Does desensitization to hexarelin occur?

Growth H o r m o n e & I G F Research 1998, 8, 141-143

D o e s d e s e n s i t i z a t i o n to hexarelin occur? A. Rahim, S. M. Shalet Departmentof Endocrinology,ChristieHospitalNHSTrust, Manchester,UK

Summary Hexarelin, a potent growth hormone (GH)-releasing peptide, is capable of causing profound GH release in normal individuals. If the GH response to hexarelin in humans becomes appreciably attenuated following long-term administration, this would seriously limit the potential therapeutic use of hexarelin and similar agents. The effect of twicedaily subcutaneous injections of hexarelin on GH release was therefore investigated over a period of 16 weeks in 12 healthy elderly individuals. The mean (_+ SEM) areas under the GH curve (AUCGH) at weeks 0, 1, 4 and 16 were 19.1 _+2.4, 13.1 _+2.3, 12.3 + 2.4 and 10.5 _+1.8 IJg/I/hour, respectively. There was a significant change in AUCGH over the study period (P = 0.0003). Further analysis showed that the decreases in AUCGHat weeks 4 and 16 were significant (P < 0.05 and P < 0.01, respectively) compared with baseline values. Four weeks after completion of the 16-week study period, hexarelin was again administered. On this occasion, AUCGHincreased significantly compared with that at week 16 (from 10.5 _+1.8 to 19.4 _+3.7 pg/I/hour; P < 0.05) and was not significantly different compared with that at week 0. These results show that attenuation of the GH response after long-term hexarelin therapy is partial and reversible. Key words: GHRP, hexarelin, GH secretion, desensitization.

INTRODUCTION Hexarelin, a potent growth hormone-releasing peptide (CHRP), is capable of causing profound growth hormone (GH) release in normal individuals after oral, intranasal, intravenous and subcutaneous administration. 1 Oral activity, together with the potential ability to induce pulsatile GH release, may make hexarelin a useful therapeutic GH secretagogue. Any appreciable attenuation of the GH response to hexarelin in humans following long-term administration will, however, seriously limit the potential therapeutic use of hexarelin and similar agents. Although hexarelin has been administered for up to 15 weeks in animals 2 and intranasally for a period of 6-10 months in seven prepubertal constitutionally short children, s the longest period to date over which the effects of hexarelinstimulated GH release have been studied in adults is 15 days. 4 The aim of the present study was to assess the effect of twice-daily subcutaneous injections of hexarelin on GH Correspondence to: S. M. Shalet,Departmentof Endocrinology,Christie Hospital NHSTrust,WilmslowRoad,ManchesterM204BX,UK.

1096-6374/98/0B01414-03 $18.00/0

© 1998 Churchill Livingstone

secretion in adults over a period of 16 weeks. The GH response to hexarelin 4 weeks after the 16-week treatment period, and the effects of hexarelin-stimulated GH release on serum levels of insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) were also assessed.

MATERIALS AND METHODS Twelve healthy elderly individuals (eight men, four women) with a median age of 68 years (range, 66-81 years) and a median body mass index of 26 kg/m a (range, 19.6-30.1 kg/m 2) were included in the study. Written informed consent was obtained from each individual, and the study was approved by the South Manchester Medical Research Ethics Committee. Before entry into the study, volunteers underwent a screening test. At 07.30-08.00 hours, after an overnight fast, an intravenous cannula was inserted into a vein in the antecubital fossa. Basal blood samples were taken for the determination of serum levels of IGF-I and IGFBP-3. A stimulation test using subcutaneous hexarelin, administered at 0 minutes at a dose of 1.5 btg/kg body © 1998 Churchill Livingstone

142 A. Rahim and S. M. Shalet

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The mean (+ SEM) AUCGH at weeks 0, 1, 4 and 16 were 19.1 +_2.4, 13.1 +_2.3, 12.3 _+2.4 and 10.5 ± 1.8 gg/l/hour, respectively (Fig. 2). The change observed in AUCcH over the study period was significant (P = 0.0003). Further analysis showed that the reductions in AUCcH seen at weeks 4 and 16 were significant (P < 0.05 and P < 0.01, respectively) compared with baseline values. At week 20, that is 4 weeks after completion of hexarelin therapy, the response to hexarelin was again measured. AUCcH increased significantly compared with AUCcH at

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weight, was then carried out, with blood samples for GH estimation being taken at -10, 0, 10, 20, 30, 40, 50, 60, 90, 120, 170 and 180 minutes. Only those individuals with a peak GH response greater than 6 gg/1 were included in the study. A peak GH response of at least 6 gg/1 was considered mandatory, as any potential reduction in GH response could be demonstrated only in those who showed a definite initial GH response to hexarelin. Individuals who were entered into the study then attended the clinic for further assessments on five occasions (Fig. 1). The procedure on each occasion was identical to that at the pre-study screening. Following the first (baseline) assessment, each individual received subcutaneous injections of hexarelin at a dose of 1.5 gg/kg body weight twice daily for 16 weeks, with the dose adjusted according to body weight at each study visit. Compliance was 100%, as injections were given by a visiting nurse. For statistical purposes, the peak GH response and area under the GH curve (AUCcH) at week 0 were taken to be the mean of the respective values obtained at the prestudy screening and the baseline study visits. Statistical analyses were performed using parametric tests. Analysis of variance with repeated measures was used to analyse the peak GH response, AUCGH, s e r u m IGF-I and serum IGFBP-3. If a significant difference had occurred over the study period, Tukey's test was used to determine between which time points significant changes had occurred. A P value of less than 0.05 was considered statistically significant.

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week 16 (from 10.5 _+ 1.8 to 19.4 _+ 3.7 gg/1/hour; P < 0.05) and was not significantly different compared with that at week 0 (19.1 + 2.4 gg/1/hour). The mean peak GH responses at weeks 0, 1, 4 and 16 were 15.9 _ 2.0, 12.8 _+2.1, 11 _ 2.0 and 10.1 _+ 1.8 gg/1, respectively. The change observed in peak GH response over the study period was significant (P = 0.006). Further analysis showed that only the reduction seen in peak GH response at week 16 was significant (P < 0.05) compared with baseline. Four weeks after completion of hexarelin therapy, the peak GH response increased significantly compared with the response at week 16 (from 10.1 _+ 1.8 to 15.3 +_ 2.9 pg/1; P < 0.05), and was not significantly different compared with that at week O. Serum IGF-I and IGFBP-3 levels did not change significantly over the 20-week study period. DISCUSSION

Hexarelin is capable of causing profound GH release in normal individuals after oral administration. ~,4 A powerful orally active GH secretagogue with the ability to cause pulsatile GH release has obvious advantages over subcutaneous GH and GH-releasing hormone (GHRH) analogues. The potential therapeutic use of hexarelin, and of similar agents, will be determined, however, by their GH-releasing capacity following chronic administration. In the present study, it has been shown that subcutaneous hexarelin, administered twice daily for a period of 16 weeks, resulted in an attenuated GH response, as evidenced by a reduction in both the peak GH response and AUCGH. Importantly, however, this reduction in hexarelin-stimulated GH release was partial, with all but one individual still releasing GH after 16 weeks of therapy. Four weeks after completion of therapy, both hexarelinstimulated AUCcH and peak GH response values had

Does desensitization to hexarelin occur? 143

increased and were not significantly different c o m p a r e d with values at week 0, suggesting that the m e c h a n i s m responsible for the attenuated GH response is not only partial but also reversible. A similar partial and reversible attenuated GH response to hexarelin has b e e n demonstrated b y Klinger et al. 3 w h o administered hexarelin intranasally, three times a day, to seven prepubertal constitutionally short children for 6 - 1 0 months. At the completion of hexarelin therapy, the m e a n peak GH response to intravenous hexarelin had decreased b y about 75% c o m p a r e d with baseline. Three m o n t h s after completion of hexarelin therapy, the m e a n peak GH response h a d increased to 50% of pretreatment levels. By c o m p a r i s o n , Ghigo et al. 4 were u n a b l e to demonstrate a change in AUCGH following 8 clays of intranasal administration of hexarelin in seven normal elderly individuals. Differences in dose (which was three times higher in the paediatric study 3 t h a n the study in adults 4) and duration of administration (6-10 m o n t h s 3 c o m p a r e d with 8 days 4) m a y account, in part, for the difference between the results obtained b y Klinger et al.3 and Ghigo et al., 4 as the route of administration was the same in b o t h studies. Ghigo et al.4 also f o u n d that the response to oral hexarelin in seven elderly w o m e n was not attenuated over 15 days of treatment. This finding is in agreement with the results presented here, where there was no significant reduction in the GH response after 1 week of therapy. The m e c h a n i s m for the decreased GH release in response to chronic hexarelin t h e r a p y remains unclear. It is likely to occur via a n u m b e r of mechanisms, including negative feedback from increased circulating GH levels. Administration of exogenous GH has b e e n s h o w n to attenuate the GH response to hexarelin, 5,s suggesting that hexarelin-stimulated GH release is subject to partial feedback inhibition b y the action of GH on somatostatin and/or GHRH. Cella and colleagues demonstrated an increased pulse frequency and amplitude of GH release following c h r o n i c s u b c u t a n e o u s a d m i n i s t r a t i o n of hexarelin twice daily in six 11- to 17-year-old beagle dogs. 2 This suggests an overall increase in circulating GH levels, which, t h r o u g h a negative-feedback effect at the hypothalamic-pituitary level, may, in part, be responsible for the attenuated GH response reported here. IGF-I acts directly at the pituitary level to inhibit basal and GHRH-induced GH secretion, and also to suppress GH gene expression. 7-1° IGF-I also appears to act at the h y p o t h a l a m i c level, where it increases somatostatin secretion. ~ In the present study, it is unlikely that IGF-I p l a y e d a m a j o r role in t h e r e d u c e d GH r e s p o n s e following chronic hexarelin therapy, as attenuation of the GH response occurred without a n y significant alteration

in circulating IGF-I levels. It is more likely that downregulation of receptor and post-receptor mechanisms, as seen after prolonged stimulation with GHRH 12 and GHRP-613 in humans, is responsible for the decrease in hexarelin-stimulated GH release. As the attenuation of the GH response after long-term hexarelin therapy, however, is partial and reversible, hexarelin and similar GH secretagogues m a y be useful therapeutic agents. REFERENCES

1. Ghigo E, Arvat E, Gianotti Let al. Growth hormone-releasing activity of hexarelin, a new synthetic hexapepfide, after intravenous, subcutaneous, intranasal, and oral administration in man. J Clin Endocrinol Metab 1994; 78: 693-698. 2. Cella SG, Cerri CG, Daniel Set al. Sixteen weeks of hexarelin therapy in aged dogs: effects on the somatotropic axis, muscle morphology, and bone metabolism. J Gerontol A Biol Sci Med Sci 1996; 51: B439-B447. 3. Klinger B, Sflbergeld A, Deghenghi R, Frenkel J, Laron Z. Desensitization from long-term intranasal treatment with hexarelin does not imerfere with the biological effects of this growth hormone-releasing peptide in short children. Eur J Endocrinol 1996; 134: 716-719. 4. Ghigo E, Arvat E, Gianotti Let al. Short-term administration of intranasal or oral hexarelin, a synthetic hexapeptide, does not desensitize the growth hormone responsiveness in human aging, Eur J Endocrinol 1996; 135: 407-412. 5. Massoud AF, Hindmarsh PC, Brook CG. Hexarelin induced growth hormone release is influenced by exogenous growth hormone. Clin Endocrinol (Oxf) 1995; 43: 617-621. 6. Arvat E, Di-Vito L, Gianotti Let al. Mechanisms underlying the negative growth hormone (GH) autofeedback on the GHreleasing effect of hexarelin in man. Metabolism 1997; 46: 83-88. 7. Ceda GP, Davis RG, Rosenfeld RG, Hoffman AR. The growth hormone (GH)-releasing hormone (GHRI-I)-GH-somatomedin axis: evidence for rapid inhibition of GHRH-elicited GH release by insulinqike growth factors I and II. Endocrinology 1987; 120: 1658-1662. 8. Ceda GP, Hoffman AR, Silverberg GD, Wilson DM, Rosenfeld RG. Regulation of growth hormone release from cultured human pituital 3, adenomas by somatomedins and insulin. J Clin Endocrinol Metab 1986; 60: 1204-1209. 9. Yamashita S, Melmed S. Insulinqike growth factor I regulation of growth hormone gene transcription in primary rat pituitary cells. J Clin Invest 1986; 79: 449-452. 10. Yamashita S, Weiss M, Melmed S. Insulin-like growth factor I regulates growth hormone secretion and messenger ribonucleic acid levels in human pituitary tumor cells. J Clin Endocrinol Metab 1986; 63: 730-735. 11. BerelowitzM, Szabo M, Frohman LA et aL Somatomedin-C mediates growth hormone negative feedback by effects on both the hypothalamus and the pituitary. Science 1981; 212: 1279-1281. 12. Davis JR, Sheppard MC, Shakespear RA, Lynch SS, Clayton RN. Does growth hormone releasing factor desensitize the somatotroph? Interpretation of responses of growth hormone during and after 10-hour infusion of GRF 1-29 amide in man. Clin Endocrinol (Oxf) 1986; 24: 135-140. 13. Huhn WC, Hartman ML, Pezzoli SS, Thorner MO. Twenty-fourhour growth hormone (GH)-releasing peptide (GHRP) infusion enhances pulsatile GH secretion and specifically attenuates the response to a subsequent GHRP bolus. J Clin Endocrinol Metab 1993; 76: 1202-1208.