SMS 201-995, an octapeptide somatostatin analogue. Assignment of the 1H 500 MHZ n.m.r. spectra and conformational analysis of SMS 201-995 in dimethylsulfoxide

The possible conformations of SMS 201-995, an active analogue of somastostatin, have been studied in dimethylsulfoxide solution by 500 MHz proton n.m.r. spectroscopy. The assignments have been made by use of 2D-correlated methods to detect long-range coupling connectivities in aromatic residues and between the alpha protons of consecutive residues. NOESY experiments enabled us to correlate amide and alpha protons of neighbouring amino acid residues, which indicate a less flexible situation than in water. Measurements of temperature coefficients of the amide protons, of NH-C alpha H coupling constants and NOE effects are in favour of one predominant conformation with a beta turn, of type II', involving amino acids Phe3 to Thr6.     

SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action

Stepwise modification of a conformationally stabilised analogue of the fragment of somatostatin which had been thought to be essential biologically active moiety has enabled us to synthesise the analogue H-(D) Phe-Cys-Phe-(D) Trp-Lys-Thr-Cys-Thr(ol) code-named SMS 201-995, which in vitro is three times more potent than the native hormone in inhibiting the secretion of growth hormone, which is highly resistant to degradation by pure enzymes and by tissue homogenates, which in vivo in rat and rhesus monkey is (depending on test system) at least 20 times more active than somatostatin, which is much longer acting, and which moreover in both species is much more selective in inhibiting the secretion of growth hormone than that of insulin. The compound is active by several routes of administration including the oral, is well tolerated both in laboratory animals and in man, and is currently undergoing preliminary clinical trial.     

Conformational analysis of two somatostatin analogues by 1H 500 MHz n.m.r. spectroscopy

A series of nine closely related somatostatin analogues, containing the hexapeptide H-Cys2-Phe3-D-Trp4-Lys5-Thr6-Cys7-NH2 sequence have been synthesized by Bauer et al. The conformational properties of two of them, showing intermediate activities between those of SMS 201-995 and somatostatin, have been studied by high field n.m.r. spectroscopy in DMSO. Assignments were made using 2D-n.m.r. methods, in particular NOESY experiments and detection of long-range connectivities in aromatic residues. In all the compounds of this series, the biologically active ones as well as the inactive ones, the n.m.r. parameters are in favour of a predominant conformation with a type II' beta turn involving amino acids Phe3 to Thr6. A clearcut correlation exists between the predominant conformation at the cystine bridge side and the activity. The presence of the exocyclic amino acids Phe1 and Thr8 (ol) plays a major role in stabilization of the active conformation.     

Treatment of acromegaly with long acting somatostatin analogue SMS 201-995

Ten acromegalic patients were treated with the somatostatin analogue SMS 201-995 (SMS) for 3-38 weeks in various doses and by different administration routines (thrice daily or multiple sc injection). Plasma GH daily profiles, plasma IGF-I, urinary GH, serum TSH, IRI and fasting blood glucose (FBG) concentrations were measured before and during SMS treatment. Plasma GH rapidly decreased within one hour in all patients and was suppressed for at least 4 h after a 50 micrograms sc injection of SMS in 8 patients. Multiple injections of 300-600 micrograms/day SMS (25-50 micrograms X 12) suppressed GH throughout the day. Plasma IGF-I was completely normalized in 4 patients, and, in all but one of the others, decreased markedly. Urinary GH decreased within the first week of treatment in all patients and normalization was obtained in 3 patients. Shrinkage of the pituitary tumor, as determined by CT or MRI, was observed in 7 of 9 patients. Other clinical improvements, such as diminution or complete disappearance of swelling of soft tissues, excessive perspiration, and headache, were observed in 7 of 8 patients. Changes in serum TSH, IRI and FBG were seen in 3-4 patients, but without any apparent clinical problems. In conclusion, SMS is a useful clinical tool for treatment of acromegaly, and a multiple sc injection method seems to be preferable.     

Conformational study of a somatostatin analogue by high-field n.m.r. spectroscopy, in aqueous solution

The cyclic analogue of somatostatin (SRIF), D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-NH2 (CTC), exhibits good affinity for both opioid and SRIF receptor systems. Its conformational properties were examined in water by high-field proton n.m.r. spectroscopy and compared with results previously obtained with structurally related analogues SMS 201-995 and Sandoz 204-090 in the same solvent. The assignments were made using 2 D-n.m.r. methods, especially long-range connectivities between neighbouring alpha protons, and between beta and aromatic protons. The 3JNH-C alpha H and delta delta/delta T values are compatible with an equilibrium between two gamma turns involving residues 2, 3 and 4 and residues 3, 4, and 5, respectively.     

Improvement of diabetes after treatment with somatostatin analogue SMS 201-995 in an acromegalic patient

The beneficial effects of long acting somatostatin analogue SMS 201-995 in an acromegalic patient affected by severe diabetes mellitus are reported. Neither human insulin alone nor human insulin plus bromocriptine allowed satisfactory metabolic control though, with the latter treatment, virtually normal plasma GH levels were reached. Conversely, addition of SMS 201-995 to insulin treatment led to normalization of blood glucose. This result was obtained with a dose of SMS 201-995 of 400 micrograms/day and only after 3 weeks of therapy.     

The use of SMS 201-995 (somatostatin analogue) in insulinomas. Additional case report and literature review

A 76-year-old man, a known case of insulinoma, was well controlled for 11 days on 50 micrograms SMS 201-995 every 12 h; clinical recovery was immediate with normalization of blood sugars and C-peptide levels. The potential value of this new drug in the management of insulinomas is illustrated by this case and by 11 additional case reports which are reviewed. A rise in C-peptide levels during treatment without concomitant hypoglycaemia might be the first indication of a loss of control.     

Hexapeptide analogue of somatostatin, Sandoz 201-456. Conformational study in dimethylsulfoxide by 1D and 2D n.m.r. methods

The conformational properties of the somatostatin analogue 201-456 (1) have been studied by high field n.m.r. in DMSO. This analogue is the base structure of nine derivates synthesized by Bauer et al. and shows a very low biological activity, although derived structures such as SMS 201-995 (2) are very potent. Our study has shown an important difference between the most stable conformation of the two compounds: although the beta turn type II' structure at the Phe3-Trp4-Lys5 level is present in both analogues, an important conformational change appears at the cystine bridge. In SMS 201-995 the beta turn/beta sheet conformation is stabilized by the additional amino-acids D-Phe1 and Thr8 (ol) through intramolecular H-bonds.     

500 MHz 1H n.m.r. of oligosaccharides of N-acetyl-lactosamine-type released from human erythrocyte glycopeptides by endo-beta-galactosidase.

500 MHz 1H n.m.r. spectroscopy has been used in structural studies of three linear and five branched oligosaccharides of N-acetyl-lactosamine-type that were released from desialylated blood group O erythrocyte glycopeptides by treatment with the endo-beta-galactosidase of Bacteroides fragilis followed by reduction. The following oligosaccharide alditols were characterized: (formula; see book)     

Food intake in rats is increased by intracerebroventricular infusion of the somatostatin analogue SMS 201-995 and is decreased by somatostatin antiserum

The chronic intracerebroventricular infusion of the somatostatin analogue SMS 201-995 resulted in a significant increase in daily food intake which was accompanied by an unexpected body weight loss. The neutralisation of central somatostatin using a specific somatostatin antiserum resulted in a significant decrease in daily food intake. These results suggest that endogenous somatostatin in the brain can drive feeding behavior and alter body weight.     

Conformation of reduced glutathione in aqueous solution by 1H and 13C n.m.r

We report on the conformation of reduced glutathione in solutions at low and physiological pH, examined with 1H and 13C n.m.r. spectroscopy. The tripeptide in 1H2O was shown to interconvert rapidly between an array of conformers; in addition, the carbon backbone of the glutamyl was more rigid than anticipated if the residue were freely mobile. This restricted motion results from interaction of the alpha-amino and alpha-carboxyl groups on the glutamyl, with the gamma-Glu-Cys peptide-carbonyl and amino, respectively. Our results support theoretical predictions of the conformation but they are at variance with previous ultraviolet spectroscopic and lower field n.m.r. studies.     

Effect of a single administration of somatostatin analogue (SMS 201-995) on GH, TSH and insulin secretion in patients with acromegaly

The effect of a long-acting somatostatin analogue SMS 201-995 on GH secretion was investigated. Eleven acromegalic patients received a single dose of 50 micrograms SMS 201-995 administered subcutaneously, and plasma GH, IGF-I, GRF, TSH, IRI and blood glucose were determined at regular intervals. Nine of 11 patients had elevated basal plasma GH levels above 5 ng/ml. In all patients, plasma GH levels fell immediately from 39.5 +/- 17.3 ng/ml (mean +/- SEM) to 4.3 +/- 1.6 ng/ml (P less than 0.05) with a maximal inhibition of 82.9 +/- 3.3% of the basal levels and the suppression persisted for about 6 h of the observation period. IGF-I and GRF levels were not apparently altered. TSH and IRI levels also rapidly fell. Blood glucose levels fell slightly by 0.5 h. Ten of 11 patients had pain at injection sites. Except for this, no side effects were observed. Our results show that the new somatostatin analogue SMS 201-995 may inhibit GH hypersecretion in acromegalic patients for significant periods, suggesting that this agent can be a useful clinical tool for the treatment of acromegaly.     

Conformational analysis of pepstatin and related renin inhibitors by 400 MHz 1H n.m.r. spectroscopy

The conformational behaviour of pepstatin (Iva-Val-Val-Sta-Ala-Sta) and of two derived renin inhibitors, Boc-Phe-Nle-Sta-Ala-Sta-OMe, 1, and Boc-Phe-Nle-X-Ala-Sta-OMe, 2 (X = -NH-CH(iPr)-CHOH-CH2-CO-) was assessed in DMSO-d6 at various temperatures and in deuteriopyridine at -35 degrees. Complete assignment of almost all proton signals was achieved by 2D COSY, 2D NOESY and selective NOE experiments. The three compounds show similar extended conformations in both solvents, with the hydrophobic lateral chains extending away from the peptide backbone. In the case of pepstatin the solvated conformation is closely related to the structure found in the crystal of the pepstatin-Rhizopus chinensis complex. Strong NOE effects and precise determination of vicinal coupling constants show the lack of large structural differences between 1 and 2 at the level of the internal Sta or X residues, which are assumed to interact with the aspartyl residues of the renin active-site. This suggests that the 100-fold lower inhibitory potency of 2 is mainly due to unfavorable close contacts of the beta-branched residue X with constituent amino acids of the enzyme.     

1H n.m.r. parameters of the N-terminal 19-residue S-peptide of ribonuclease in aqueous solution

The 1H n.m.r. chemical shifts and the spin-spin coupling constants of the N-terminal 19-residue S-peptide of ribonuclease A have been measured in a 10 mM solution in D2O, pD 3.0, 27 degrees, at 300 MHz. The titration parameters for end groups Lys-1 and Ala-19 and side chains Lys-1, Glu-2, Lys-7, Glu-9, Arg-10, His-12 and Asp-14 have been determined at 90 MHz. An assignment of observed signals to individual residue protons based upon characteristic shifts, spectral analysis, double resonance, titration shifts and comparison with the spectrum of C-peptide (N-terminal 13-residue) is proposed. Differences in the observed chemical shifts, pKa's and titration shifts with reference to those proposed as "random coil" parameters are not large enough to assume the existence of a significant population of secondary structure in the conditions studied. The H alpha chemical shifts differences can be accounted for by the Phe-8 phenyl ring current for an extended peptide backbone conformation and appropriate values for the torsion angles chi 1 Phe-8 and chi 2 Phe-8.     

Central action of somatostatin analog, SMS 201-995, to stimulate gastric acid secretion in rats

The effects of intracisternal and intravenous injections of the somatostatin analog, SMS 201-995, on gastric acid secretion were investigated in rats with pylorus ligation or gastric cannula. Intracisternal injection of SMS 201-995 induced a dose-related (0.1-0.3 microgram) and long-lasting stimulation of gastric acid output with a peak response at 3 h postinjection in conscious, pylorus-ligated rats. Intracisternal SMS 201-995 increased histamine levels in the portal blood, whereas plasma gastrin levels were not modified. Atropine, cimetidine and adrenalectomy abolished the stimulatory effect of intracisternal SMS 201-995 (0.3 microgram). SMS 201-995 (0.03 microgram), microinjected unilaterally into the dorsal vagal complex, increased gastric acid output in urethane anesthetized rats. SMS 201-995, injected intravenously at 0.5 microgram, did not alter gastric secretion, whereas higher doses (5-20 micrograms) resulted in a dose-related inhibition of gastric acid secretion in conscious pylorus-ligated rats. These data indicate that SMS 201-995, a selective ligand for somatostatin-1 receptor subtype, induces a centrally mediated stimulatory effect on gastric acid secretion in rats. The central action involves the parasympathetic system, muscarinic and H2 receptors as well as adrenal-dependent pathways.     

Effect of subcutaneous injection of a long-acting analogue of somatostatin (SMS 201-995) on plasma thyroid-stimulating hormone in normal human subjects

SMS 201-995 (SMS), a synthetic analogue of somatostatin (SRIF) has been shown to be effective in the treatment of the hypersecretion of hormones such as in acromegaly. However, little is known about the effects of SMS on the secretion of thyroid-stimulating hormone (TSH) in normal subjects. In this study, plasma TSH was determined with a highly sensitive immunoradiometric assay, in addition to the concentration of SMS in plasma and urine with a radioimmunoassay, following subcutaneous injection of 25, 50, 100 micrograms of SMS (4 subjects/dose) or a placebo (6 subjects) to normal male subjects, at 0900 h after an overnight fast. The plasma concentrations of SMS were dose-responsive and the peak levels were 1.61 +/- 0.09, 4.91 +/- 0.30 and 8.52 +/- 1.18 ng/ml, which were observed at 30, 15 and 45 min after the injection of 25, 50 and 100 micrograms of SMS, respectively. Mean plasma disappearance half-time of SMS was estimated to be 110 +/- 3 min. Plasma TSH was suppressed in a dose dependent manner and the suppression lasted for at least 8 hours. At 8 hours after the injection of 25, 50 and 100 micrograms of SMS, the plasma TSH levels were 43.8 +/- 19.4, 33.9 +/- 9.4 and 24.9 +/- 3.2%, respectively, of the basal values. The results suggest that SMS suppresses secretion of TSH from the normal thyrotrophs in man and thus also that attention should be paid to possible hypothyroidism during the long-term treatment of patients such as those with acromegaly with this potent analogue of SRIF.     

Calcium and praseodymium complexes in solution. 1H n.m.r. conformational study of the model tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine

The synthetic tetrapeptide acetyl-aspartyl-valyl-aspartyl-alanine (Ac-DVDA) is a model of the calcium binding site of proteins such as carp parvalbumin, thermolysin and calmodulin. 1H n.m.r. spectra of the tetrapeptide are presented and assigned for D2O and DMSO solutions to determine the conformational mobility. The resonance of the two aspartyl side chains could be completely analysed and the vicinal coupling (C alpha H-C beta H and NH-C alpha H) indicated that the free peptide has considerable conformational mobility. The Ca(II) complex generates a different 1H n.m.r. spectrum for the aspartyl resonances at neutral pH. The solution conformation of Pr(III) complex of Ac-DVDA has been investigated using induced chemical shifts. The observed trends in the magnitude of the shift ratios and the rotamer population suggest that the metal ion binds predominantly to both carboxylates of two aspartyl residues in a bidentate fashion. We discuss the consistency of the differentiated spectra for aspartyl residues in the complex with the stepwise binding of Ca2+ to the carrier.