[Medical treatment of early spontaneous miscarriages: a prospective study of outpatient management using misoprostol]. / Traitement médical des fausses couches spontanées précoces. Etude prospective d'une prise en charge ambulatoire à l'aide du misoprostol.

OBJECTIVES: Medical treatment using misoprostol has been recommended as an alternative to surgical evacuation for spontaneous miscarriages in the first trimester, in order to avoid anesthesia, a surgical operation and hospitalization. Our aim was to assess the efficacy and the safety of vaginal misoprostol in out-patient management of early miscarriages. MATERIAL AND METHODS: We conducted a prospective cohort study including patients with a spontaneous miscarriage of less than 14 weeks gestation. Exclusion criteria were hemorrhagic miscarriages, gestational sac larger than 40 mm and/or cranio-caudal length of the embryo of more than 30 mm. Our protocol used 4 intravaginal tablets (800 microg) of misoprostol on Day 1, out-patient follow-up, clinical and ultrasound reassessment on Day 2. If the intra-uterine antero-posterior diameter on ultrasound examination was greater than 15 mm (or the gestational sac persisted), a second dose of misoprostol was administered or surgical evacuation was performed (failure of medical treatment). RESULTS: One hundred two patients were included, of whom 30 had an inevitable or threatened miscarriage and 72 had a missed abortion. The success rate defined by complete expulsion of the products of conception without resorting to surgical evacuation and without short-term complications, was 78.4% (80 patients). Two patients were lost to follow-up at 48 hours. Complete expulsion occurred within 2 days in 94% of cases. There were 13 emergency consultations, including 8 for expulsion pain and five surgical evacuations (3 for hemorrhagic expulsion, 2 for hemorrhagic retention). All together, there were 15 surgical evacuations for retention of the products of conception after 1 or 2 doses of misoprostol. There was one case of endometritis (1%). Thirty-four patients were hospitalized, 22 for surgical evacuation and 12 for expulsion pain. CONCLUSION: It is possible to use misoprostol as a single vaginal dose (800 microg) as out-patient treatment, since it gives satisfactory efficacy (80%) and is sufficiently safe (5% needing emergency surgery). However, out-patient management should only be performed after explaining the treatment and its risks clearly to the patient.

[Complications of vacuum extractor deliveries]. / Complications des accouchements assistés par ventouse.

OBJECTIVES: To describe maternal and neonatal complications following deliveries assisted by vacuum extraction and to compare outcomes with those obtained after spontaneous vaginal delivery. We wanted to know if vacuum extractor was a risk factor by itself. MATERIALS AND METHODS: We conducted a retrospective study of two years activity involving 4524 deliveries of which 845 (18.7%) were vacuum extractor assisted. We precisely defined maternal and neonatal complications to compare their rates in spontaneous vaginal delivery and vacuum extractor groups. RESULTS: There were 1333 maternal complications and 114 neonatal complications. The adjusted risks of maternal complications were significantly higher in the vacuum extractor group for simple vaginal tears (OR=3.0; p<0.001), the simple perineal tears (OR=1.8; p<0.001) and third degree perineal tears (OR=2.7; p<0.01). For neonatal complications, the difference was significant for cephalhematomas (OR=10; p<0.001) and scalp abrasions (OR=53; p<0.001). No cases of skull fracture or subgaleal subaponeurotic hemorrhage were recorded. CONCLUSION: Our rates of maternal and neonatal complications after vacuum extractor were similar to those described in the literature. We have been able to show that vacuum extraction is itself a risk factor for third degree perineal tears and cephalhematoma. However, these complications are so infrequent that the advantages of this method of extraction argue in favor of wide use in obstetrics.

Evaluation of enkephalinase inhibition in the living mouse, using [3H]acetorphan as a probe.

A novel in vivo binding test was developed in order to evaluate the degree of occupancy of enkephalinase (EC 3.4.24.11), a membrane-bound metallopeptidase, in cerebral and peripheral tissues of mice treated with enkephalinase inhibitors. The probe selected for this purpose was the prodrug [3H]acetorphan, a lipophilic diesterified derivative of the potent enkephalinase inhibitor thiorphan readily releasing the latter by tissue hydrolysis. In order to validate the in vivo binding assay, [3H]thiorphan binding to membranes was first studied in vitro. [3H]Thiorphan binding to cerebral and peripheral tissues (lung and kidney) was saturable over a low nonspecific binding, occurring with a KD of 0.6 nM consistent with the Ki of the compound as enkephalinase inhibitor. [3H]Thiorphan binding varied largely among various tissues and was highly correlated with the catalytic activity of enkephalinase, thus indicating a selective labeling of the peptidase. After the i.v. administration of [3H]acetorphan a large fraction of the radioactivity remained bound to membranes isolated by a rapid filtration assay. Bound radioactivity mainly corresponded to [3H] thiorphan as identified by high-performance liquid chromatography analysis of kidney membranes, whereas unchanged [3H]acetorphan was not detectable. In vivo binding generated by [3H]acetorphan was saturable, with maximum binding sites values which were in rather good agreement with corresponding maximum binding sites values of [3H]thiorphan binding in vitro, particularly in brain. Specific in vivo binding was calculated as the difference between total and a generally low, nonspecific binding evaluated in mice receiving a large dose of nonlabeled acetorphan. Specific in vivo binding varied largely among tissues and generally reflected the abundance of enkephalinase molecules in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)

Labelling and exploration of the active site of enkephalinase (EC 3.4.24.11) in kidney membranes with [3H]thiorphan as ligand.

[3H]Thiorphan, a potent inhibitor of enkephalinase (EC 3.4.24.11), was used to label the enzyme in membranes from rat kidney cortex an to explore its specificity at the active site. [3H]Thiorphan binding occurred reversibly, with low non-specific binding and to a single class of sites. The dissociation constant, determined by either kinetics or saturation studies was approximately 0.4 nM. The ratio of the maximal velocity of enkephalinase with enkephalins as substrates to the maximal binding of [3H]thiorphan was consistent with the catalytic constant of the enzyme. Enkephalinase inhibitors competed with [3H]thiorphan and had inhibitory constants in agreement with the corresponding values derived from measurement of the enzyme catalytic activity, whereas inhibitors of other metallopeptidases were ineffective. The inhibitory potencies of a series of systematically varied oligopeptides regarding [3H]thiorphan binding and enkephalinase activity were also highly correlated. Structure-activity relationships among competitors indicated that the main subsites of enkephalinase are: (1) the hydrophobic pocket in P'1, the requirements of which are best satisfied by aromatic amino acid side chain residues (2) the P'2 subsite, the requirements of which are best satisfied by amino acids with a short, uncharged side chain and a free terminal carboxyl group. This novel binding assay should facilitate the exploration of the active site of enkephalinase and the development of new inhibitors.

Characterisation of two probes for the localisation of enkephalinase in rat brain: [3H]thiorphan and a 125I-labeled monoclonal antibody.

We studied the binding of two radioactive probes, i.e. [3H]thiorphan and a 125I-labeled monoclonal antibody raised against the rabbit kidney enzyme, to enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase) from rat cerebral membranes. [3H]Thiorphan binding at equilibrium to striatal membranes was monophasic with a KD (0.7 nM) and a pharmacology consistent with a selective labeling of the enzyme. The ratio of Vmax/Bmax was in the same range as the Kcat of the enzyme purified from peripheral tissues. The monoclonal antibody immunoprecipitated to a similar extent the solubilised enkephalinase activity and [3H]thiorphan binding sites from striatum. The regional distributions of binding sites for the two probes established either on isolated membranes or autoradiographic sections were highly heterogeneous and similar to that of enkephalinase activity. Hence the two probes appear to label membrane-bound enkephalinase in rat brain but, from a technical point of a view, the 125I-monoclonal antibody is a more sensitive and flexible tool.

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit.

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.

[Inhibition of the evoked liberation of (Met5)enkephalin from slices of globus pallidus in the presence of thiorphan and bestatin]. / Inhibition de la libération évoquée de (Met5)enképhaline de coupes de globus pallidus en présence de thiorphan et de bestatine.

(Met5) enkephalin released from slices of Rat globus pallidus by 30 mM or 50 mM K+ is hydrolysed to the extent of 85% before reaching the incubation medium. When thiorphan and bestatin, two peptidase inhibitors, are added, this inactivation process is completely prevented but the peptide release from tissues is significantly diminished. The latter effect being reversed in the presence of an opioid antagonist, naloxone, in moderate concentration, the existence of an auto-inhibition process regulating enkephalin release is suggested.

Peptidases involved in the inactivation of exogenous and endogenous enkephalins.

Among the various cerebral enzyme activities able to hydrolyse the enkephalins into inactive fragments only two seem responsible for the metabolism of the endogenous opioid peptides: a dipeptidylcarboxypeptidase ("enkephalinase"), and a bestatin-sensitive aminopeptidase. Their inhibition by thiorphan and bestatin results in an antinociceptive effect observed in tests in which the nociceptive stimulation is probably accompanied by a concomittent release of enkephalins.

Inhibition of enkephalin metabolism by, and antinociceptive activity of, bestatin, an aminopeptidase inhibitor.

In the presence of thiorphan an 'enkephalinase' inhibitor, bestatin an aminopeptidase inhibitor of bacterial origin potently inhibited the hydrolysis of [3H][Leu5]enkephalin by slices from rat striatum with an IC50 value of about 0.2 microM whereas puromycin was approximately 1000 times less potent on this preparation. In vivo bestatin or thiorphan (but not puromycin) significantly protected [3H][Met5]enkephalin administered intracerebroventricularly to mice from hydrolysis and co-administration of these two peptidase inhibitors resulted in a strong reduction in the appearance of hydrolysis products in brain. In a parallel fashion the antinociceptive activity of [Met5]enkephalin in the mouse hot-plate test was additively potentiated by bestatin and thiorphan but not by puromycin. Finally both bestatin and thiorphan themselves displayed antinociceptive properties on either the hot-plate jump test or the phenyl-benzo-quinone writhing test. It is concluded that a bestatin-sensitive aminopeptidase activity together with the 'enkephalinase' activity plays a critical role in the inactivation of both exogenous and endogenous enkephalins.

Enkephalin metabolism in brain and its inhibition.

Various cerebral peptidases are able to hydrolyse the enkephalins into inactive fragments. Among these enzymes enkephalin-dipeptidylcarboxypeptidase ("enkephalinase") inhibited by thiorphan and a bestatin-sensitive aminopeptidase activity seem to play a key-role as "inactivating neuropeptidases". Their inhibition, in vitro as well as in vivo, leads to a protection of endogenous enkephalins and to antinociceptive effects.

Selective protection of methionine enkephalin released from brain slices by enkephalinase inhibition.

Methionine enkephalin release was evoked by depolarization of slices from rat striatum with potassium. In the presence of 0.1 microM thiorphan [(N(R,S)-3-mercapto-2-benzylpropionyl)glycine], a potent inhibitor of enkephalin dipeptidyl carboxypeptidase (enkephalinase), the recovery of the pentapeptide in the incubation medium was increased by about 100 percent. A similar effect was observed with the dipeptide phenylalanylalanine, a selective although less potent enkephalinase inhibitor. Inhibition of other known enkephalin-hydrolyzing enzymes--aminopeptidase by 0.1 mM puromycin or angiotensin-converting enzyme by 1 microM captopril--did not significantly enhance the recovery of released methionine enkephalin. These data indicate that enkephalinase is critically involved in the inactivation of the endogenous opioid peptide released from striatal neurons.