cp < 0.01 vs peptide alone.

cp < 0.01 vs peptide alone.

tation. The recent important studies by Dickson et al. (36) demonstrate that GHRP-6 acts directly on the hypothalamus in vitro. Recently reported in vitro studies of Korbonits et al. failed to demonstrate that GHRP increased or decreased GHRH or SRIF release from the hypothalamus (39). Furthermore, peptidomimetics of the GHRPs did not induce a reproducible rise of GHRH and/or fall in SRIF hypophyseal portal blood in vivo (40).

Mechanism of action studies by Cheng in 1989 (29) demonstrated that GHRP-6 did not activate the adenyl cyclase cAMP pathway, but together with GHRH, synergistically raised intracellular cAMP levels by acting through the protein kinase C pathway. In 1983, we also reported that neither DTrp2 nor DTrp3 in vitro raised pituitary cAMP or cGMP levels (9). Later results of Adams et al. (41) and Mau et al. (42) demonstrated that although GHRH stimulated the cAMP pathway GHRP-6 stimulated the phospholipase-C IP3 (inositol triphosphate) pathway. In vitro results have supported the role of GHRP as a functional SRIF antagonist at the molecular level in that the peripheral membrane of the somatotroph is depolarized by GHRP by blocking the K+ channels and inhibiting hyperpolarization by SRIF (43,44). Intracellular Ca2+ is raised via voltage-activated L-type channels and by release from intracellular stores (44,45). Recently, details of these studies were discussed by Smith et al. (40) and Chen (46).

In 1989, our group, together with Michael Thorner's and as Ilson et al. at Smith-Kline Beecham, found that GHRP-6 very effectively released GH in normal young men (47,48).

There was a small concomitant transient rise of serum PRL and Cortisol, both of which were still within the normal range. Similar to that found in animal models, i.e., rats, monkeys, and cows, the combined administration of GHRP-6 and GHRH on GH release was synergistic in humans. These results underscore that, in humans also, GHRP and GHRH act differently. Another important property of the GHRPs was revealed when Huhn and Thorner et al. (49) and Jaffe and Barkan et al. (50) independently demonstrated that continuous iv infusion of GHRP-6 administered for 24-36 h to normal young men increased the amplitude of the spontaneous pulsatile secretion of GH. Because the GH response to GHRP-6 was readily desensitized after repeated administration to rats (21), as well as by continuous administration during perifusion of dispersed rat pituitary cells (18), these results in humans were surprising. However, the results of Clark and Robinson in conscious rats suggested that continuous infusion of GHRP-6 to humans might increase the amplitude of the spontaneous GH pulsatility and that this could occur despite desensitization of the GH response (32).

Between 1991-1997, a series of detailed and noteworthy studies were performed with the very potent GHRP-6-like hexapeptide hexarelin, HisD2MeTrpAlaTrpDPheLysNH2 that had been developed by Dengheni et al.: The effects of hexarelin essentially paralleled those of the other GHRPs (51,52). Also, during this time, Walker and Bercu (53) reported the results of chronic administration of GHRP-6 to rats. They investigated the corrected effects of GHRP-6+GHRH co-administration, relationships to endogenous GHRH, TRH and GnRH secretion as well as secretion of PRL, body weight gain, and effect on serum lipids and hepatic mRNA levels for low-density lipoproteins (LDLs).

In 1992, a seminal accomplishment and a major GHRP milestone was the development of a substituted benzolactam peptidomimetic L-692,429 by Merck and Co. (54). This was a special achievement because a peptidomimetic agonist was developed from a peptide agonist. In contrast, the development of a peptidomimetic antagonist from a peptide agonist is not such an unusual event. Undoubtedly this peptidomimetic will catalyze efforts to develop other peptidomimetic agonists that mimic the actions of small peptide hormones. A point of note has been the finding that the peptides and peptidomimetics act on the same receptor and activate GH release by the same intra-cellular signal transduction pathway (55). An important improvement of the benzolactam GH secretagogue was reported by the Merck group in 1995. This spiroindoline derivative [MK-0677 (L-693,191)] is more potent, has higher oral bioavailability («60%) and increases pulsatile GH secretion with an associated increase of serum IGF-I levels during chronic oral administration to normal younger and older subjects (56).

Also, in 1995, highly potent GHRPs were developed by the Genentech (57,58) and Novo Nordisk groups (59,60). These GHRPs were developed primarily from the DTrp2,3 type of GHRP with an aromatic core in the center of the molecule and special functional groups at each end. The Genentech group has reported potent GHRPs that are low in molecular weight ranging from 496 to 508. Gradually, small partial peptide GHRPs are being developed with more substitutions of the amino acids by organic chemical nonpeptide groups. Besides the four or more major types of GHRPs, there are now three major chemical classes of GHRPs, i.e., peptide, partial peptide, and peptidomimetics. Regardless of the broad range of the GHRP SARs, all of them appear to act on the same receptor and by the same molecular mechanism(s). What is different among these GH secretagogues is the pharmacokinetics. In principle, the pharmacokinetics do not alter the action on GH release, but MK-0677, with a more prolonged serum half-life, appears advantageous in terms of increasing pulsatile GH secretion and serum IGF-I levels after oral administration. These same results have been observed with continuous infusion of GHRP-6 and GHRP-2 (49,50,61,62).

In 1996, another seminal milestone was accomplished by the Merck group by cloning the MK-0677 receptor and characterizing it as the GHRP receptor (63). This is a new seven transmembrane domain G-protein coupled receptor. Anatomically it has been localized in the hypothalamic arcuate nucleus and the infundibulum as well as in the pituitary on the somatotrophs. All of the various types and classes of GHRPs specifically bind to the transfected cloned receptor with high affinity. Genomic analysis of the receptor supports the presence of a single highly conserved gene in human, chimpanzee, swine, bovine, rat, and mouse genomic DNA.

The SARs of GHRP strongly support that the putative native GHRP-like hormone is a peptide. Because of the substitution of unnatural D amino acid stereoisomers in the GHRPs, it is probable that the amino acid sequence of the putative native GHRP-like hormone will not closely simulate the sequence(s) of the current peptide GHRPs. In regard to how GHRP releases GH, it is well established that it acts on both the hypothalamus and pituitary (27). What is still unanswered is the relative importance of the action of GHRP at these two anatomical sites as well as the type of action(s) GHRP has on the hypothalamus, i.e., increased GHRH and/or decreased SRIF release or even the seemingly likely possibility of increased release of a yet unidentified factor. It has been postulated that the hypothalamic action of GHRP involves the release of U-factor (unknown factor) which in part mediates its effect on GH release (27). The latter has been proposed because of an inability to explain the action of low dose GHRP via an effect on GHRH or SRIF release or as a functional SRIF-antagonist. Sequential events of the GHRP story also were outlined in 1996 (64).

What has become gradually more apparent is that the type of action(s) induced by GHRP is probably dose dependent. High dosages are considered to reflect a pharmacological action and low dosages presumably a physiological action of a putative endogenous GHRP-like hormone. Conceptual models of the role of the putative GHRP system in the physiological regulation of GH secretion can be categorized in terms of three different types, hypothalamic, pituitary, and hypothalamic-pituitary (27). Because GHRP acts on both the hypothalamus and pituitary, the hypothalamic-pituitary model is the most logical choice, but this model is particularly difficult to envision without knowing more about the hypothalamic action of GHRP and to what degree the quality and quantity of this effect is dosage-dependent.

Unusual and unexpected effects of GHRP in humans have been exemplified by the not infrequent unique actions of this new class of GH secretagogues. Figure 4 shows that each of the three GHRPs, GHRP-6, -1 and -2, increasingly released more GH in normal young men than GHRH when 1 ^g/kg of the peptides was administered by iv bolus injection. Data recorded in Fig. 5 demonstrate another important aspect ofthese three initial GHRPs in that even though they are peptides they very effectively release GH after oral administration in normal young men. Figure 6 shows the high reproducibility and marked effect on GH release of four different oral formulations of GHRP-2 including small tablets at a dosage of 10 mg in normal young men. The low and consistent serum concentration of GHRP-2 after oral administration supports the consistency of the GH effect as well as the high potency of the peptide.

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