics, especially in collection, transport, and storage. Nevertheless, DBS applicability demands specific studies for each of the analytes proposed for testing. Here, GHRP-2 has been selected as a representing member of the growth hormone-releasing peptides (GHRPs) family to provide further evidence of DBS suitability for GHRPs abuse detection in sport testing. An analytical procedure to extract GHRP-2 and its main metabolite (AA-3) from DBS and to detect them by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. The method has been validated for the detection of GHRP-2. Specificity and identification capabilities have been assessed in agreement with antidoping guidelines. The low AA-3 levels found in DBS samples prevented its effective application for the determination of this metabolite. The limit of detection (LoD) for GHRP-2 has been established at 50 pg/ml. Long-term stability (>2 years) has been confirmed. The procedure has been successfully applied to actual DBS samples from an administration study with a single intravenous dose of GHRP-2 (100 μg) being detected up to 4 h after drug injection. GHRP-2 concentrations have been higher in venous blood DBS than in capillary blood DBS. Despite the observed differences, a similar detection window has been achieved independently of the type of blood used. In summary, this study provides specific evidence supporting DBS usefulness to detect GHRP-2, and potentially other GHRPs family members, for antidoping tests.

The objective of this study was to investigate the effects of growth hormone-releasing peptide-2 (GHRP-2) and cysteamine (CS) administration on growth performance in yaks with growth retardation and try to elucidate its regulatory mechanisms. Trial 1, thirty-six 1-year-old Qinghai high plateau yaks (body weight 38-83.2 kg) were randomly chosen for body weight and jugular blood samples collection. The relationship between body weight and serum GHRH (P < 0.05, R = 0.45), GH (P < 0.05, R = 0.47), IGF-1 (P < 0.05, R = 0.62) was significantly correlated in yaks colonies with lighter body weights. Trial 2, fifteen 1-year-old Qinghai high plateau yaks with growth retardation (average body weight 54.8 ± 8.24 kg) were randomly selected and assigned to negative control group (NG), GHRP-2 injection group (GG) and cysteamine feeding group (CG), with 5 yaks per group. Another five 1-year-old Qinghai high plateau yaks with normal growth performance (average body weight 75.3 ± 2.43 kg) were selected as positive control group (PG). The average daily gain (ADG) of the GG and CG were significantly higher than those in the PG and NG (P < 0.05). Both GHRP-2 and CS administration significantly enhanced the myofiber diameter and area of skeletal muscle (P<0.05). GHRP-2 significantly enhanced the serum GH and IGF-1 levels (P < 0.05), and up-regulated GHR, IGF-1 and IGF-1R mRNA expression in the liver and skeletal muscle (P < 0.05), enhanced the mRNA expression of PI3K, AKt and mTOR in the skeletal muscle (P<0.05). CS significantly reduced the serum SS levels and the hypothalamus SS mRNA expression (P < 0.05), and enhanced GHR and IGF-1 mRNA expression in the liver (P < 0.05), decreased the mRNA expression of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) mRNA (P < 0.05).

GHRP-2 is a synthetic agonist of ghrelin receptor. GHRP-2 has similar physiological functions with ghrelin. In our previous study, ghrelin (i.c.v.) could induce analgesic effect through an interaction with GHS-R1α and with the central opioid system in the acute pain in mice. To date, the function of GHRP-2 in pain processing was not understood. Therefore the aim of this study was to investigate the effects of GHRP-2 on pain modulation at supraspinal level in mice using the tail immersion test. Intracerebroventricular (i.c.v.) administration of GHRP-2 (0.1, 0.3, 1, 3 and 10 nmol/L) produced a concentration- and time-related antinociceptive effect. This effect could be fully antagonized by GHS-R1α antagonist [d-Lys(3)]-GHRP-6, indicating that the analgesic effect induced by GHRP-2 is mediated through the activation of GHS-R1α. Interestingly, naloxone, naltrindole and nor-binaltorphimine, but not β-funaltrexamine, could also block the analgesic effect markedly, suggesting that δ- and κ-opioid receptor is involved in the analgesic response evoked by GHRP-2. Moreover, i.c.v. administration of GHRP-2 potentiated the analgesic effect induced by morphine (i.c.v., 1 nmol/L) and this potentiated effect could not be reversed by [d-Lys(3)]-GHRP-6. Thus these findings may be a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation. Furthermore, GHRP-2 may be a promising peptide for developing new analgesic drugs.

The arginine + GHRH test has been established as an alternative to the insulin tolerance test (ITT) for the diagnosis of adult GH deficiency (AGHD). However, the glucagon, arginine, and GH releasing peptide-2 (GHRP-2) test are recommended as alternatives in Japan. The objective of this study was to evaluate the arginine and GHRP-2 tests as alternatives to the ITT for the diagnosis of AGHD in a Japanese population. Three stimulation tests (ITT, arginine test, and GHRP-2 test) were conducted in 71 pre-operative adult patients with pituitary tumors (age, 18-65 years). The peak GH responses to each test were examined. The peak GH responses were significantly lower with the ARG test (median 4.43 μg/L) (p < 0.0001) than with the ITT (median 9.38 μg/L), and the peak GH responses with the GHRP-2 test (median 28.88 μg/L) were higher (p < 0.0001). However, among the AGHD patients, there was no significant difference between the peak GH responses to the ARG test and the ITT. The sensitivities and specificities of the ARG / GHRP-2 tests compared to the ITT for the diagnosis of severe AGHD (peak GH responses to ITT ≤ 1.8 μg/L) were 93.8% / 81.3% and 85.5% / 94.5%, respectively. The arginine and GHRP-2 stimulation tests are acceptable alternatives to the ITT for the diagnosis of AGHD in Japanese patients. The method and criterion for the diagnosis of AGHD should be reconsidered and adjusted to each population.

Black market products of a pharmaceutical nature and nutritional supplements have received substantial and increasing attention because of potential performance enhancement in elite and non-professional sports. In addition, improved general health is claimed for non-competing individuals. The risks and foreseeable dangers of the uncontrolled use of highly potent and non-approved pharmaceutical compounds in healthy individuals are of considerable concern. In the present case report, the emerging drug candidate GHRP-2 with verified growth-hormone-releasing properties was identified and quantified in tablets offered as an over-the-counter nutritional supplement. The impact of this orally active peptide on the hGH/IGF-axis has been established for several years and its illicit use in elite sports has been assumed. As a releasing factor for hGH, GHRP-2 belongs to the list of substances prohibited by the World Anti-Doping Agency (WADA). Unfortunately, to date there is no routinely performed assay for the determination of these peptides potentially occurring in biological fluids of competing athletes, but the present data will facilitate the implementation by providing principle analytical information on liquid chromatographic and mass spectrometric behaviour. Qualitative identification of the target analyte after extraction from the tablet matrix was performed by high resolution/high accuracy mass spectrometry after liquid chromatographic separation under consideration of the accurate masses and the ratios of the protonated molecules and their fragment ions derived from their collisionally induced dissociation. Quantitative results were obtained by means of liquid chromatography coupled to a triple quadrupole mass spectrometer and linear regression using an external calibration curve (with GHRP-2 reference compound) adjusted via internal standard (Hexarelin). Hereby, the content of GHRP-2 was determined with approximately 50 µg per tablet.

Sex steroids are prominent regulators of pulsatile GH secretion.

Recent reports suggest that Atrogin-1 and MuRF1, E3 ubiquitin ligases, play a pivotal role in muscle atrophy. In the present study, effect of Growth Hormone Releasing Peptide-2 (GHRP-2), a GH secretagogue receptor (GHS-R) agonist, on the expressions of Atrogin-1 and MuRF1 in vivo rat muscles was examined. Dexamethasone administration increased Atrogin-1 mRNA level in rat soleus muscle. The increased mRNA level of Atrogin-1 was significantly attenuated by GHRP-2. In addition, GHRP-2 decreased MuRF1 mRNA level irrespective of the presence of dexamethasone. Although IGF-I is a well-known protective factor for muscle atrophy, GHRP-2 did not influence plasma IGF-I levels and IGF-I mRNA levels in muscles. To clarify a direct effect of GHRP-2, differentiated C2C12 myocytes were used. Ten micrometer dexamethasone increased both Atrogin-1 and MuRF1 mRNA levels in C2C12 cells. GHRP-2 attenuated dexamethasone-induced expression of them dose-dependently and decreased the basal level of MuRF1 mRNA. The suppressive effect on the expressions of Atrogin-1 and MuRF1 by GHRP-2 was blocked by [D-Lys(3)]-GHRP-6, a GHS-R1a blocker, suggesting the effect of GHRP-2 was mediated through GHS-R1a. Taken together, GHRP-2 directly attenuates Atrogin-1 and MuRF1 mRNA levels through ghrelin receptors in myocytes.

Chronic arthritis induces hypermetabolism and cachexia. Ghrelin is a gastrointestinal hormone that has been proposed as a treatment to prevent cachexia. The aim of this work was to examine the effect of administration of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) to arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with GHRP-2 (100 microg/kg) or with saline for 8 days. Arthritis induced an increase in serum ghrelin (P < 0.01) and a decrease in serum concentrations of leptin (P < 0.01), whereas GHRP-2 administration increased serum concentrations of leptin. GHRP-2 increased food intake in control rats but not in arthritic rats. However, in arthritic rats GHRP-2 administration ameliorated the external symptoms of arthritis, as it decreased the arthritis score (10.4 +/- 0.8 vs. 13.42 +/- 0.47, P < 0.01) and the paw volume. In addition, circulating IL-6 and nitrites/nitrates were increased by arthritis, and GHRP-2 treatment decreased the serum IL-6 levels (P < 0.01). To elucidate whether GHRP-2 is able to modulate IL-6 release directly on immune cells, peritoneal macrophage cultures were incubated with GHRP-2 or ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor. Both GHRP-2 (10(-7) M) and ghrelin (10(-7) M) prevented endotoxin-induced IL-6 and decreased nitrite/nitrate release from peritoneal macrophages in vitro. These data suggest that GHRP-2 administration has an anti-inflammatory effect in arthritic rats that seems to be mediated by ghrelin receptors directly on immune cells.

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.

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 (GH)-releasing hormone (GHRH) and GH-releasing peptides (GHRPs) stimulate the release of GH through their specific receptors on somatotropes. Combined GHRH and GHRP administration causes a synergistic GH release in vivo by an unknown mechanism. The current study focuses on the direct action of GHRH and GHRP on several molecular targets in somatotropes.

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.

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.

There is a difference between the sheep and rat somatotrophs in the response to growth hormone-releasing peptide-2 (GHRP-2), which raises the question of what the response may be in human somatotrophs. In the present study, cells were obtained from seven human acromegalic tumours and the effects of GHRP-2 were studied. Cells were dissociated and kept in primary culture for 1-3 weeks before experimentation. Application of GHRP-2 for 30 min induced a significant increase in GH secretion from the cultured cells from all seven tumours whereas human GH-releasing hormone (hGHRH) at a dose of 10 nM induced a significant GH release in only four of seven tumours. The intracellular levels of cAMP in all seven tumours were significantly increased by both 10 nM GHRP-2 and GHRH, but the response to GHRH was significantly higher than the response to GHRP-2. The adenylyl cyclase inhibitor, MDL 12330A, blocked the effect of GHRH and GHRP-2 on intracellular cAMP levels, whereas the Ca2+ channel blocker Co2+ (0.5 mM) did not attenuate the cAMP response. For the tumours in which GH secretion was increased by GHRH and GHRP-2, the cAMP antagonist Rp-cAMP blocked the GH response to GHRH but not to GHRP-2. When a protein kinase A (PKA) inhibitor (H89) was applied, GHRH stimulated GH release was blocked, but cAMP accumulation was not affected. The response to GHRP-2 was not altered by H89. Calphostin C [a protein kinase C (PKC) inhibitor] reduced the effect of GHRP-2 on the secretion of GH but did not affect the response to GHRH. Both GHRH and GHRP-2 increased the intracellular Ca2+ concentration in a concentration-dependent manner. We conclude that (1) GHRH increases GH secretion from human GH tumours via the cAMP pathway whereas GHRP-2 increases GH secretion mainly via the PKC pathway; (2) GHRH increases cAMP (without GH release) in a subset of tumours whereas GHRP-2 increases cAMP levels (slightly) and GH secretion in all tumours; and (3) GHRP-2 and GHRH do not act on the same receptor on human somatotrophs derived from acromegalic tumours.

In the present study, the effect of GHRP-2 on GH release was evaluated in rat primary pituitary cells, and the results were compared with those elicited by GHRP-6. In the rat system, GHRP-2, like GHRP-6, acts synergistically with GRF to release GH. Co-administration of GHRP-2 and GHRP-6 at maximal concentrations had no further effect on GH release than either one alone. The GHRP's were able to desensitize cells to each other, but not to GRF. The effect of GHRP-2 was inhibited by Peptide Antagonist, but was not affected by a GRF antagonist. In conclusion, GHRP-2 was found to stimulate GH release from rat pituitary cells via the same receptor and mechanism as GHRP-6, despite the structural difference between the peptides.

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.

The newly synthesized GH-releasing peptide, GHRP-2 (D-Ala-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2), was studied in somatotroph-enriched populations of ovine pituitary cells in primary culture. Nystatin-perforated whole-cell recordings were made on identified somatotrophs after 4-14 days of culture. Using a standard bath solution (containing Na+, Ca2+) and an electrode solution containing K+ in current-clamp recordings, GHRP-2 (10 nM) depolarized the membrane potential of the cells triggering a burst of action potentials. Voltage-clamp recordings indicated that GHRP-2 produced a slowly inactivated inward current with a slight reduction in outward current. The inward current was blocked by the Ca2+ channel blocker, Co2+ (0.5 mM). Ca2+ currents were then isolated using tetraethylammonium bath solution and an electrode solution containing Cs+. Ovine somatotrophs possess transient (T type) and long lasting (L type) Ca2+ currents. The L type current was abolished by addition of nifedipine (3 microM) to the bath solution and T type current was isolated on this basis. Current-voltage relationships indicated an increase in both T and L type Ca2+ currents in response to GHRP-2. The voltage-dependent inactivation curve for T type Ca2+ current was shifted towards a less negative level by the peptide. Intracellular free Ca2+ concentration ([Ca2+]i) in somatotroph-enriched populations was specifically increased by GHRP-2 but this effect was totally abolished by blockade of membrane Ca2+ channels. These data show that GHRP-2 causes an influx of Ca2+ leading to an increase in [Ca2+]i in ovine somatotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)