A bioactive somatostatin analog without a type II' beta-turn: synthesis and conformational analysis in solution.

A cyclic somatostatin analog [structure: see text] (1) has been synthesized. Biological assays show that this compound has strong binding affinities to somatostatin hsst2 and hsst5 receptor subtypes (5.2 and 1.2 nM, respectively, and modest affinity to hsst4 (41.1 nM)). Our conformational analysis carried out in DMSO-d6 indicates that this compound exists as two structures arising from the trans and cis configurations of the peptide bond between Phe7 and N-alkylated Gly8. However, neither conformer exhibits a type II' beta-turn. This is the first report of a potent bioactive somatostatin analog that does not exhibit a type II' beta-turn in solution. Molecular dynamics simulations (500 ps) carried out at 300 K indicate that the backbone of compound 1 is more flexible than other cyclic somatostatin analogs formed by disulfide bonds.

A novel somatostatin analogue prevents early renal complications in the nonobese diabetic mouse.

BACKGROUND: PTR-3173 (S) is a novel somatostatin analogue that has been found to exert a prolonged inhibitory action on the growth hormone (GH)-insulin-like growth factor (IGF)-I axis, but not on insulin secretion. We investigated the potential effect of this agent on the development of markers of diabetic nephropathy in the nonobese diabetic (NOD) mouse model of insulin-dependent diabetes. METHODS: Female diabetic NOD mice treated with PTR-3173 (DS group) or saline (D) and their control groups of nonhyperglycemic age-matched littermates (C) and C mice treated with PTR-3173 (CS) were sacrificed three weeks after onset of diabetes. RESULTS: Serum GH was elevated in the D group, decreased in the DS group, and unchanged in the CS group. Serum IGF-I was significantly decreased in both the D and DS groups. Kidney weight, glomerular volume, albuminuria, and creatinine clearance were increased in the D animals and showed a trend toward normalization in the DS animals. Renal extractable IGF-I protein and IGFBP1 mRNA were increased in the D group and normalized in the DS group. CONCLUSIONS: GH antagonism by PTR-3173 has a blunting effect on renal/glomerular hypertrophy, albuminuria, and glomerular filtration rate (GFR) in diabetic NOD mice. This phenomenon is apparently associated with the prevention of renal IGF-I accumulation. Thus, modulation of GH effects may have beneficial therapeutic implications in diabetic nephropathy.

Novel long-acting somatostatin analog with endocrine selectivity: potent suppression of growth hormone but not of insulin.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is a natural cyclic peptide inhibitor of pituitary, pancreatic, and gastrointestinal secretion. Its long-acting analogs are in clinical use for treatment of various endocrine syndromes and gastrointestinal anomalies. These analogs are more potent inhibitors of the endocrine release of GH, glucagon, and insulin than the native SRIF; hence, they do not display considerable physiological selectivity. Our goal was to design effective and physiologically selective SRIF analogs with potential therapeutic value. We employed an integrated approach consisting of screening of backbone cyclic peptide libraries constructed on the basis of molecular modeling of known SRIF agonists and of high throughput receptor binding assays with each of the five cloned human SRIF receptors (hsst1-5). By using this approach, we identified a novel, high affinity, enzymatically stable, and long-acting SRIF analog, PTR-3173, which binds with nanomolar affinity to human SRIF receptors hsst2, hsst4, and hsst5. The hsst5 and the rat sst5 (rsst5) forms have the same nanomolar affinity for this analog. In the human carcinoid-derived cell line BON-1, PTR-3173 inhibits forskolin-stimulated cAMP accumulation as efficiently as the drug octreotide, indicating its agonistic effect in this human cell system. In hormone secretion studies with rats, we found that PTR-3173 is 1000-fold and more than 10,000-fold more potent in inhibiting GH release than glucagon and insulin release, respectively. These results suggest that PTR-3173 is the first highly selective somatostatinergic analog for the in vivo inhibition of GH secretion, with minimal or no effect on glucagon and insulin release, respectively.

Design, synthesis, and biological activities of potent and selective somatostatin analogues incorporating novel peptoid residues.

We report the synthesis, bioactivity, and structure-activity relationship studies of compounds related to the Merck cyclic hexapeptide c[Pro6-Phe7-d-Trp8-Lys9-Thr10-Phe11], L-363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with arylalkyl peptoid residues. We present a novel approach utilizing beta-methyl chiral substitutions to constrain the peptoid side-chain conformation. Our studies led to molecules which show potent binding and increased selectivity to the hsst2 receptor (weaker binding to the hsst3 and hsst5 receptors compared to L-363, 301). In vivo, these peptoid analogues selectively inhibit the release of growth hormone but have no effect on the inhibition of insulin. The biological assays which include binding to five recombinant human somatostatin receptors carried out in two independent laboratories and in vivo inhibition of growth hormone and insulin provide insight into the relationship between structure and biological activity of somatostatin analogues. Our results have important implications for the study of other peptide hormones and neurotransmitters.

A backbone-cyclic, receptor 5-selective somatostatin analogue: synthesis, bioactivity, and nuclear magnetic resonance conformational analysis.

Cyclo(PheN2-Tyr-D-Trp-Lys-Val-PheC3)-Thr-NH2 (PTR 3046), a backbone-cyclic somatostatin analogue, was synthesized by solid-phase methodology. The binding characteristics of PTR 3046 to the different somatostatin receptors, expressed in CHO cells, indicate high selectivity to the SSTR5 receptor. PTR 3046 is highly stable against enzymatic degradation as determined in vitro by incubation with rat renal homogenate and human serum. The biological activity of PTR 3046 in vivo was determined in rats. PTR 3046 inhibits bombesin- and caerulein-induced amylase and lipase release from the pancreas without inhibiting growth hormone or glucagon release. The major conformation of PTR 3046 in CD3OH, as determined by NMR, is defined by a type II' beta-turn at D-Trp-Lys and a cis amide bond at Val-PheC3.

The anticonvulsant effect of deprenyl on pentylenetetrazol-induced seizures in Lewis rats.

There is recent evidence that deprenyl may have anticonvulsant action in a rat kindling model of epilepsy as well as in a maximal electroshock model. We therefore investigated the effect of deprenyl on the brain sensitivity threshold to pentylenetetrazol (PTZ)-induced maximal seizures in Lewis rats, in a model that provides pharmacodynamic information free of pharmacokinetic interference. The novel finding of this investigation was the anticonvulsant effect of deprenyl following repetitive administration whereas a single deprenyl dose did not affect the PTZ concentrations required to induce maximal seizures. The data suggests that the mechanism of this effect is not associated with the dopaminergic activity of deprenyl since pretreatment with both bromocriptine (a dopamine D2 agonist) and haloperidol (dopamine antagonist) did not affect the seizure threshold, whereas levodopa caused a proconvulsant effect. It was also concluded that the mechanism is not related to changes in acetylcholine levels since prolonged pretreatment with deprenyl did not attenuate the brain sensitivity to pilocarpine-induced seizures. The fact that long term administration of deprenyl was needed to produce its anticonvulsant effect may indicate that the anticonvulsant effect of deprenyl may be due to changes in levels of certain endogenous compounds or down or up-regulation of relevant receptor/effector units.

Differential effects of various antiinflammatory drugs on theophylline neurotoxicity.

The purpose of the present investigation was to evaluate whether antiinflammatory drugs affect the pharmacodynamics of theophylline-induced seizures. Adult male Lewis rats were treated with either dexamethasone (DEX), hydrocortisone (HYD), ibuprofen (IBU), or mefenamic acid (MFA), for 4 consecutive days. On the fourth day they received a constant infusion of theophylline (2 mg/min IV) until the onset of maximal seizures. Then, blood and cerebrospinal fluid (CSF) were obtained for theophylline concentration determinations by HPLC. It was found that pretreatment with the corticosteroids DEX and HYD elevated the CSF theophylline concentration required to induce maximal seizures in comparison to the untreated rats (242 +/- 6, 232 +/- 6, and 203 +/- 10 mg/l, respectively, n = 10, p < 0.05). MFA also increased the CSF theophylline concentration at that end-point in comparison to the controls (p < 0.01), whereas pretreatment with IBU had no effect (280 +/- 10 MFA, 225 +/- 9 IBU vs. 220 +/- 8 controls, n = 12). The data suggests that concomitant treatment with antiinflammatory drugs, together with theophylline, do not increase the risk for theophylline-induced seizures. Moreover, in certain cases they may elevate the seizure threshold and protect against these hazardous episodes.

Cyclosporine enhances theophylline neurotoxicity in rats.

Treatment with cyclosporine may be associated with adverse central nervous system (CNS) effects as well as with the potentiation of effects of certain other drugs. In particular, theophylline-induced seizures, which are often fatal and occur unpredictably over a wide range of serum theophylline concentrations, may be precipitated. To study this interaction, adult rats that were injected with cyclosporine or placebo (50 mg/kg in a single dose or on each of four consecutive days) received a constant infusion of theophylline (2 mg/min iv) until the onset of maximal seizures. At that time, blood, cerebrospinal fluid (CSF), and brain tissue samples were obtained for theophylline concentration determinations by HPLC, as well as for measurement of several biochemical parameters in the serum. Consecutive cyclosporine administration (but not a single dose) reduced serum protein levels. There was a small increase in theophylline sensitivity after a single dose of cyclosporine. The CSF theophylline concentrations at the onset of seizures were 215 +/- 10 vs 202 +/- 5 mg/L (P < 0.04); however, sequential cyclosporine treatment resulted in significant lowering of the CSF theophylline concentrations required to produce convulsions (231 +/- 8 vs 191 +/- 10, P < 0.001). Likewise, the drug concentrations at the onset of convulsions in both the brain and serum were significantly lower in cyclosporine-treated rats than in control animals. Thus, cyclosporine treatment may be a predisposing factor for theophylline toxicity and increase the risk for generalized seizures.