Two dipeptidyl peptidase IV (DPPIV, DPP4)-related proteins, DPP8 and DPP9, have been identified recently [Abbott, Yu, Woollatt, Sutherland, McCaughan, and Gorrell (2000) Eur. J. Biochem. 267, 6140-6150; Olsen and Wagtmann (2002) Gene 299, 185-193; Qi, Akinsanya, Riviere, and Junien (2002) Patent application WO0231134]. In the present study, we describe the cloning of DPP10, a novel 796-amino-acid protein, with significant sequence identity to DPP4 (32%) and DPP6 (51%) respectively. We propose that DPP10 is a new member of the S9B serine proteases subfamily. The DPP10 gene is located on the long arm of chromosome 2 (2q12.3-2q14.2), close to the DPP4 (2q24.3) and FAP (2q23) genes. The active-site serine residue is replaced by a glycine residue in DPP10, resulting in the loss of DPP activity. The serine residue is also replaced in DPP6, which lacks peptidase activity. DPP8 and DPP9 share an identical active site with DPP4 (Gly-Trp-Ser-Tyr-Gly). In contrast with the previous results suggesting that DPP9 is inactive, we show that DPP9 is a DPP, hydrolysing Ala-Pro-(7-amino-4-methyl-coumarin) with similar pH-specificity and protease-inhibitor-sensitivity to those of DPP4 and DPP8. Northern-blot analysis shows that whereas DPP8 and DPP9 are widely expressed, DPP10 is expressed mainly in the brain and pancreas. DPP6, which has the highest amino acid identity with DPP10, has been shown previously [Nadal, Ozaita, Amarillo, de Miera, Ma, Mo, Goldberg, Misumi, Ikehara, Neubert et al. (2003) Neuron 37, 449-461] to associate with A-type K(+) channel subunits, modulating their transport and function in somatodendritic compartments of neurons. It is possible that DPP10 is involved in similar functions in the brain. Elucidation of the physiological or pathophysiological role of DPP8, DPP9 and DPP10 and characterization of their structure-function relationships will add impetus to the development of inhibitor molecules for pharmacological or therapeutic use.

Cysteine proteinases are important not only in the intracellular catabolism of peptides and proteins and in the processing of prohormones and proenzymes, but also in the penetration of normal human tissue by malignant cells and possibly microorganisms, including viruses. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. We have synthesized peptide derivatives mimicking the proposed proteinase-binding centre of cystatin C and find that they irreversibly inhibit cysteine proteinases. Several bacteria produce proteinases, so we tested a tripeptide derivative (Z-LVG-CHN2) for in vitro anti-bacterial activity against a large number of bacterial strains belonging to thirteen different species. It was found to inhibit specifically the growth of all strains of group A streptococci. The susceptibility of these human pathogens to the peptide was compared with that to well-established anti-streptococcal antibiotics such as tetracycline and bacitracin. Moreover, the peptide was active in vivo against group A streptococci: mice injected with lethal doses of these bacteria were cured by a single injection of Z-LVG-CHN2. The cysteine proteinase produced by group A streptococci was isolated and found to be inhibited by Z-LVG-CHN2; moreover, excess proteinase relieved the growth inhibition caused by the peptide derivative, suggesting that the antibacterial activity of Z-LVG-CHN2 is due to inhibition of this cysteine proteinase. This strategy of blocking proteinases with peptide derivatives that mimic naturally occurring inhibitors could be useful in the construction of new agents against other microorganisms, including viruses.

A series of antagonists of gonadotropin-releasing hormone (GnRH) of the general formula Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph/4Amf(P)-D4Aph/D4Amf(Q)-Leu-ILys-Pro-DAla-NH2 was synthesized, characterized, and screened for duration of inhibition of luteinizing hormone release in a castrated male rat assay. Selected analogues were tested in a reporter gene assay (IC50 and pA2) and an in vitro histamine release assay. P and Q contain urea/carbamoyl functionalities designed to increase potential intra- and intermolecular hydrogen bonding opportunities for structural stabilization and peptide/receptor interactions, respectively. These substitutions resulted in analogues with increased hydrophilicity and a lesser propensity to form gels in aqueous solution than azaline B [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Atz)-D4Aph(Atz)-Leu-ILys-Pro-DAla-NH2 with Atz = 3'-amino-1H-1',2',4'-triazol-5'-yl, 5], and in some cases they resulted in a significant increase in duration of action after subcutaneous (s.c.) administration. Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorotyl)-D4Aph(carbamoyl)-Leu-ILys-Pro-DAla-NH2 (acetate salt is FE200486) (31) and eight other congeners (20, 35, 37, 39, 41, 45-47) were identified that exhibited significantly longer duration of action than acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2] (6) when administered subcutaneously in castrated male rats at a dose of 50 microg in 100 microL of phosphate buffer. No correlation was found between retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.0 (a measure of hydrophilicity) or affinity in an in vitro human GnRH report gene assay (pA2) and duration of action. FE200486 was selected for preclinical studies, and some of its properties were compared to those of other clinical candidates. In the intact rat, ganirelix, abarelix, azaline B, and FE200486 inhibited plasma testosterone for 1, 1, 14, and 57 days, respectively, at 2 mg/kg s.c. in 5% mannitol (injection volume = 20 microL). Based on the information that 31, 33, 35 and 37 were significantly shorter acting than acyline or azaline B after intravenous administration (100 microg/rat), we surmised that the very long duration of action of the related FE200486 (for example) was likely due to unique physicochemical properties such as solubility in aqueous milieu, comparatively low propensity to form gels, and ability to diffuse at high concentrations in a manner similar to that described for slow release formulations of peptides. Indeed, in rats injected s.c. with FE200486 (2 mg/kg), plasmatic concentrations of FE200486 remained above 5 ng/mL until day 41, and the time after which they dropped below 3 ng/mL and plasma LH levels started rising until full recovery was reached at day 84 with levels of FE200486 hovering around 1 ng/mL. Additionally, FE200486 was less potent at releasing histamine from isolated rat mast cells than any of the GnRH antagonists presently described in preclinical reports.

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With the aim of developing inhibitors of vasopressin- and oxytocin-induced uterine activity, 17 analogues of 1-deamino-oxytocin were synthesized by the solid-phase method. Modifications were made at positions 2, O-methyltyrosine (Tyr(OMe] and O-ethyltyrosine (Tyr(OEt],D-Tyr,D-Tyr(OEt),D-Trp; 4, Val,Thr and 8, Orn,Cit,Arg,D-Arg. The analogues were tested for antiuterotonic activity in vitro and in vivo in the rat and in vitro on myometrial strips from non-pregnant women and pregnant women at term. Their selectivity was also investigated in blood pressure and antidiuretic bioassays in rats. Results were compared with those from an original antiuterotonic analogue 1-deamino-2-Tyr(OEt)-oxytocin (d(OEt)-oxytocin). In the rat in vitro and in vivo all analogues possessed higher antiuterotonic activity than d(OEt)-oxytocin. The negative logarithm of the molar concentration of the antagonist which reduced the effect of a dose of agonist to that of half the dose (pA2) was between 7.6 and 8.9 for all the new inhibitors compared with 7.2 for d(OEt)-oxytocin. The highest pA2 value was found for 1-deamino-2-Tyr(OMe)-8-Orn-oxytocin (8.9 +/- 0.2, S.E.M.) and 1-deamino-2-Tyr(OEt)-4-Thr-8-Orn-oxytocin (8.9 +/- 0.6). In myometrium from non-pregnant women the most potent peptide was 1-deamino-2-D-Tyr(OEt)-4-Thr-8-Orn-oxytocin (17.2 +/- 2.0 times more potent that d(OEt)-oxytocin). In myometrium from pregnant women the inhibitory effects of the majority of the analogues were less pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)

The growth hormone (GH) releasing ability of GH-releasing factor (GRF) and a GH-releasing hexapeptide, GHRP, have been studied in anaesthetized and conscious male and female rats. The GH responses to GHRP in anaesthetized rats were inconsistent, and this peptide was much less potent than GRF. Continuous iv infusions of GRF or GHRP both caused an initial GH release which was not maintained, and further GH release could be elicited by injection of GRF during an infusion of GHRP and vice versa. In contrast, conscious rats were much more sensitive to GHRP. Infusions of GHRP or GRF both caused an initial GH release. With GRF infusions, GH release continued in the normal episodic pattern whereas with GHRP infusion, GH secretion remained elevated over baseline and the normal pulsatile rhythm was disrupted. Plasma GH levels fell after stopping GHRP infusion, without an immediate resumption of normal GH pulsatility. Conscious male rats responded intermittently to injections of GRF given iv every 45 min, but when such serial injections of GRF were given during a continuous iv infusion of GHRP, the GH responses to GRF became regular and more uniform. These results suggest that GHRP prevents the normal cyclic refractoriness to GRF in male rats by disrupting cyclic somatostatin release. The greater potency of GHRP in conscious rats may also depend on the release of endogenous GRF since passive immunization with an anti-GRF serum reduced the plasma GH response to GHRP infusion. Thus in the conscious animal, GHRP may release GH by complex actions at both a hypothalamic and pituitary level.Although the hypothalamic peptide, growth hormone-releasing factor (GRF) was discovered in 1982 (1,2), other substances with the ability to stimulate the release of growth hormone (GH) had previously been described, and of these, a pentapeptide derived from the enkephalin structure was shown to release G H specifically and separately from opiate activity (3-5). Further analogue design based on conformational analysis led to a hexapeptide, His-DTrp-Ala-Trp-DPhe-Lys-NH,, termed G H R P which was shown to be a potent and specific G H secretagogue in a number of in vitro and in vivo models (6-11). The apparent dissimilarity between both the structure and the size of this hexapeptide and the Structure of the endogenous G R F and its smallest active fragments, together with the much higher potency of G R F compared with G H R P in several test systems, has resulted in very few recent studies of the properties of these small G H R P peptides.

Summary. A competitive inhibitor of the action of oxytocin on the uterus, l‐deamino‐2‐D‐Tyr‐(OEt)‐4‐Thr‐8‐Orn‐oxytocin, was studied for the first time in 13 patients with established, uncomplicated premature labour. Intravenous infusion of 10–100 μg/min of the analogue was given for 1–10 h and the effect was monitored by external cardiotoco‐graphy. In all women an inhibition of uterine activity was observed, and in the majority of patients infused with 25 μg/min and a total dose of about 5 mg or more of the drug total inhibition of uterine contractions was achieved. There were no effects on the maternal and fetal pulse rates, nor were there any other side‐effects. The results of this preliminary study support the concept of an increased concentration of uterine oxytocin receptors being aetiologically important in uncomplicated premature labour. They also suggest that the present oxytocin antagonist could be an interesting therapeutic alternative in the condition, primarily because of the marked selectivity of its effect.

The side effects typically associated with the clinical profiles of opioid -receptor agonists have driven continuing efforts to identify novel efficacious analgesics, including agonists acting at opioid receptors. Unfortunately, the therapeutic potential of agonists seems limited by significant central nervous system side effects. Opioid agonists, however, exhibit potent peripherally mediated antihyperalgesic and antinociceptive effects, suggesting that a peripherally acting agonist may be efficacious in pain control with a more desirable safety profile than that associated with currently available opioids. Here, we report an all D-amino acid tetrapeptide characterized as a novel, highly selective opioid receptor agonist. FE200041 (D-Phe-D-Phe-DNle-D-Arg-NH 2 ) showed selectivity for the human opioid receptor of greater than 30,000-and 68,000-fold versus human opioid receptor and human ␦-opioid receptor receptors, respectively, and efficacious agonist activity using in vitro tissue assays. FE200041 produced local, peripheral antinociception in the hindpaw ipsilateral, but not contralateral, to injection. Antinociceptive effects of FE200041 in the mouse acetic acid writhing assay lasted over 60 min and were antagonized by naloxone and by selective , but not , opioid receptor antagonists. FE200041 significantly inhibited acetic acid writhing and inhibited formalin-induced flinching in rats. FE200041 did not elicit sedation or motor impairment after systemic administration at a dose 10-fold higher than that needed to achieve antinociception. FE200041 is thus a potent peripherally restricted opioid agonist with no demonstrable side effects typical of agonists with central nervous system activity and with unprecedented selectivity for the opioid receptor. The pharmacology of this compound suggests the possibility of therapeutic application.