Growth hormone-releasing peptide and its analogues

ELSEVIER

Growth Hormone-Releasing Peptide and Its Analogues Novel Stimuli to Growth Hormone Release Marta Korbonits and Ashley B. Grossman GHRPs are oligopeptides with GH-releasing effects in humans when given by either parenteral or oral routes; in addition, nonpeptide pharmacologic analogues have recently been synthesized. Although the exact mechanism of action of these agents 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. GHRPs and their analogues may have a potential role in the treatment of short stature in children or in other situations of GH deficiency, such as adult GH deficiency, obesity, catabolic states, and even normal old age. (Trends Endocrinol Metab 1995;6:43-49)

Although the principal GHRH was not identified and sequenced until 1982 (Guillemin et al. 1982, Rivier et al. 1982), some years prior to this a new series of compounds had been identified, which demonstrated GH-releasing ability at the level of the pituitary (Momany et al. 1981). Such compounds were not apparently major endogenous releasing peptides; however, subsequent research into their nature and activity has led to a number of findings that may well be relevant to clinical practice. This review summarizes recent developments in our understanding of these compounds, known jointly as GHRPs, as well as the current nonpeptide pharmacologic analogues, L-692,429 and L-692,585.

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GHRPs

tide, GHRP-6, stimulates GH secretion in vitro (Badger et al. 1984, Sartor et al. 1985a) and in vivo in a number of different species (Walker et al. 1990, Malozowski et al. 1991), and is also able to stimulate GH release in the human (Ilson et al. 1989, Bowers et al. 1990 and 1992, Hartman et al. 1992). More recent studies have also shown that, in appropri-

The first GHRP identified, GHRP-6 (Figure l), was derived from the pentapeptide Marta Korbonits and Ashley B. Grossman are at the Department of Endocrinology, St, Bartholomew’s Hospital, London EClA 7BE, England.

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met-enkephalin through theoretic lowenergy conformational calculations, computer modeling, structural modification, and biologic studies (Momany et al. 1984). Although such compounds were based on an opioid peptide, the modifications required to induce the specific pituitary GH-releasing activity did not appear to be dependent on opiate receptors (Codd et al. 1988). Subsequent work has concentrated mainly on modifications of this basic hexapeptide structure. The original pep-

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ate doses, GHRP-6 is active at stimulating GH secretion in humans when given orally (Hartman et al. 1992). Analogues of GHRP-6 Following the original synthesis of GHRP6, a second generation of GHRPs was

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developed, initially the heptapeptide GHRP-1 (Ala-His-D-fiNal-Ala-Trp-D-PheLys-NH,) (Bowers et al. 1991a). Two further hexapeptides were then synthesized, GHRP-2 (D-Ala-D-PNal-Ala-Trp-DPhe-Lys-NH,) and “Hexarelin” (His-D2Methyl-Trp-Ala-Trp-D-Phe-Lys-NH,) (Deghenghi et al. 1992, Bowers 1993). Most recently, a number of nonpeptide GH secretagogues have been produced, including the substituted benzolactam, L-692,429 (Cheng et al. 1993) and its 2-hydroxyl propyl derivative L-692,585 (Jacks et al. 1994) (Figure 1). These newer GHRPs and nonpeptide secretagogues appear to be approximately 2-3 times more potent than the original GHRP-6, but in general appear to be otherwise qualitatively similar to GHRP6 in their activity (Bowers 1993) although there are discordant findings [see later here; Wu et al. (1994)]. Molecular mechanisms of action. In rat pituitary cells, GHRH appears to bind to specific GTP-linked receptors in the plasma membrane, activation of which results in the stimulation of adenylate cyclase activity and the consequent generation of CAMP (Lussier et al. 1991). The increase in CAMP leads to the opening of voltage-dependent calcium channels (VDCCs), and the large and rapid increase in intracellular calcium promotes GH release via exocytosis (Lussier et al. 1991). Conversely, the inhibitory effect of somatostatin involves inhibition of adenylate cyclase activity, a fall in intracellular calcium, and a consequent reduction in intracellular calcium concentration (Lussier et al. 1991, Pong et al. 1991, Bilezikjian and Vale 1983). In contrast, although the precise details of the mechanism of action of the GHRPs remain unknown, they do not appear to involve the generation of CAMP (Cheng et al. 1989, Akman et al. 1993). GHRPs induce an increase in intracellular calcium within pituitary cells (Akman et al, 1993), and their activity can be blocked by chelation of extracellular calcium or by the use of calcium channel blockers (Sartor et al. 1985a). GHRP- 1 produces a rapid and sustained increase in intracellular calcium in rat somatotrophs, prob-

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NH2

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(--q

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this does not appear to be the predominant mode of action of the GHRPs, GHRP-6 has been reported to amplify CAMP levels stimulated by GHRH (Cheng et al. 1989). Similar results were also found for the nonpeptide analogue L692,429 by the same group (Cheng et al. 1993), but could not be reproduced with GHRP-1 (Akman et al. 1993), and thus the involvement of CAMP remains speculative. Interest has more recently turned to other second messengers, such as the protein kinase C pathway. It has been suggested that GHRP-6 and L-692,429 may act, at least in part, through protein kinase C (Cheng et al. 1991), although data on GHRP-1 are not concordant with this speculation. Depolarization with a high concentration of potassium chloride stimulates GH release from somatotrophs, whereas somatostatin hyperpolarizes somatotroph membranes. It has been shown that GHRPs also depolarize rat somatotroph membranes, which in

demonstrated after the administration of “Hexarelin” to the infant rat (Locatelli et al. 1994). Thus, most data suggest that the GHRPs and their nonpeptide analogues operate through common mechanisms; this is quite distinct from the CAMP pathway involved in the action of GHRH and does not directly involve the GHRH receptor. Changes in intracellular calcium are involved in this process, but the precise second messenger(s)

II

0

CH3

I

II 0

Figure 1. The structure of GHRP-6 (top panel) and of L-692,429 (bottom panel).

ably occurring via influx of calcium through VDCCs; this rise in intracellular calcium concentration could be blocked by nifedipine, an inhibitor of such channels (Akman et al. 1993). The entry of external calcium through L-type VDCCs appears to be essential for the GHsecretory activity of GHRP-6. It has also been shown that GHRP-I and GHRP-6 are able to depolarize rat somatotroph cell membranes, leading to opening of VDCCs (Pong et al. 1991). Nevertheless, although both GHRH and GHRP-6 appear to enhance transmembrane calcium currents, the peptides may have discordant effects on the kinetic properties of such currents, suggesting that the molecular basis for their activity is quite different and probably involves different second messenger systems (Wu et al. 1994). Somatostatin is equally effective at inhibiting the rise in intracellular calcium induced by either GHRH or GHRP- 1. With regard to CAMP, although

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There is also evidence to suggest that GHRH and GHRP act on different receptor groups on the pituitary cell membrane: (a) GHRP does not compete with GHRH-binding sites in a radioreceptor assay (Thorner et al. 1994). (b) In vitro, competitive antagonists of GHRP and GHRH do not block the response evoked by the other peptide (Cheng et al. 1989, Thorner et al. 1994). [There are certain discordant results: in a recent study it was reported that the release of GH induced by GHRP-2 from bovine pituitary cells was blocked by the specific GHRH antagonist, [Ac-Tyrl,D-Arg*]GHRH (l-29) (Wu et al. 1994). Paradoxically, this GHRH antagonist did not affect the responses to earlier generations of GHRPs.] (c) There is homologous, but not heterologous, desensitization to GHRP and GHRH in vitro (Badger et al. 1984, Sartor et al. 1985b, Blake and Smith 1991, Wu et al. 1994) and in vivo (Clark et al. 1989, Robinson et al. 1992). (d) With the use of the reverse hemolytic plaque assay, GHRP was shown to increase the number of somatotrophs releasing GH without affecting the amount of GH secreted per cell (Goth et al. 1992), whereas GHRH stimulates both the number and amount of secreted GH per cell (Chao et al. 1988). For comparison, somatostatin has been reported to decrease the number of cells secreting GH without affecting the amount of GH secreted per cell. It has clearly been shown that GHRH stimulates not only the release but also the synthesis of GH from pituitary cells. This effect involves CAMP activation but appears to be independent of its GH-releasing activity, as it does not depend on changes in intracellular calcium concentration (Barinaga et al. 1985). There are currently few reports on the effects of the GHRPs on GH synthesis, but in one recent preliminary study an elevation in GH mRNA was

TH2 il 7” ii TH2 H2N\C/c\NH/c\C,NH,c/c\NH~cH,c,NH~cH/c\NH/CH I

turn activates VDCCs (Pong et al. 1991, Akman et al. 1993).

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involved remains

elusive

(Sartor

et al.

1985a, Akman et al. 1993).

Site of action. The original studies on the GHRPs suggested a major mode of action at the level of the pituitary; however, in more recent years it has been suggested that GHRPs also have direct effects on the hypothalamus (Bowers et al. 1991b). Specific binding sites have been demonstrated in both the hypothalamus and pituitary (Codd et al. 1988, Sethumadhavan et al. 1991). The in vitro release of GH by GHRP-6 is greater from hypothalamopituitary incubates than from the isolated pituitary alone (Bowers et al. 1991 b). It has been difficult to identify which site of action is of major importance, but activity at both sites appears to be generally accepted. PITUITARY SITE OF ACTION. All of the different GHRP analogues have been shown to act directly on the pituitary in vitro (Bowers et al. 1984, Akman et al. 1993, Cheng et al. 1993, Wu et al. 1994), although in many instances these effects in vitro were much less than has been seen in vivo, suggesting an additional site of action, presumably involving the hypothalamus. A recent report demonstrated GH-releasing effect from both sparsely granulated (type I) and heavily granulated (type II) rat pituitary cell types in vitro (Lindstrom and Savendahl 1994, P. Lindstrom personal communication 1994). A mode of action at the level of the pituitary is also shown by the stimulatory effect of GHRP-6 on GH release in animals subjected to medial hypothalamic ablation or in hypophysectomized rats bearing pituitary transplants, and also in sheep that had undergone hypothalamopituitary disconnection in vivo (Mall0 et al. 1993, Fletcher et al. 1994). Interestingly, GHRP-6 was reported not to cause GH release in children with pituitary stalk transection, although such release could be induced by GHRH (Hayashi et al. 1993). These results would suggest that the predominant site of action of GHRPs is the hypothalamus, at least in the human. The effect of GHRP is also attenuated by

the simultaneous administration of somatostatin, and augmented by the administration of antisomatostatin antibody in vivo and in vitro (Bowers et al. 1984 and 199 1b). Although somatostatin produces hyperpolarization of somatotrophs and inhibits Ca2+ influx, GHRPs

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produce depolarization and facilitate Ca*+ inflow, suggesting that these agents behave as functional antagonists of somatostatin at the level of the pituitary. SYNERGISM OF GHRPS

WITH GHRH ON

THE PITUITARY. It has been almost univer-

sally found that GHRP synergizes with GHRH in causing GH release in vivo (Bowers et al. 1990, Peiialva et al. 1993a), although it still remains unclear whether the primary synergism occurs at the level of the pituitary or the hypothalamus. Various groups have found that GHRH has merely an additive effect with GHRH on the pituitary in vitro (Sartor et al. 1985a, Blake and Smith 1991, Wu et al. 1994), whereas others have reported direct synergism for both GHRP-6 (Cheng et al. 1989) and L-692,429 (Cheng et al. 1993) on pituitary cells or in hypothalamopituitary stalk-transected pigs (Hickey et al. 1994). An alternative approach to this problem has been the use of the mutant strain little mouse (lit/lit), which has a point mutation in the GHRH receptor and thus does not respond to GHRH. In this model, GHRP-6 is unable to stimulate GH release either in vitro or in vivo. This interesting finding suggests that GHRP-6 requires the presence of activity at GHRH receptors to be effective, although it still leaves open the question of whether this synergism occurs at the level of the pituitary or the hypothalamus. In the dwarf rat, where the precise mutation is currently unknown, GHRPs are still able to elicit a rise in GH (Clark et al. 1989). EFFECT ON THE HYPOTHALAMUS. Although the evidence described here suggests that GHRPs may act, at least in part, at the level of the hypothalamus, there are various possible mechanisms by which they may operate: One early explanation for the hypothalamic mechanism of action of GHRP-6 is that it may release endogenous GHRH by activating opiate receptors. This was suggested by the fact that GHRP-6 was originally derived from met-enkephalin, and that morphine and a variety of opiates, including the en-

dogenous opioid peptides, may stimulate release of GH by activating hypothalamic opiate receptors, while being devoid of GH-releasing activity on the pituitary. Several studies have shown, however, that the opiate antagonist naloxone does not influence the effect of GHRPs on GH

release in vitro (Codd et al. 1988, Sethumadhavan et al. 1991), or in vivo in animals (Sartor et al. 1985b) or in humans (M. Korbonits and G.M. Besser unpublished data). Furthermore, data that show an inverse correlation between opiate receptor binding and GH-releasing activity with a number of GHRP analogues (Codd et al. 1988) as well as the synergistic effects of GHRPs with opioid agonists (Bowers et al. 1991b), also suggest that the GH-releasing effect of GHRPs does not occur via an interaction with opiate receptors. A second possibility is that GHRPs may stimulate the release of GHRH independent of an opioid mechanism. Thus, in the sheep, intravenous injection of “Hexarelin” elevated GHRH levels in hypophyseal portal blood, while no change in portal somatostatin was detected (Guillaume et al. 1994). In addition, systemic or intracerebroventricular administration of GHRP-6 or the nonpeptide GH secretagogues cause activation, in terms of electric excitation and the expression of c-fos, of putative GHRH neurons in the rat arcuate nucleus (Dickson et al., 1995). Furthermore, preliminary results have given indirect evidence that L-692,585 requires the presence of GHRH in order to reveal its effects (Hickey et al. 1994), and antibody against GHRH attenuates the GH-releasing effect of GHRP (Clark et al. 1989, Bowers et al. 199ib). These animal studies are moderately convincing, however, data in the human do not support the notion that the principal effect of the GHRPs is via stimulation of endogenous hypothalamic GHRH. GHRP is able to potentiate GH release in response to a maximally stimulating dose of GHRH (Bowers et al. 1990, Petialva et al. 1993a), whereas in the majority of studies the GH response to the GHRPs alone is considerably greater than that to GHRH (Ilson et al. 1989, Bowers et al. 1990, Petialva et al. 1993a; see also later here). There are also data demonstrating synergism between GHRP and GHRH in animals (Malozowski et al. 1991). Furthermore, results from infant rats have suggested that the effect of “Hexarelin” cannot be abolished with anti-GHRH antibody, demonstrating independence of GHRH in this particular model (Locatelli et al. 1994). Finally, our recent data from rat hypothalamic explants do not indicate that GHRPs are able to acutely stimulate

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maximal doses of GHRH (8 1 f 30 mu/L) (Bowers et al. 1990). Furthermore, the GHRPs are effective via the subcutaneous, intranasal, or oral routes of administration (Hayashi et al. 1991, Bowers et al. 1991a and 1992, Hartman et al. 1992, Ghigo et al. 1994). The bioavailability of some of these drugs, however, particu-

hypothalamuy

Figure 2. Suggested hypothetical mechanisms of GHRPs. Full lines indicate possible stimulatory pathways, and broken lines indicate inhibitory pathways (ss, somatostatin). GHRH release in vitro (M. Korbonits and A.B. Grossman unpublished observations). There

appears

to be little doubt that

GHRPs do not work by inhibition of somatostatin release, as originally suggested (Clark et al. 1989, DeBell et al. 1991) as the release of somatostatin from rat hypothalamic explants is either unchanged (M. Korbonits and A.B. Grossman unpublished data) or even stimulated (Hao et al. 1988); the infusion of GHRP-6 in the human decreases the TSH and PRL responses to TRH, also suggesting a stimulatory rather than an inhibitory role in somatostatin release (Jaffe et al. 1993). Thus, although it is possible that the stimulation of somatostatin is a consequence of increased endogenous GHRH, there is no evidence for GHRPs stimulating GH via the inhibition of somatostatin release. It is clear that GHRH may be necessary for GHRPs to exert their full effect; however, none of the previously suggested mechanisms can fully explain the synergistic effects of GHRH with GHRP. Bowers and colleagues (199Ib) have therefore speculated that the GHRPs stimulate an undefined endogenous hypothalamic factor, the U-factor, which interacts in combination with GHRH on the pituitary to release GH. This factor requires the presence of GHRH in order to exert an effect on the pituitary (Bowers et al. 1991b). The probable existence of specific binding sites in the pituitary and the hypothalamus for the GHRPs also suggests the existence of an endogenous ligand (Codd et al. 1988, Sethumadhavan et al. 1991). This putative

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factor is not a ligand for any GHRP receptor, but must exist as a separate agent. Further studies are clearly needed in order to identify and define such a substance (if it exists). In conclusion, GHRPs almost certainly have a dual site of action, at both the pituitary and the hypothalamus, and appear to stimulate GH release by a specific, non-GHRH receptor mechanism. GHRH is required, however, for the GHRPs to exert their full effects. The hypothesized various mechanisms of action of GHRPs are demonstrated in Figure 2.

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Human Studies

Normals

GHRPs have been found to be potent GH-releasing agents in healthy young males (Ilson et al. 1989, Bowers et al. 1990 and 1992, Hartman et al. 1992), females (Peiialva et al. 1993b), children (Peiialva et al. 1993b, Laron et al. 1993), and in the elderly (Thorner et al. 1994). In contrast to data in the rat (Mall0 et al. 1993) most studies in the human have not been able to show any clear sex differences (Bowers 1993). In healthy young males, intravenous administration of 1 pg/kg of GHRP-6, GHRP-1, GHRP-2, and “Hexarelin” stimulated serum GH levels to peak levels of 137 f 31 mu/L, 132 f 20 mu/L, 156 f 14 mu/L, and 107 f 2 1 mU/L, respectively (mean f SEM) (Bowers et al. 1990, 1991a, and 1994, Ghigo et al. 1994). A 15-min infusion of 1 mg/kg L-692,429 led to a mean peak GH level of 165 f 30 mu/L (Gertz et al. 1993). These levels are considerably higher than those seen after

larly the peptide analogues, are dramatically reduced after oral administration. Chronic administration of the GHRPs causes a sustained rise in serum GH and IGF-I levels (Jaffe et al. 1993), with a maintained elevation of GH pulse amplitude. Similarly, the chronic administration of GHRP to rats resulted in an increase in animal weight, confirming the absence of desensitization with longterm administration (Bowers et al. 1984). As mentioned here, the coadministration of GHRH with GHRP demonstrates a remarkable synergistic effect on GH release (Figure 3). This is also demonstrable in situations of diminished GH secretion, as in the elderly (Thorner et al. 1994), in obesity (Cordido et al. I993), and in children with short stature (Pihoker et al. 1994; see later here). The poor GH response to GHRH in obese patients can be counteracted by coadministration of GHRP (Cordido et al. 1993), but not in patients with Cushing’s disease (Leal et al. 1994). GHRP-6 can stimulate GH release in acromegalic patients, and again synergism is seen with GHRH (Hanew et al. 1994). Effects in Children with Short Stature Owing to the remarkable and sustained effects of the GHRPs on GH release, their potential role as therapeutic agents in children with short stature has been investigated. Most studies have confirmed that GHRPs are potent stimuli to GH release in children with short stature, with the results being more consistent than those seen with other stimuli to GH release (Bowers et al. 1992, Hayashi et al. 1993, Laron et al. 1993, Dieguez et al. 1993, Pihoker et al. 1994), although the response may still be variable, with some children responding to GHRP but not to GHRH or responding to GHRH but not to GHRP (Hayashi et al. 1993). Furthermore, intranasal GHRP-2 has been shown to synergize with intravenous GHRH (Pihoker et al. 1994). In spite of these positive findings, GHRP has been reported to be unable to stimulate GH release in patients with stalk

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transection (Hayashi et al. 1993) suggesting that a further hypothalamic factor is required for GHRP to exert its

0 GHRH 0 GHRP-6 A GHRH+GHRP-6

effect in the human. Alternatively, it is possible that the characteristics of the pituitary cell population change after stalk transection, so that GHRP-responsive cells are no longer present. In children with so-called GH neurosecretory dysfunction, the GH response to GHRP-6 was similar to that in normal children, and greater than in children with idiopathic GH deficiency (Dieguez et al. 1993).

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Adverse Effects and Specificit) Mild clinical adverse effects have been reported with GHRP-6, such as facial flushing and mild sweating (Bowers et al. 1990 and 199ib, DeBell et al. 1991, Hartman et al. 1992), and with “Hexarelin” some slight drowsiness has additionally been reported (Ghigo et al. 1994). No clinical side effects have been reported with GHRP-1 in children (Laron et al. 1993). Although the GHRPs appear to specifically stimulate GH release in rats (Bowers et al. 1984) and monkeys (Malozowski et al. 1991) occasional changes in serum cortisol, ACTH, and PRL have been found in some clinical studies. Thus, activation of the pituitaryadrenal axis has been reported after intravenous administration of GHRP-6, GHRP-1, “Hexarelin,” and L-692,429. A rise in serum PRL has been reported with GHRP-6, GHRP-1, “Hexarelin,” and the nonpeptide compounds as well (Ilson et al. 1989, Bowers et al. 1990, Hayashi et al. 1991, Gertz et al. 1993, Ghigo et al. 1994).

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Diagnostic and Therapeutic Implications

It is well known that the standard tests for GH stimulation (insulin tolerance test, arginine, glucagon, and so on) produce a high percentage of false positive and false negative results and are often considered to be unreliable (Hindmarsh and Brook 1992). Thus, a reliable, easy, and safe provocative test is much needed. GHRPs might have a role on their own or in combination with other stimuli in the diagnosis of GH insufficiency. The ability of the GHRPs to stimulate GH release in children with short stature, with such stimulation also being seen after oral administration of

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Minutes Figure 3. Mean + SEM of GH secretion in five normal subjects challenged on separate days with either (0) GHRH 100 &kg i.v.; or (0) GHFW6 100 &kg i.v.; or (A) GHRH plus GHRP-6 100 @kg i.v. of each. Redrawn and reproduced from Lea1et al. (1994) with the permission of the authors and the publishers.

the nonpeptide analogues, suggests that these agents may play a role in the treatment of short stature. It is particularly important that no evidence of desensitization with prolonged use has been recorded. Enthusiasm should be tempered, however, with the cautionary note that the treatment of children with idiopathic short stature with GH remains controversial, with current data suggesting that any influence on final height attained is unlikely to be major. Nevertheless, the possible use of such agents in other situations of GH deficiency, such as adult GH deficiency, obesity, catabolic states, and even in normal old age, clearly warrants further therapeutic trials.

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Conclusions

Although the pharmaceutical companies are clearly interested in this new class of novel GH-releasing agents, to the neuroendocrinologist they are particularly exciting as probes to the presence of novel hypothalamic GH regulatory factors. Whether such agents interact directly with second messenger pathways or through classic receptor-mediated mechanisms, their elucidation will clearly lead to a quantum leap forward in understanding the growth axis.

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Acknowledgment

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