GROWTH HORMONE SECRETAGOGUES: FOCUS ON THE GROWTH HORMONE-RELEASING PEPTIDES

Pharmacological Research, Vol. 36, No. 6, 1997

GROWTH HORMONE SECRETAGOGUES: FOCUS ON THE GROWTH HORMONE-RELEASING PEPTIDES VITTORIO LOCATELLIU and ANTONIO TORSELLO Department of Pharmacology, School of Medicine, Uni®ersity of Milan, Milan, Italy Accepted 13 October 1997

This review systematically analyses recent knowledge of the biology of the growth hormone-releasing peptides. Many years before native GHRH had been isolated and sequenced, the synthesis of an enkephalin analog, devoid of any opioid activity but capable of specifically releasing GH from in ®itro pituitaries, prompted the design of a number of structurally interrelated GHRPs with improved GH-releasing activity. Nowadays, GHRPs are the most effective GH-secretagogues known and could be used profitably in humans with GH hyposecretory disturbances to promote a pattern of GH secretion that mimics physiology in a better way than the exogenously administered GH. Q 1997 The Italian Pharmacological Society KEY

WORDS:

GHRPs, GHRP-6, GH, GHRH, somatostatin.

INTRODUCTION: SEARCHING MIMICS FOR A PHYSIOLOGICAL PATTERN OF GH SECRETION The availability of recombinant human growth hormone ŽGH. in unlimited amounts has generated renewed interest in GH therapy. GH replacement may reverse some of the bodily changes that are likely caused by its decline during aging w1, 2x. GH may also be useful for treating GH-deficient children w3x, improving wound healing in burned patients w4x, acting as an adjuvant to gonadotropins to trigger ovulation w5, 6x, preventing osteoporosis w7x, improving bodily functions in GH deficient adults w8x and attenuating the devastating effects of catabolic illnesses w4, 9x. Finally, since GH also stimulates T cell development, it may be of value in the treatment of T cell deficiency syndromes w10x. Current methods for administering GH, however, are not ideal. In fact, since GH is a large polypeptide hormone devoid of oral bioavailability it needs parenteral administration. This results in non-physiologic and sustained plasma GH concentrations, so that GH replacement is therefore frequently associated to side-effects, such as fluid retention, carpal tunnel syndrome and glucose intolerance w11x. Under physiologic conditions, the pituitary secretes GH according to a pulsatile pattern of release that is under a CNS control operated by two hypothalamic neurohormones, GH-releasing hormone ŽGHRH. and somatostatin w12x. Properly delivered GHRH could elicit the pulsatile release of GH in U

Correspondance to: Vittorio Locatelli, Department of Pharmacology, via Vanvitelli 32, 20129 Milano, Italy.

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physiologic amounts, thus optimizing its biologic effects and preventing those side-effects caused by persistently elevated plasma GH levels. Unfortunately, replacement with GHRH is hampered by the short half-life and lack of oral bioavailabilty of the peptide, which makes it mandatory for a parenteral route of administration. In the past few years, the search for compounds alternative to either GH or GHRH, endowed with oral bioavailability and high effectiveness, has resulted in the development of a new family of oligopeptides, named growth hormone-releasing peptides ŽGHRPs.. This family now comprises a number of different short peptidyl and non-peptidyl molecules, some of which have been already proven to be effective in humans.

THE DISCOVERY OF THE GROWTH HORMONE-RELEASING PEPTIDES Many years before native GHRH had been isolated and sequenced w13, 14x, the work by Bowers et al. w15x led to the synthesis of an enkephalin analog, devoid of any opioid activity, which was capable of specifically releasing GH from in ®itro pituitaries w16 x. Though this pentapeptide, Tyr] DTrp] Gly]Phe]Met]NH 2 , had a relatively weak potency as GH releaser and was active only in ®itro, it served as a model for designing new molecules with much greater activity. As described by Momany et al. w17x, the design approach consisted of using ‘structural concepts derived from conformational energy calculations in Q1997 The Italian Pharmacological Society

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conjunction with peptide synthesis and biological activity data’. Unique and key to the succes of this approach, was the insertion of structural data derived from conformational energy calculations into the design cycle before synthesis of the peptide, as well as after obtaining experimental data. By this approach, a number of structurally interrelated GHRPs were designed and these novel molecules showed an improved GH-releasing activity. Among th e m , G H R P -6 Ž H is ] D T r p ] A la ] T r p ] DPhe]Lys]NH 2 . was considered particularly suited because of its potent and specific GH-releasing effect exerted directly on the pituitary. The GH-releasing activity of GHRP-6 was demonstrated both in ®itro and in ®i®o in a wide range of species, i.e. monkeys, sheep, pigs, chicks, steers, rats w18]22x, as well as man w23, 24x. Moreover, GHRP-6 was active orally in rats, dogs, monkeys and humans w25, 26x. Interestingly, GHRP-6 was 5.3]30 times more effective in monkeys than in rats or dogs. In man, 1 m g kgy1 of GHRP-6 administered as an intravenous bolus injection released more GH than the physiological releasing hormone GHRH-44 given at the same dosage and by the same route w24, 27x. In healthy men, comparable amounts of GH were released after i.v. administration of 1 m g kgy1 GHRP-6 or 300 m g kgy1 oral GHRP-6 w26x. In healthy volunteers, marked GH release also occurred after intranasal administration of 30 m g kgy1 of GHRP-6; when 15 m g kgy1 GHRP-6 were administered intranasally every 8 h for 3 days, serum GH and IGF-I levels increased significantly w28x. In these studies, the stimulation of GH release appeared to be specific and without associated adverse effects, the only exception being mild sweating that occurred occasionally. In dogs, a 16-week administration of a huge dose of a GHRP-6 analog, Hexarelin, blunted the GH response to acute Hexarelin administration which, however, was easily restored following treatment withdrawal. Despite this effect, the administration of Hexarelin augmented the indices of spontaneous pulsatility of GH, reduced bone resorption, improved some biochemical and morphological muscular indices, though it did not induce changes of plasma IGF-I levels w29x.

THE STRUCTURE–ACTIVITY RELATIONSHIP A very specific configuration of selected aromatic rings is required for all these peptides to specifically stim ulate G H release. The pentapeptide, Tyr]DTrp]Gly]Phe]Met]NH 2 , served as the starting model for the development of more potent GHRPs. Substitution of tyrosine in position 1 with histidine and the addition of lysine in position 6 resulted in peptides active in ®i®o to release GH. As described by Momany et al. w17x, the in ®itro and in

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®i®o activity results primarily from the charged side chain of the N-terminal histidine and the central aromatic residues. Acetylation of the N-terminus reduces the activity w17x. The C-terminal lysine significantly enhances the in ®i®o activity, but is not a strict requirement for the overall activity, since its substitution does not change significantly the in ®itro activity. GHRP-6 is the prototype starting from which many different analogs ranging from seven to three amino acid residues ŽGHRP-1, GHRP-2, Hexarelin, etc.. have been synthesized and shown to be active also in man. Recently, new peptides with reduced molecular size and complexity have been synthesized in the hope of understanding the minimal and tridimensional structure required for the GHRPs. Among them a pentapeptide ŽG-7039., a tetrapeptide ŽG-7134., a pseudotripeptide ŽG-7502. and a rigid heptapeptide ŽG-7203. were shown to be active at the putative GHRP receptor expressed in anterior pituitary cells. After 45 min of continous exposure to these compounds, homologous desensitization developed, but the cells remained sensitive to GHRH w30x. Another tetrapeptide, named Tetrarelin w31x, was shown to be active in ®i®o. Also cyclic analogs of GHRPs have been developed and shown to be very active both in ®itro and in ®i®o w32x. Although GHRPs were initially introduced as specific GH-secretagogues, small rises in serum cortisol and prolactin ŽPRL. occurring in humans after i.v. infusion, bolus injection or intranasal administration of peptidyl GHRPs were disclosed w24x. Stimulation of GH and PRL secretion from rat primary pituitary cell cultures has also been reported w32x. The real impact of these observations in situations of long-term GHRP therapy is presently unknown. Despite the potential of affecting the secretion of more than one anterior pituitary hormone, at present GHRPs are the most effective GH-secretagogues known and could be beneficially used in humans with GH hyposecretory disturbances to promote a pattern of GH secretion that mimics physiology in a better way than the exogenously administered GH. The strategy of stimulating GH secretion by activating the endogenous pituitary machinery is an attractive alternative that has also been pursued with the synthesis of non-peptidyl mimics of GHRP6, a research that has led to the discovery of a novel series of compounds, the benzolactam secretagogues w33x. A lead compound, biphenylcarboxylic acid ŽL158,077., identified through direct screening of nonpeptidyl templates, stimulated GH release from rat primary pituitary cultures in a dose-dependent manner. Replacement of 29-carboxylic acid with a tetrazole gave the more potent analog L-158,432. Further resolution of the racemic center at C-3 resulted in the isolation of L-692,429, which proved to be the first highly tolerated, non-peptidyl GH secretagogue

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in animal and man and the spiropiperidine L-163,191 ŽMK-677. that is characterized by high oral bioavailability. These newly developed molecules are very effective in eliciting GH release, even if they also stimulate in ®i®o the release of ACTH and PRL and MK-677 also increases fasting blood glucose and insulin concentrations w34]36x. Although their oral bioactivity is much greater than that of the peptidic secretagogues, clinical use of these non-peptidyl molecules is still currently hampered by a lack of understanding of the pharmacodynamic profile required for optimal efficacy.

MECHANISM(S) OF ACTION: CENTRAL VS PERIPHERAL SITE A large body of evidence suggests that GHRPs may act directly on the pituitary, but proof has also been provided that these compounds may exert their action at the hypothalamic level w37]39x. In fact, specific binding sites for GHRPs have been demonstrated in both the pituitary and hypothalamus w40]42x. The identification of the precise siteŽs. and mechanismŽs. of GHRPs’ action is of major importance. From numerous reports in the literature, the greater effectiveness of GHRPs in in ®i®o than in ®itro models suggests a major hypothalamic site of action w37, 43, 44x. This would not contradict the finding that GHRPs are still capable of releasing GH in rats, sheep and pigs bearing surgical disconnection of the hypothalamo]pituitary unit w44]48x. However, in these instances the extent of the GH release elicited by GHRPs is reduced when compared to that occurring in intact animals, depending on the species and time elapsed since the estabilishment of the disconnection. It is of interest, in this context, that GHRPs failed to stimulate GH release in children with transection of the pituitary stalk, which were instead responsive to GHRH w28, 49]51x. Overall, these results would indicate that the hypothalamus is the primary site of action of GHRP-6 at least in humans, rats, sheep and pigs.

The hypothalamic mechanisms In this section we will review, briefly, the possible mechanismŽs. underlying the GH-releasing activity of GHRPs. GHRH In sheep, direct measurement of hypothalamic neurohormones released into the hypophyseal portal blood showed that Hexarelin increased GHRH levels, while no changes were detected for somatostatin w52x. Supporting a GHRH involvement was the finding that GHRPs partially loose their ability to stimulate GH release in adult rats passively immunized against GHRH w38, 53x. Following this line, systemic or intracerebroventricular administration of GHRP-6 or the non-peptide

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analogs caused c-fos accumulation and electric excitation of putative GHRH neurons located in the arcuate nucleus w54x. In contrast, other data would deny the obligatory involvement of GHRH. GHRPs were in fact capable of increasing further in dogs and humans the GH release induced by a maximally effective dose of GHRH w24, 55, 56x and, reportedly, the GH response to GHRPs alone is considerably greater than that to GHRH w24, 27, 55x. Furthermore, we have documented in neonatal rats the complete lack of GHRH and somatostatin involvement in the GH releasing activity of GHRP-6 and Hexarelin w43x and shown a clearcut, though attenuated, GH release following the same peptides also in 10-day-old pups passively immunized against GHRH since birth w43, 47x. Overall, these data indicate that GHRH activation may represent an intermediate, though not obligatory, step of GHRPs action, that an intact hypophyseal stalk is mandatory for a maximal GH response to occur and, finally, that the pituitary component of the action of these compounds is of marginal importance. Somatostatin The involvement of somatostatin in GHRPs’ action is less controversial than that of GHRH. Experiments in adult rats passively immunized against somatostatin have shown that this neurohormone is not directly involved in the mediation of the GHRPs’ action w57x and similar conclusions have been reached in experiments performed in the infant rat w43x. Other experiments would indicate that GHRPs may even stimulate somatostatin secretion from hypothalamic fragments w58x. Somatostatin, however, could play an indirect role by functioning as a GHRP antagonist at the pituitary level w38x. In conscious rats, continuous sub-threshold GHRP-6 infusion along with repeated injections of GHRH induced GH responses that were uniform and greater in magnitude than those of rats given GHRH alone. Furthermore, between repeated GHRH boli, serum GH concentrations remained elevated above baseline suggesting that the GHRP infusion had reduced the inhibitory influences that somatostatin exerts on the pituitary. It has also been shown that in the rat a long acting somatostatin analogue ŽSMS 201-995. given i.c.v. 20 min before centrally administered GHRP-6 completely blocks GH release w59x. Since i.c.v. SMS 201-995 did not affect GH release stimulated by i.v. GHRH, it was surmised that somatostatin may have acted as a functional antagonist of GHRP-6 at a central site. Data obtained in humans also point to GHRP antagonism of somatostatin effects. In fact, the GH-releasing effect of Hexarelin was partly refractory not only to inhibition by substances stimulating hypothalamic somatostatin release but even to a dose of exogenous somatostatin fully effective in suppressing the GH response to GHRH w60]62x. Moreover,

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administration of GH only blunted the GH-releasing activity of Hexarelin, but abolished that of GHRH w63, 64x. In view of the negative GH autofeedback mechanism likely occurring via somatostatin, these results would indicate that Hexarelin counteracted the GH-induced somatostatinergic hyperactivity. Also the results obtained with oral administration of the non-peptidyl analog MK-677 support the hypothesis that these GH secretagogues may functionally antagonize somatostatin action w36x. The U factor Though many reports have clearly shown that GHRH is necessary for GHRPs to exert their full effect, none of them has clearly indicated a mechanismŽs. that could account satisfactorily for the synergistic effect of GHRH and GHRPs. In this connection, Bowers et al. w37x have postulated that stimulation by GHRPs of an unknown endogenous hypothalamic factor Žnamed U factor., which in combination with GHRH would have been able to stimulate the pituitary to release GH. Our own experiments showed the inability of GHRPs to stimulate GH synthesis in rats with hypothalamic ablation w47x and, instead, the stimulatory effects of GHRPs in rats with selective GHRH deficiency w65x favour the existence of a mechanism mediated by an endogenous hypothalamic non-GHRH factor, whose existence and nature are, however, very elusive. Further studies are warranted to demonstrate the actual existence and nature of this mechanism.

The pituitary mechanisms Effects on GH secretion It has been clearly demonstrated that GHRH and GHRPs act on the pituitary through different mechanisms and, likely, via distinct receptors. Somatotrophs that are maximally stimulated with GHRPs can release additional GH in response to GHRH and viceversa w66, 67x. Moreover, homologous, but not heterologous, desensitization is observed after continuos exposure of the pituitary to GHRPs or GHRH w38, 66, 68, 69x. In this vein, further evidence may derive from binding studies. The affinity of GHRPs for the GHRH receptor is in the range of 2 = 10y5 M, thus hardly reconciling with the reported high potency of the GHRPs in ®i®o. At the beginning of the studies with GHRPs it was thought that GHRP-6 was acting on the GHRH receptor because it failed to elicit GH release in the litrlit mouse, an animal model with a point mutation in the GHRH receptor w70x. However, against this view data obtained in cultures of human somatotropinomas showed a GH responsiveness to GHRP-6 in all adenomas, whereas only half of them responded to GHRH w71x. As an alternative possibility, Elias et al. w30x have suggested that despite the presence of mutually exclusive binding sites for GHRH and GHRPs, in the pituitary the post-receptor activity triggered by GHRH may be instrumental to GHRPs action.

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In contrast to the marked synergy observed in ®i®o, only additive or synergistic effects are evident when GHRPs and GHRH are coincubated in ®itro w66, 67, 72x. It is widely recognized that the GH release induced by GHRH is paralleled by an increase in cAMP titers in the pituitary, followed by the opening of voltage-dependent calcium channels ŽVDCCs.. The ensuing rapid increase in intracellular calcium concentrations promotes the release of GH. In contrast, GHRP-6 alone is devoid of such effects on cAMP w67, 69x, but a synergistic increase of cAMP levels occurs when GHRP-6 and GHRH are coadministered w67x. Although the precise definition of the molecular mechanismŽs. subserving the action of GHRPs is still lacking, these compounds elicit an increase in intracellurar calcium derived by an extracellular source, since it can be blocked by the chelation of extracellular calcium or incubation with calcium channel blockers w73, 74x. Accordingly, it has also been shown that GHRP-1 and GHRP-6 induce depolarization of rat pituitary cell membranes, leading to opening of VDCCs w75x and it has been proposed that GHRP-6 and L-692,429 may act, at least in part, through activation of the protein kinase C pathway w76, 77x. More recently, Pong et al. w42x have demonstrated in porcine and rat pituitary membranes a specific binding site for GHRPs, distinct from that for GHRH and with the properties of a new G-protein-coupled receptor and Van der Ploeg et al. w78x have reported the cloning of a receptor for GHRP-6 and non-peptidyl GH secretagogues. Studies on pituitary and hypothalamic membrane preparations have revealed the existence of multiple binding sites with different affinities for GHRPs and non-peptidyl compounds. In the rat pituitary, MK677 would bind preferentially to an high affinity site Žkd in the pM range., whereas medium ŽnM range. and low affinity Ž m M range. sites would primarily bind GHRP-6 w40]42x. In pig pituitaries only the high and low affinity sites were expressed w42, 79x. Similar results were also obtained in hypothalamic membrane preparations. In all, an abundant experimental evidence indicates that the GHRPs and their non-peptidyl analogues operate through common cellular mechanisms, which involve the activation of a receptorŽs. which is not that of GHRH and the activation of intracellular mechanisms different from the cAMP pathways. Although the GHRP receptor Žor, most likely, one of its subtypes. has been cloned in man, rat and swine w78, 80x, we presently ignore the existence and the nature of the purported endogenous GHRP ligand. Effects on GH synthesis The synthesis of GH in the pituitary is mainly under the positive control of GHRH w12x. This effect involves cAMP activation but appears to be independent from changes in

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intracellular calcium concentrations w81x. It was therefore of interest investigating whether GHRPs may play a role not only in the control of GH release but also of its biosynthesis. To date, there are only very few reports about GHRPs effects on GH mRNA levels. In our study, a 5-day treatment with Hexarelin Ž150 m g kgy1 , s.c., twice daily. did not modify GH mRNA levels in the pituitary of normal adult male rats w65x. We repeated a similar treatment schedule in rats with surgical ablation of the mediobasal hypothalamus ŽMBH. and in intact rats with two ectopic pituitaries transplanted under the kidney capsule w82x. Our aim was to assess whether GHRPs effects on GH mRNA levels could be unmasked in an experimental condition of reduced GH mRNA levels. Also in this instance, however, the 5-day Hexarelin treatment failed to increase GH mRNA level in both the pituitary of MBH-ablated rats and in the ectopic pituitaries. A different approach was that of studying the effect of GHRPs treatment in infant rats, in which both GHRP-6 and Hexarelin are very effective stimulators of GH secretion w43, 47, 83x. In these pups a 3]10-day treatment with GHRP-6 or Hexarelin consistently primed the pituitary to the GH response elicited by the acute GHRP challenge, but no effect was detected on pituitary GH mRNA levels. However, in pups treated since birth with an antiserum against GHRH, whose pituitary GH mRNA levels were significantly reduced as compared to controls, a 5-day treatment with GHRP-6 or Hexarelin restored GH mRNA levels to control values ŽFig. 1. w43, 65x. Similarly, in young-adult male rats given an antiserum against GHRH for 15 days, a 10-day treatment with Hexarelin restored to control values the reduced GH mRNA levels ŽFig. 2. w65x. Overall, these findings indicate that GHRPs can increase GH mRNA levels independently of endogenous GHRH; this effect would not be present in normal rats, being possibly masked by the overwhelming action of GHRH. The ineffectiveness of GHRPs to restore GH mRNA levels in the pituitary of rats with surgical disconnection of the hypothalamo]pituitary unit and in the ectopic pituitaries re-emphasizes the concept that GHRPs action is exerted mostly on the hypothalamus and requires the anatomical and functional integrity of the hypothalamo]pituitary unit.

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Fig. 1. Infant rats. Effects of 3, 5 or 10 days of treatment with Hexarelin on GH mRNA levels. Pups were given normal rabbit serum ŽNRS. or an antiserum against GHRH ŽGHRH-Ab. starting from day 1 and then on days 2, 4, 6, 8 and 10. Individual groups of 16 infant rats were treated daily with Hexarelin Ž80 m g kgy1, s.c., b.i.d.. for 10 days Žpost-natal days 1]10., 5 days Žpost-natal days 6]10., and 3 days Žpost-natal days 8]10. or isovolumetric amounts of physiological saline for 10 days Žpost-natal days 1]10; shown as 0 days of Hexarelin treatment .. GH mRNA levels were determined by Northern blot. Data are expressed as the mean " SEM of six determinations. I, NRS; U B, GHRH-Ab. P - 0.05 ®s the indicated group Žreproduced with permission from Ref. w65x..

In ®i®o, the main site of action for ACTH and cortisol stimulation is not the pituitary w30, 87x nor the adrenals because these effects were abolished by stalk transection in the pig w44x or hypophysectomy in the dog w88x, thus indicating a hypothalamic mechanism. Interestingly, some GHRPs mantained their prolactin releasing effect on mammosomatotroph adenomas both in ®itro w89x and in ®i®o w90x. This was not the case for Hexarelin, which did not stimulate the release of prolactin in patients with idiopathic hyperprolactinemia, thus implying the existence of a partial resistance to GHRPs in this condition w90x. The precise mechanismŽs. underlying these effects is still unknown.

EFFECTS ON THE SECRETION OF OTHER PITUITARY HORMONES

CONCLUSIONS

Although predominant, the activity of GHRPs is not fully specific for GH release. It has been reported that peptidyl and non-peptidyl GHRPs have been shown to elicit in ®itro a small but consistent rise of PRL w32x and in ®i®o of PRL, ACTH and cortisol in man w24, 34, 84, 85x, dogs w35x, pigs w68x and rats w86x.

GHRPs are synthetic peptides endowed with a strong GH-releasing activity in a variety of mammalian species, including humans. Their effect is most likely occurring through the interaction with the receptor of an endogenous hypothalamic regulator of GH

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3.

4.

5. 6.

Fig. 2. Young-adult male rats. Effects of 5 or 10 day treatments with Hexarelin on GH mRNA levels. Twenty rats were given NRS or GHRH-Ab Ž500 m lrrat, i.p.. starting on day 1 and then on day 2, 4, 6, 8, 10, 12 and 14. In both treatment groups, five rats were treated daily with Hexarelin Ž80 m g kgy1 , s.c., b.i.d.. for 10 days Žexperimental days 6]15., five with Hexarelin for 5 days Žexperimental days 11]15. and the last 10 with isovolumetric amounts of physiological saline for 10 days Žexperimental days 6]15.. GH mRNA levels were determined by Northern blot. Data are expressed as the mean " SEM of five deU terminations. I, NRS; B, GHRH-Ab. P- 0.05 ®s the Ž indicated group reproduced with permission from Ref. w65x..

7.

8.

9. 10.

11.

secretion, whose nature still remains elusive. Studies on GHRPs’ mechanism of action have led to a better understanding of the multifactorial regulation of the growth hormone axis. Because of a marked GH-releasing effect even after oral administration, GHRPs may prove clinically useful for the treatment of GH hyposecretory states.

12. 13.

14.

ACKNOWLEDGEMENTS Personal studies included in this review were supported by grants from Europeptides, CNR and MURST. The authors wish to thank Drs Romano Deghenghi, Roberta Grilli, Margherita Guidi, Marina Luoni, Francesca Schweiger, Elena Bresciani, Silvano G. Cella and Eugenio E. Muller for their ¨ excellent help in the realization of these researches.

15.

16.

17.

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