Jerzy Trojnar

Inhibition of uterine contractions of premature labour with an oxytocin analogue. Results from a pilot study

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.

Novel d-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral κ-opioid receptors,

Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/>1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (>1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.

Cystatin C Based Peptidyl Diazomethanes as Cysteine Proteinase Inhibitors: Influence of the Peptidyl Chain Length

The peptidyl diazomethanes Cbz-Gly-CHN2, Boc-Val-Gly-CHN2, H-Leu-Val-Gly-CHN2, Cbz-Leu-Val-Gly-CHN2 and Cbz-Arg-Leu-Val-Gly-CHN2, with peptidyl portions modelled after the proposed cysteine proteinase interacting N-terminal segment of human cystatin C, were synthesized. Their efficiency as cysteine proteinase inhibitors was tested against papain, human cathepsin B and bovine cathepsin B. All, except Cbz-Gly-CHN2, were found to be irreversible inhibitors of the tested enzymes. Each addition of an amino acid residue to their peptidyl portions resulted in an increased inhibition rate of all three enzymes. These data suggest that the arginyl residue of the tetrapeptidyl diazomethane, and also the corresponding arginyl residue in native cystatin C, interact with a S4 substrate pocket subsite of both papain and cathepsin B. The most efficient inhibitor, Cbz-Arg-Leu-Val-Gly-CHN2, inhibited papain and cathepsin B with rate constants of the same order of magnitude as those for L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane (E-64). The high water-solubility of Cbz-Arg-Leu-Val-Gly-CHN2 allowing it to be dissolved to molar concentrations without use of non-physiological additives, makes it suitable for in vitro and in vivo cysteine proteinase inhibition studies.

Synthesis of cysteine proteinase inhibitors structurally based on the proteinase interacting N-terminal region of human cystatin C.

Fibronectin contains two latent gelatinolytic enzymes, FN-gelatinase and FN-laminase that can be activated in the presence of Ca2+ from the purified cathepsin D-produced 190-kDa fibronectin fragment. The results of this work show that Achromobacter collagenase cleaves fibronectin and generates an active FN-gelatinase. In contrast to the cathepsin D digest, the collagenase digest directly exhibits gelatinolytic activity without additional activation. The gelatinolytic activity of the total collagenase digest can be inhibited by phenylmethanesulfonyl fluoride, a serine proteinase inhibitor and by pepstatin A, an aspartic-acid proteinase inhibitor. FN-laminase activity, when assayed with its synthetic substrate GPAGPR and also with laminin was revealed after separation of the collagenase digest of fibronectin on heparin Ultrogel. FN-gelatinase and FN-laminase activities were found in heparin unretained and heparin strongly retained fractions. These results have demonstrated that in contrast to cathepsin D, Achromobacter collagenase activates two matrix-degrading proteinases from fibronectin, FN-Gelatinase und FN-Laminase.

A New Peptide for Human Parvovirus B19 Antibody Detection

A serological assay for human parvovirus B19 was developed. Linear overlapping synthetic peptides were synthesised according to parts of open reading frames 1 and 2. A region at the N-terminus of viral protein VP2 detected serological reactivity in indirect enzyme-linked immunosorbent assays for IgG and IgM with known seropositive human sera. A cyclized peptide taken from this region, amino acids 284-307, gave the best selective reactivity with seropositive and seronegative sera. The peptide assay appears suitable for further studies of B19 infections and their complications.

THE EFFECT ON THE HUMAN UTERUS OF TWO NEWLY DEVELOPED COMPETITIVE INHIBITORS OF OXYTOCIN AND VASOPRESSIN

Abstract. in order to develop inhibitors of vasopressin (VP) and oxytocin (OXY) action on uterine activity, 1‐deaminat‐ed vasotocin derivatives with modifications at positions 2, 4 and 8 were developed. Two of the most effective analogues in the rat, l‐deamino‐2‐D‐Tyr(OEt)‐4‐Val‐8‐Orn‐vasotocin (dE‐VVT) and l‐deamino‐2‐D‐Tyr(OEt)‐4‐Thr‐8‐Orn‐vaso‐tocin (dE‐TVT) were now tested on human nonpregnant myometrium obtained at hysterectomy in fertile age and on pregnant myometrial tissue obtained at elective cesarean section. the effect was compared with that of a previously synthesized analogue l‐deamino‐Tyr(OEt)‐oxytocin (dE‐OXY) which has already been tested in nonpregnant and pregnant women in vivo. Both of the new analogues competitively inhibited the action of the posterior pituitary hormones. on the nonpregnant uterus dE‐VVT was about five times and dE‐TVT almost twenty‐five times more potent than dE‐OXY in inhibiting the effects of VP. on pregnant myometrium, dE‐TVT inhibited oxytocin action about as effectively as a five‐fold stronger concentration of dE‐OXY, and dE‐VVT slightly less. A moderate agonistic effect of dE‐OXY on pregnant myometrium was found, whereas it was minimal with dE‐VVT and not detectable at all with dE‐TVT. It appears that these two analogues, particularly dE‐TVT, would be interesting for clinical testing both in dysmenorrhea, where increased VP secretion could be of etiological importance, and in premature labor where an increased myometrial concentration of OXY receptors has been demonstrated.

Vasotocin analogues which competitively inhibit vasopressin stimulated uterine activity in healthy women

Summary. Three analogues of posterior pituitary hormones, 1 ‐ deamino ‐ 2 ‐ D ‐ Tyr(OEt) ‐ 4 ‐ Val ‐ 8 ‐ Om ‐ vasotocin(dE ‐ VVT), l‐deamino‐2‐D‐Tyr(OEt)‐4‐Thr‐8‐Orn‐vasotocin(dE‐TVT) and 1‐deamino‐2‐D‐Tyr(OEt)‐oxytocin(dE‐OXY) were compared for their inhibitory effects on vasopressin (VP)‐induced uterine activity in healthy women. At menstruation, during recording of intrauterine pressure (18 recording sessions in 11 women), intravenous infusion of lysine vasopressin (LVP, 1 ng/min/kg/body weight) induced an increase of the uterine activity and dysmenorrhoea‐like symptoms. Intravenous injections of all analogues (10 μg/kg body weight) caused relief of symptoms and inhibition of uterine activity, dE‐TVT was the most effective and dE‐OXY was least active. With dE‐TVT almost complete inhibition of contractions was seen during the first 10 min after injection. The duration of effect was also greatest with that analogue (40–50 min). Only dE‐OXY had an agonist effect on spontaneous uterine activity. Pharmacokinetic studies of intravenous dE‐TVT (10 ng/kg body weight) showed that the plasma half‐life was approximately 16 min and the clearance 30 1/h. The bioavailability of 100 ng/kg given intra‐nasally was about 5·5%. Further studies are recommended.

Immunosuppressive activity of antamanide and some of its analogues.

In connection with our discovery of a strong immunosuppressive activity of cyclolinopeptide A (CLA), we investigated immunosuppressive properties of antamanide and a number of its analogues, including symmetrical antamanide, and compared them with the activities of cyclosporin A and CLA. The peptides were investigated by using plaque forming cell (PFC), graft-versus-host (GvH), delayed type hypersensitivity (DTH), and autologous rosette formation cell (ARFC) tests. Antamanide and symmetrical antamanide exhibit an immunosuppressive activity lower than CLA. Linear antamanide fragments are also active. At higher concentrations of the latter peptides, toxic effects occur.

The synthesis of a new class of oxytocin antagonists.

The synthesis of a new class of oxytocin antagonists, with significantly modified C-terminal part, is described. The chemistry of the Mitsunobu reaction was applied to obtain the key derivatives. In spite of the extensive modifications of previously described compound F792, the peptides retain biological activity as oxytocin antagonists.

Effects of vasopressin on the human non-pregnant uterus: studies with analogues of different vasopressor potencies.

The uterotonic effects of arginine8-vasopressin (AVP) have been studied on uterine strips from non-pregnant women. Concentration-dependent contractions could be recorded over a 10 min period in the presence of AVP (5.5.10(-10)-3.10(-7) M); the most reproducible recordings were obtained with tissue from the inner part of the myometrium. Analogues of AVP and oxytocin (OT), modified at positions 1 (2-hydroxy-3-mercaptopropionic acid, deamino-3-mercaptopropionic acid), 2 (Phe), 4 (Arg, Val), 7 (Sar) or 8 (Orn) were synthesized and tested for uterotonic activity on human and rat uterine strips, and for vasopressor and antidiuretic activity in the rat in vivo. There was a positive correlation between the activity of these analogues on non-pregnant human myometrial tissue with that in the rat vasopressor assay (r = 0.86, P less than 0.01) but none with their activity in the antidiuretic assay. For example, [Mpa1,D-Arg8]vasopressin had more than twice the antidiuretic activity of AVP but less than 0.2% of its pressor or human uterotonic potency (Mpa = 3-mercaptopropionic acid). Correspondingly, the specific pressor analogue [Hmp1,Phe2,Orn8]OT was as potent as AVP on the human uterus, but had less than 3% of its antidiuretic activity (Hmp = 2-hydroxy-3-mercaptopropionic acid). There was no correlation between the uterotonic activities of AVP or its analogues when non-pregnant human and rat tissues were compared, indicating that rat uterine tissue is a poor guide when testing analogues intended for clinical use in non-pregnant women.(ABSTRACT TRUNCATED AT 250 WORDS)