Immune enhancing effect of a growth hormone secretagogue.

Growth hormone (GH) has been known to enhance immune responses, whether directly or through the insulin like growth factor-1, induced by GH. Recently a nonpeptidyl small m.w. compound, a GH secretagogue (GHS), was found to induce the production of GH by the pituitary gland. In this study, we examined the effect of GHS in immunological functions of 5- to 6-wk-old and 16- to 24-month-old mice. In young mice, we observed a significant increase in PBLs, but T and B cell-proliferative responses were not consistently enhanced. The old mice, treated with GHS for 3 wk, did not show increases in peripheral lymphocytes, but they exhibited a statistically significant increase in thymic cellularity and differentiation. When inoculated with a transplantable lymphoma cell line, EL4, the treated old mice showed statistically significant resistance to the initiation of tumors and the subsequent metastases. Generation of CTL to EL4 cells was also enhanced in the treated mice, suggesting that GHS has a considerable immune enhancing effect, particularly in the old mice. We have also found that GHS promoted better thymic engraftment in bone marrow transplant of SCID mice. We found more cycling cells in the spleens of treated mice, suggesting that GHS may exert its immune enhancing effect by promoting cell division in lymphoid cells. These observations ascribe to GHS a novel therapy possible for aging, AIDS, and transplant individuals, whose immune functions are compromised.

Nipecotic and iso-nipecotic amides as potent and selective somatostatin subtype-2 receptor agonists.

N-Substituted nipecotic and iso-nipecotic amides of beta-methylTrpLys tert-butyl ester were found to be novel, selective and potent agonists of the somatostatin subtype-2 receptor in vitro. For example iso-nipecotic amide 8a showed high hsst2 binding affinity (Ki = 0.5 nM) and good selectivity (h5/h2 = 832).

Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to humans.

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.

Thiazole-derived potent, highly bioavailable short duration growth hormone secretagogues.

Replacement of the phenyl in 3 with a 2-pyridyl or 4-thiazolyl group resulted in increased potency in the rat pituitary cell GH release assay and in beagles.

Potent, orally bioavailable somatostatin agonists: good absorption achieved by urea backbone cyclization.

Backbone cyclization of urea-based somatostatin agonists resulted in novel, orally bioavailable agonists. Binding assays confirmed that the resulting conformationally constrained cyclic ureas retained the potency of their acyclic counterparts. SAR studies subsequently led to highly potent analogs, selective for receptor subtype 2, and having good oral bioavailability.

Increases in circulating insulin-like growth factor I levels by the oral growth hormone secretagogue MK-0677 in the beagle are dependent upon pituitary mediation.

It has been well established that the spiroindoline sulfonamide MK-0677 stimulates GH secretion from the pituitary both in vitro and in vivo. MK-0677 has also been shown to increase serum insulin-like growth factor I (IGF-I) and cortisol levels in vivo; these increases are assumed to be driven by the increased serum GH and ACTH levels, respectively. However, such increases could also be due to a direct stimulatory action of MK-0677 at the level of the liver and adrenal cortex. To address this possibility, we investigated whether MK-0677 increased IGF-I and cortisol levels in hypophysectomized dogs. Baseline GH, IGF-I, and cortisol responses to MK-0677 (1 mg/kg, orally) were initially determined. Hypophysectomy (hypox; n = 7) or sham surgery (sham; n = 5) was then carried out. Six days postsurgery, the GH and cortisol responses to MK-0677 were reevaluated in each dog. In addition, each dog was treated with porcine GH (PST; 0.1 IU/kg, s.c.) to confirm the responsiveness of the GH-IGF-I axis. The mean peak GH increases in response to MK-0677 in the presham dogs (83.7 +/- 19.2 ng/ml), post-sham dogs (108 +/- 26.2 ng/ml), and pre-hypox dogs (121.2 +/- 13.6 ng/ml) were not significantly different. Mean peak GH levels were unchanged after MK-0677 administration in the hypox dogs (2.3 +/- 0.7 ng/ml). Before surgery, serum IGF-I levels increased to 243 +/- 27 and 224 +/- 47 ng/ml in the sham and hypox groups, respectively, after MK-0677 administration. Surgery was associated with a marked (> or =50%) decrease in serum IGF-I levels. MK-0677 administration increased IGF-I levels in the sham dogs from 78 +/- 14 to 187 +/- 31 ng/ml, whereas IGF-I levels remained unchanged (17.7 +/- 2.4 ng/ml) in the-hypox dogs. PST treatment increased IGF-I levels in the sham dogs from 162 +/- 30 to 325 +/- 32 ng/ml. In the hypox dogs PST treatment restored IGF-I to physiological levels (from 17.7 +/- 2.4 to 199 +/- 41 ng/ml). Cortisol was increased after MK-0677 administration 3.7-fold in the pre-sham, 3.6-fold in the post-sham, and 3.6-fold in the pre-hypox dogs, but no increase was seen in the post-hypox dogs. ACTH GEL administration (2.2 U/kg, i.m.) to hypox dogs returned cortisol to normal physiological levels, demonstrating the functional integrity of the adrenal cortex. This study demonstrates that the GH secretagogue MK-0677 does not directly stimulate an increase in serum IGF-I or cortisol levels, but depends upon the presence of an intact pituitary.

A potent, orally bioavailable benzazepinone growth hormone secretagogue.

The identification of L-739,943 (8b), a potent, orally bioavailable benzolactam growth hormone secretagogue, is obtained from zwitterionic L-692,429 through modification of its amino acid side chain and replacement of the acidic 2'-tetrazole with the neutral and potency enhancing 2'-(N-methylaminocarbonylamino)methyl substituent. L-739,943 is orally active for the release of growth hormone in beagle dogs at doses as low as 0.5 mg/kg. Oral bioavailability in dogs of 8b is 24% at a dose of 2 mg/kg with a mean drug Cmax of 145 +/- 46 ng/mL. L-739,943 represents a significant breakthrough in terms of both potency and oral bioavailability as compared to the prototype benzolactam L-692,429.

Potent 3-spiropiperidine growth hormone secretagogues.

Systematic SAR studies of the different regioisomers and homologues of the spiro(indane-1,4-piperidine) moiety in the growth hormone secretagogue L-162,752 are presented. Among them, spiro(3H-1-benzopyran-2,3-piperidine) was found to afford secretagogues with low nanomolar in vitro activity.

Synthesis and biological activities of phenyl piperazine-based peptidomimetic growth hormone secretagogues.

A new class of potent, orally active phenyl piperazine-based GH secretagogues have been discovered from attempts to mimic the arrangement of the phenyl substituent in the spiroindanyl piperidine and spiroindoline sulfonamide privileged structures of 4 and 1, respectively. The best of these compounds, 18 (EC50 = 2.8 nM) is nearly as potent as MK-0677 for releasing GH from rat pituitary cells.

Repeat administration of the GH secretagogue MK-0677 increases and maintains elevated IGF-I levels in beagles.

We have reported that MK-0677 is a novel, orally active GH secretagogue that stimulates an immediate and long-lasting increase in serum GH levels in dogs. Significant elevations in IGF-I levels were associated with the increased GH secretion. Cortisol secretion was also increased following MK-0677 administration. In the current study, we determined the effect of repeat oral administration of MK-0677 on GH, IGF-I and cortisol levels; we also investigated if the GH and cortisol responses to MK-0677 are influenced by circulating IGF-I concentrations. Following the initial oral administration of MK-0677, GH secretion (area under the time-response curve (AUC) ng/ml per h) was increased 7.9- to 9.8-fold (1.0 mg/kg), 5.6-fold (0.5 mg/kg) or 3.9-fold (0.25 mg/kg). With repeat MK-0677 administration, the GH response was decreased by 41-77%; GH concentrations remained significantly above control in the 0.5 mg/kg and 1.0 mg/kg groups. Individual beagle GH profiles indicated that the increased GH concentration was associated with an amplified GH pulsatile profile. Serum IGF-I levels were significantly increased over control levels at all dosage levels by 480 min on the first day of MK-0677 administration. With repeated administration, IGF-I levels were increased up to 126% and remained elevated through 14 days, the longest treatment period evaluated. While daily MK-0677 administration appeared to increase IGF-I levels over 24 h, as evidenced by significant increases in the pretreatment IGF-I levels on days 4-14, no such increase was noted with alternate day MK-0677 administration; thus the dosage regimen modulated circulating IGF-I levels. MK-0677 stimulated increases in cortisol secretion (AUC microgram/dl per h) on the first day of treatment. A decreased cortisol response was observed following repeated daily treatment with MK-0677; in contrast, with alternate day treatment, no decrease in cortisol response to MK-0677 occurred. A marked increase in circulating IGF-I concentrations following administration of exogenous GH resulted in a significant decrease in both the GH and cortisol response to MK-0677 compared with control animals. Our findings suggested, therefore, that circulating IGF-I concentrations regulate GH and cortisol response to MK-0677. In summary, chronic oral administration of MK-0677 was associated with significant increases in GH and IGF-I levels that were maintained for the duration of the treatment. The GH profile following MK-0677 administration consisted of episodic increases above control. Compared with day 1, repeated daily treatment with MK-0677 resulted in an attenuated GH response that was associated with an increase in circulating IGF-I levels. The cortisol response was similarly reduced during chronic MK-0677 treatment, suggesting that IGF-I mediated negative feedback on both the GH and cortisol axes. The fact that similar attenuation of the GH and cortisol responses to MK-0677 on day 1 was observed if IGF-I levels were increased by treating animals with exogenous GH suggested that the attenuated response to MK-0677 that occurred during chronic treatment was mediated by increases in IGF-I rather than desensitization to MK-0677. Thus, a regulatory feedback loop apparently prevents hyperstimulation of the GH axis by MK-0677. We conclude that MK-0677 offers the potential of an orally active GH secretagogue that can maintain elevated IGF-I levels when administered chronically.

Quaternary heterocyclylamino beta-lactams. V. L-640,876 treatment of induced enterotoxigenic colibacillosis (scours) in calves and piglets.

A new semisynthetic cephalosporin antibiotic designated L-640,876, 7-beta-(1-benzylpyridinium-4-yl)amino-3-[( (1-methyl-1H-tetrazol-5-yl)thio] methyl)ceph-3-em-4-carboxylate, was highly active in vitro against 110 enteropathogenic strains of Escherichia coli and Salmonella species of animal origin. The MIC90 was 0.125 microgram/ml for the E. coli strains, 2 micrograms/ml for the S. choleraesuis strains and 4 micrograms/ml for the S. typhimurium strains. In colostrum-fed calves infected with E. coli strain B44, L-640,876 administered by gavage at 30 mg/calf (0.67 mg/kg) twice a day for 3 days, starting at 20-hour post-inoculation, eliminated the diarrhea and reduced the mortality from 82% in the infected, nonmedicated calves to 11% in the infected, medicated calves (P less than 0.05). In colostrum-fed piglets infected with E. coli strain P155, L-640,876 administered by gavage at 12.5 or 20 mg/piglet (10 or 16 mg/kg) twice a day for 3 days, starting at 6-hour post-inoculation, eliminated the diarrhea and reduced the mortality from 79% in the infected, nonmedicated to 25% in the infected, medicated piglets (P less than 0.05). Thus, L-640,876 was highly effective in restoring the calves and piglets to good health by eliminating diarrhea and reducing mortality.

Cephamycin C treatment of induced enterotoxigenic colibacillosis (scours) in calves and piglets.

Cephamycin C is a beta-lactam antibiotic that has broad gram-negative activity and is resistant to degradation by beta-lactamases and safe for use in animals. In colostrum-fed calves infected with Escherichia coli strain B44, cephamycin C administered by gavage at 31.3 to 1,000 mg per calf (0.75 to 24 mg/kg) twice a day for 6 days starting at 20 h post-inoculation eliminated the diarrhea and reduced the mortality from 90% in infected, nonmedicated calves to 14% in infected, medicated calves (P < 0.01). Comparable results were obtained with a shorter treatment regimen (30 mg of cephamycin C per calf [0.71 mg/kg] twice a day for 3 days). In colostrum-fed piglets infected with E. coli strain P155 and housed in cages, cephamycin C administered prophylactically by gavage at 12.5 mg per piglet (10.4 mg/kg) twice a day for 4 days completely prevented both diarrhea and mortality, whereas nonmedicated piglets had 100% diarrhea and all died. When eight doses of cephamycin C were given therapeutically starting at 6 h post-inoculation, mortality was reduced from 79 to 23% (P < 0.02), and diarrhea was eliminated in the surviving medicated piglets by 4 days post-inoculation. In infected suckling piglets, cephamycin C administered therapeutically by gavage at 12.5 mg per piglet twice a day for 3 days starting at 6 h post-inoculation, diarrhea and mortality were reduced (P < 0.05): infected, nonmedicated piglets had 87% diarrhea and 75% mortality, whereas infected, medicated piglets had 25% diarrhea and 31% mortality. All surviving medicated piglets had solid feces by 2 days post-inoculation. Thus, cephamycin C was highly effective in restoring the calves and piglets to good health by eliminating diarrhea and reducing mortality.

Anticoccidial and tolerance studies in the chicken with two 6-amino-9-(substituted benzyl)purines.

Initial assays of 6-amino-9-(2-chloro-6-fluorobenzyl) purine (MK-302) and 6-amino-9-(2,6-dichlorobenzyl)purine (coded L-628,914) showed potential as anticoccidial agents on the basis of broad-spectrum activity and safety. In battery efficacy studies, dietary levels of 60 to 70 p.p.m. and above MK-302 and 45 to 60 p.p.m. L-628,914 proved to have excellent broad-spectrum anticoccidial activity in chickens given heavy exposure to virulent field isolates of coccidia. Eight-week floor-pen tolerance trials showed that the maximum tolerated diet concentration (MTC) of MK-302 was approximately 95 p.p.m. while the MTC of L-628,914 was approximately 60 p.p.m. Dietary relationships (p.p.m. MK-302:p.p.m. L-628,914 for equivalent effects) derived from the efficacy and tolerance results were 1.2:1 and 1.6:1 respectively and clearly demonstrated a higher therapeutic ratio for MK-302.