EP-51216

The search for inappropriately high growth hormone (GH) titers in plasma has been widely used to detect GH abuse, despite many shortcomings especially related to the pulsatile nature of GH secretion. Hence, the need for new anti-doping strategies. In the present study dogs were used to evaluate the ability of recombinant human GH (rhGH) to affect canine GH (cGH) release ensuing after somatostatin (SS) infusion withdrawal (SSIW) – a purported stimulus for the release of endogenous GH-releasing hormone (GHRH) – or the cGH response to administration of a GH-releasing peptide (GHRP). In the SSIW experiments, 8 beagle dogs of either gender (4–6 years old) were given a subcutaneous bolus injection of physiological saline (0.1 ml/kg) or, alternatively, rhGH (0.2 IU/kg s.c.) 60 min before the starting a continuous infusion of SS (4 µg/kg g h i.v.) of 1.5 h duration. In the dogs given a saline bolus, SSIW was followed by a ‘rebound’ rise in plasma cGH levels. In contrast, in dogs which had received the bolus injection of rhGH, the cGH rise elicited by SSIW was completely abrogated. In the set of experiments with a GHRP challenge, 13 dogs of either gender (3–12 years old) received the following treatment schedule at least 15 days apart: (1) a single bolus injection of rhGH (0.2 IU/kg s.c.); (2) rhGH (0.05 IU/kg s.c.) daily for 12 days; (3) rhGH (0.2 IU/kg s.c.) on alternate days for 12 days, and (4) rhGH (0.2 IU/kg s.c.) daily for 12 days. For each treatment schedule, before treatment, during treatment (24 h from the previous rhGH injection) and 1, 5 and 10 days after treatment, all dogs received an intravenous injection of a GHRP, EP51216 (125 µg/kg). In all treatments under baseline conditions, a single injection of EP51216 elicited an abrupt rise in plasma cGH. Twenty-four hours after the injection of an acute bolus of rhGH, the C_max and AUC_0–90 of the GHRP-stimulated cGH response were significantly lower than the baseline cGH response. Five days later, there was a trend in the C_max and AUC_0–90 towards complete restoration of the original values. One, 5 and 10 days after the end of the daily treatment with rhGH (0.05 IU/kg s.c.), no significant changes in the GHRP-stimulated cGH responses vs. the baseline GH response were recorded. In contrast, treatment with rhGH at a dose of 0.2 IU/kg s.c., on either alternate or daily administration, markedly reduced the GHRP-stimulated cGH responses evaluated after 3 and 5 rhGH injections. One day after the last rhGH injection, the EP51216-stimulated cGH response was still significantly reduced when compared with that present under baseline conditions. Five and 10 days following termination of rhGH treatment on alternate days, no significant differences in the C_max and AUC_0–90 of the cGH responses to EP51216 were present. Differently, following the end of daily rhGH treatment, a marked inhibition in the C_max of the cGH response to EP51216 was still present at 1 and 5 days, though not at 10 days. In conclusion, these studies show that a single administration of rhGH can abrogate the cGH response ensuing SSIW or acute stimulation by a GHRP. The inhibitory effect of rhGH on the cGH response to GHRP is present even 5 days after termination of a short-lived treatment with rhGH at a dose (0.2 IU/kg) which, in the dog, is undoubtedly lower than that used in humans for doping purposes. Extrapolation of these preclinical results to humans may pave the way for the development of a new rhGH anti-doping test.

GH-releasing peptides (GHRPs), a class of small synthetic peptide and non-peptide compounds, act on specific receptors at both the pituitary and the hypothalamic level to stimulate GH release in both humans and other animals. GHRPs, like corticotropin-releasing hormone (CRH), also possess acute ACTH- and cortisol-releasing activity, although the mechanisms underlying the stimulatory effect of GHRPs on the hypothalamo-pituitary-adrenal (HPA) axis are still unclear. In recent years, studies in humans and other animals have provided evidence that the rebound GH rise which follows withdrawal of an infusion of somatostatin (SS) (SSIW) is due, at least in part, to the functional activation of GH-releasing hormone (GHRH) neurons of the recipient organism. Unexpectedly, in humans, SS infusion, at a dose inhibiting basal GH secretion, has been associated with an activation of the HPA axis, leading to the hypothesis that this response was mediated, at least in part, by a central nervous system ACTH-releasing mechanism activated by the SS-induced decrease in GH secretion. Interestingly, the rebound GH rise which follows SSIW was magnified by the administration, before SS withdrawal, of a GHRP, implying that the SSIW approach could also be exploited to investigate in vivo the functional interaction in the process of GH and/or ACTH/cortisol secretion between endogenous GHRH (and/or other ACTH-releasing mechanisms) and GHRPs. In the present study, six young beagle dogs were given, on different occasions, at the beginning and at the end of a 3-h i.v. infusion of SS or saline (SAL), a bolus of physiological SAL or a GHRP compound, EP51216. SSIW induced a GH rebound rise without affecting plasma cortisol concentrations, while the withdrawal of SAL infusion was ineffective on either hormone paradigm. Administration of EP51216 at the beginning of SAL infusion evoked release of both GH and cortisol, whereas EP51216 administration at the withdrawal of SAL infusion evoked somatotroph and cortisol responses which were reduced in amplitude and duration. SS infusion significantly reduced the secretion of GH elicited by EP51216 but did not affect the rise of plasma cortisol levels. Interestingly, SSIW resulted in a marked enhancement of the somatotroph and cortisol responses evoked by EP51216. The marked rise of plasma GH levels induced by the GHRP after SSIW recalled that occurring after acute combined administration of recombinant human GHRH and EP51216, implying that exogenously delivered GHRP had synergized with the endogenous GHRH release triggered by SSIW. In contrast, acute combined administration of GHRH and the GHRP induced a cortisol response not different from that induced by GHRP alone, indicating that endogenous GHRH release was not involved in the enhanced cortisol response following EP51216 administration after SSIW. Similarly, the direct involvement of endogenous CRH could be ruled out, since i.v. administration of ovine CRH after SSIW evoked cortisol peak levels not different from those evoked by CRH at the withdrawal of SAL infusion. In conclusion, enhancement of the GH response to EP51216 alone by SSIW, to an extent reminiscent of that following combined administration of GHRH and EP61216, reinforces the view that SSIW elicits release of endogenous GHRH. Further studies are indeed necessary for a better understanding of the mechanisms underlying the enhanced cortisol response, since from now on the involvement of endogenous GHRH or CRH can be ruled out.