Role of Mifepristone in Trial of Labour after Previous Caesarean Delivery in Intrauterine Fetal Death.

When a baby dies in utero, the options are either to wait for spontaneous labour or to induce it. An obstetrician, encounters with a perplexity of choosing a management plan when this worst situation of IUFD coalesced by history of previous caesarean delivery. The ideal drug for the termination should not only be efficacious and cost-effective, but also be convenient enough to avoid operative interference arising from a wasted pregnancy. The study was aimed to evaluate efficacy, safety and compliance of oral mifepristone in trial of labour in case of IUFD after previous caesarean section. This was a cross sectional descriptive type of observational study conducted in the Department of Obstetrics and Gynaecology, Mymensingh Medical College Hospital, Bangladesh from February 2018 to August 2018. Total 50 patients were selected purposively based on inclusion criteria and diagnosed as IUFD with previous caesarean delivery. The patients were received mifepristone once and reviewed after 48 hours and those who were not attained favourable Bishop's score were counseled for mechanical induction. Antibiotics and analgesia were administered according to requirement. Data analysis was done using SPSS version 22.0. All the 50 women received 200 mg oral mifepristone. Forty-four 44(88.0%) women was delivered vaginally among them 18(36.0%) were delivered following mifepristone induction only and 26(52.0%) required additional induction method. The earliest induction to delivery interval following mifepristone was 13 hours. Twenty eight (63.6%) cases were discharged within 72-120 hours. After 48 hours following induction there was significant improvement of Bishop's score. In this study 6(12.0%) out of 50 cases were reasoned for laparotomy and blood transfusion was required for them. There was no statistically significant difference according to gestational age in mode of delivery (p>0.05). There was no difference observed in mean induction to delivery interval between second and third trimester at 5% level of significance (p>0.05). In this study, the women showed drastic improvement in cervical score following induction with mifepristone and decreased repeat caesarean rate. Eventually, the length of agony of receiving dead baby was cut short without much more ailments. Hence, mifepristone may be considered as a safe, efficacious, convenient and cost-effective induction agent for labour induction in women with dead fetus in utero in previously scarred uterus.

Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and cooperativity.

Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed in a reconstituted system using progesterone, testosterone, 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and alpha-naphthoflavone (ANF) as substrates. CYP3A4 wild type displayed sigmoidal kinetics of ANF 5,6-oxide formation and 7-BFC debenzylation. Analysis of 12 mutants with significant steroid hydroxylase activity showed a lack of positive correlation between ANF oxidation and stimulation of progesterone 6beta-hydroxylation by ANF, indicating that ANF binds at two sites within CYP3A4. 7-BFC debenzylation was stimulated by progesterone and ANF, and 7-BFC did not inhibit testosterone or progesterone 6beta-hydroxylation. Correlational analysis showed no relationship between 7-BFC debenzylation and either progesterone or testosterone 6beta-hydroxylation. These data are difficult to explain with a two-site model of CYP3A4 but suggest that three subpockets exist within the active site. Interestingly, classification of the mutants according to their ability to oxidize the four substrates utilized in this study suggested that substrates do bind at preferred locations in the CYP3A4 binding pocket.

Identification and functional characterization of eight CYP3A4 protein variants.

The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the population studied was found to be heterozygous for one of these variants. In a bacterial heterologous expression system, two mutants, R130Q and P416L, did not result in detectable P450 holoprotein. One mutant, T363M, expressed at significantly lower levels than wild-type CYP3A4. G56D, V170I, D174H and M445T were not significantly different when compared with wild-type CYP3A4 in expression or steroid hydroxylase activity. L373F displayed a significantly altered testosterone metabolite profile and a four-fold increase in the Km value for 1'-OH midazolam formation. The results suggest a limited contribution of CYP3A4 protein variants to the inter-individual variability of CYP3A4 activity in Caucasians. Some variants may, however, play a role in the atypical response to drugs or altered sensitivity to carcinogens.

The importance of SRS-1 residues in catalytic specificity of human cytochrome P450 3A4.

The structural basis for the regioselective hydroxylation of Delta-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6beta-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2beta-OH rather than 6beta-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S(50) values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the V(max) values for 2beta-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by alpha-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6beta-hydroxylation.

Structure-function analysis of human cytochrome P450 3A4 using 7-alkoxycoumarins as active-site probes.

The oxidation of a series of seven alkyl ethers of 7-hydroxycoumarin by cytochrome P450 3A4 (CYP3A4) has been studied to probe the active site of the enzyme. TLC of the reaction mixture showed formation of metabolites other than 7-hydroxycoumarin. The separation and characterization of the different metabolites of the C4 to C7 compounds were achieved using a combination of TLC, HPLC, and gas chromatography-electron impact mass spectra. Among the 7-alkoxycoumarins, 7-hexoxycoumarin was found to be the most suitable candidate for investigating the active site of cytochrome CYP3A4, due to the well-separated metabolite peaks on TLC and HPLC. 7-hexoxycoumarin was found to produce three side-chain hydroxylated products besides 7-hydroxycoumarin: 7-(5-hydroxyhexoxy)coumarin, 7-(4-hydroxyhexoxy)coumarin, and 7-(3-hydroxycoumarin). The substitution of residues from substrate recognition sites -1, -4, -5, and -6 of CYP3A4 showed a strong influence on the product profile of 7-hexoxycoumarin, the most prominent effects observed with mutants at residues 119, 301, 305, 370, 373, and 479. The docking of 7-hexoxycoumarin into a molecular model of CYP3A4 also confirmed the presence of these residues within 5 A of the substrate. A comparative study of cytochrome P450 2B1 showed that the active-site mutants F206L, T302V, V363A, and S478G but not V363L exhibited a dramatic decrease in total 7-hexoxycoumarin hydroxylation. The study suggests that although the electronic nature of the substrate is important, enzymatic constraints significantly contribute to CYP3A4 selectivity.

The role of distal histidine in peroxidase activity of myoglobin--transient-kinetics study of the reaction of H2O2 with wild-type and distal-histidine-mutanted recombinant human myoglobin.

The distal histidine has been proposed to play a crucial role in the reaction of peroxidases with hydroperoxide. Myoglobin (Mb), due to its close similarity with peroxidases, also reacts with various peroxides. The effect of mutation of the distal histidine on the peroxidase activity of Mb has been investigated by stopped-flow kinetics of the reaction of hydrogen peroxide with wild-type Mb and [Gly64]Mb. The results indicate kinetic and mechanistic differences in the formation of peroxide compounds from these two forms of Mb. The rate of reaction of H2O2 with the wild-type Mb decreased 8-9-fold on mutation of the distal histidine to glycine ([Gly64]Mb). A second slow phase was observed for the reaction of H2O2 with [Gly64]Mb, but was not observed in the corresponding reaction with wild-type Mb. It is suggested that the decrease in the rate of the reaction on mutation is due to the absence of a general acid-base catalyst. The effect of pH on the rate of reaction of H2O2 with wild-type Mb and [Gly64]Mb is contrasting. While the rate of the Mb [corrected] reaction decreased for wild-type Mb at higher pH, probably due to the acid-alkaline transition of Mb at higher pH, the rate of reaction was found to increase at higher pH for mutant Mb. The increase in the rate of the reaction is suggested to be due to an increase in the ionization of H2O2 at higher pH, the rate-determining step being the formation of the intermediate Fe-O-O-H complex and not the subsequent step of oxo-ferryl complex formation. The thermodynamic parameters calculated from the temperature-dependent study showed that the enthalpy of binding of H2O2 with Mb is positive, indicating that the process is endothermic. The apparent energy of activation of the reaction of H2O2 with Mb was found to be higher than that of peroxidases, suggesting that this may be oue of the reasons for the slower rate of the reaction of H2O2 with Mb compared with peroxidases.

Chlorambucil-induced seizures.

BACKGROUND: Anecdotal reports of chlorambucil-induced seizures have sporadically appeared, mainly in the nononcologic literature. The majority of cases have occurred in patients treated with high dose therapy and in children with nephrotic syndrome. Because of its rarity, oncologists and hematologists may not be aware of this potential complication. METHODS: Two elderly patients with a remote history of seizures had generalized tonic-clonic seizures 3 days after chlorambucil therapy was initiated. A MEDLINE search was performed of previously reported cases and additional cases were found in the bibliographies of retrieved articles. RESULTS: In addition to the 2 new cases presented here, there have been 28 reported cases of chlorambucil-induced seizures. Underlying diseases included nephrotic syndrome (n = 12 cases), solid tumors (n - 10 cases), non-Hodgkin's lymphoma (n = 3 cases), and chronic lymphocytic leukemia (n = 1 case). Five cases were secondary to accidental overdose. Sixteen of 30 patients were younger than 18 years; 11 had nephrotic syndrome, 1 had choriocarcinoma, and 4 accidentally ingested the medication. Nine of 14 adults received high dose chlorambucil in Phase I-II studies or as part of a conditioning regimen prior to bone marrow transplantation for solid tumors, 3 were on intermittent pulse therapy, 1 was on daily low dose administration of chlorambucil, and 1 patient had an accidental poisoning. Two patients had recurrent seizures when they were rechallenged with chlorambucil. CONCLUSIONS: A relatively high incidence of chlorambucil-induced seizures in children with nephrotic syndrome may be due to an increased sensitivity in childhood or altered pharmacokinetics. In adults without a seizure history, seizures were observed only in patients treated with high dose chlorambucil; however, in adults with a seizure history, lower doses as used in pulse therapy also caused seizures. In the latter group of patients, daily low dose chlorambucil or, more likely, an alternative drug may be the safest approach to therapy.

Steady-state and picosecond-time-resolved fluorescence studies on the recombinant heme domain of Bacillus megaterium cytochrome P-450.

The conformational changes associated with the interaction of sodium laurate with the recombinant heme domain for cytochrome P-450BM3 have been investigated by steady-state and picosecond-time-resolved fluorescence spectroscopy. The steady-state quenching experiments show that while all the five tryptophan residues are accessible to acrylamide in the free enzyme as well as the enzyme x substrate complex, the number of tryptophan residues accessible to ionic quenchers decreases on interaction of the substrate with the enzyme. This indicates that some of the tryptophan residues move towards the core of the protein on interaction with the substrate. The number of tryptophan residues accessible to the solvent as determined by the calculation of the solvent-accessible area for the free enzyme agrees with the values obtained by the quenching experiments. The time-resolved fluorescence studies carried out by means of the time-correlated single-photon-counting technique show that the fluorescence-decay curve is best fitted to a three-exponential model (0.2, 1.0 and 5.4 ns). Lifetime distributions, as recovered by the maximum-entropy method, agree with the discrete exponential model. The binding of the substrate does not lead to any significant change in the lifetime components of the enzyme, indicating that the tryptophan residues are possibly away from the substrate-binding domain. The decay-associated emission spectra and the magnitudes of amplitude of different lifetimes indicate that the shortest lifetime component (tau1) originates from the three tryptophan residues that are completely or partially accessible to the solvent, and tau2 originates from the tryptophan residues that are buried in the core of the enzyme and not accessible to the solvent. X-ray crystallographic data and solvent-acessible-area calculations have been used to identify these residues.