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Biomedical subjects

D J Back

Publications and source records attributed to D J Back.

At least 91 records · Page 5Linked to original sources
PubMed1993

Effect of chlormethiazole on hepatic monooxygenases activity in vivo.

Chlormethiazole is a strong inhibitor of cytochrome P-450-dependent monooxygenases in isolated human liver microsomes. To assess its effect in vivo, we measured the pharmacokinetic parameters of antipyrine (1.2 g orally) and tolbutamide (0.5 g i.v.) before and after administration of chlormethiazole 314 mg b.d. for 2 days to 8 healthy volunteers. The elimination of neither substance was affected, indicating that chlormethiazole did not inhibit in vivo the cytochrome P-450 isozymes responsible for the elimination of antipyrine and tolbutamide.

Antipyrine↗
PubMed1993

Pharmacokinetics and protein binding of 3-ketodesogestrel and gestodene in the serum of women during 6 cycles of treatment with two low dose oral contraceptives.

The serum concentrations of 3-ketodesogestrel (KDG) and gestodene have been measured in 30 and 31 women respectively who took low dose oral contraceptives containing 30 micrograms ethinylestradiol together with either 150 micrograms desogestrel or 75 micrograms gestodene for 6 months. On days 1, 10 and 21 of the first third and sixth treatment cycles blood samples were drawn at 0, 0.5, 1, 1.5, 2, 3, 4 and 24 h. KDG and gestodene levels were measured by radioimmunoassays and were evaluated for Cmax (peak serum concentration), tmax (time to Cmax), and AUC (area under the curve) to 4 and 24 h. The overall total gestodene concentrations were higher and the accumulation of the steroid throughout a cycle greater than that of KDG. For example, the AUC0-4 of gestodene increased in cycle 1 by a factor of 2.8 (day 10 vs. day 1) and 3.6 (day 21 vs. day 1) compared to 2.3 and 2.6 for KDG. The higher concentration of gestodene reflects a lower volume of distribution than KDG, and is consistent with gestodene binding to sex hormone binding globulin (SHBG) with a higher affinity than KDG. Concentrations of KDG and gestodene were higher on day 1 of cycles 3 and 6 than on day 1 of cycle 1. The serum concentrations of KDG and gestodene during multiple dosing cannot be predicted on the basis of single dose pharmacokinetics.

Adolescent↗
PubMed1993

Influence of gestodene and desogestrel as components of low-dose oral contraceptives on the pharmacokinetics of ethinyl estradiol (EE2), on serum CBG and on urinary cortisol and 6 beta-hydroxycortisol.

A randomized controlled clinical trial was undertaken over a 6-month treatment period with two low-dose combined oral contraceptives (OC) to investigate whether the metabolism and elimination of ethinyl estradiol (EE2) is differently influenced by the two progestational components gestodene (G) and desogestrel (D), an issue which has been very controversial recently. The two formulations contained 30 micrograms EE2 each, together with either 75 micrograms G or 150 micrograms D. Of the 40 young women recruited for each formulation, 31 of each group were available for statistical evaluation. The pharmacokinetics of serum EE2 were studied on day 1, 10 and 21 of cycle 1, 3 and 6. There were no significant differences between the two groups in any cycle with respect to parameters measured. This was true for the distinct intracyclical rise in the mean EE2 serum levels from day 1 to day 10 and the smaller further increase between day 10 and day 21, with no change in this respect between the cycles studied. Respective changes were seen with regard to the area under the EE2 serum concentration curve up to 4 and 24 hours (AUC0-4 and AUC0-24), cmax and tmax of serum EE2. The estrogen-dependent corticoid-binding globulin (CBG) increased similarly in the two groups intracyclically and slightly also intercyclically at all times tested. Except for the first treatment cycle, urinary excretion of cortisol and 6 beta-hydroxycortisol displayed a tendency to lower values intracyclically as well as intercyclically, again with no differences between the two groups. Also, the 6 beta-hydroxycortisol-to-cortisol ratio was not different between the groups, showing a slight tendency to rise from about 4 at the beginning of the medication to around 5.5 at the end of the 6th treatment cycle in both groups. It is concluded that G and D as components of low-dose OCs exert comparable effects on the metabolism and elimination of EE2.

Adult↗
PubMed1993

Absence of an effect of high vitamin C dosage on the systemic availability of ethinyl estradiol in women using a combination oral contraceptive.

Previous studies in small numbers of women have suggested that the administration of gram quantities of ascorbic acid interferes with the conversion of ethinyl estradiol (EE2) to its sulfates, leading to higher blood levels of EE2. The possibility of such potentiation has been investigated in 37 women using a combination monophasic oral contraceptive (30 micrograms EE2 and 150 micrograms levonorgestrel) for two consecutive cycles. Concomitant daily administration of 1 g ascorbic acid taken 1/2 hour before OC intake, was randomly assigned to the first or second cycle of OC use. On the first and 15th day of OC intake, blood samples were drawn 11 times over a 12-hour interval and Cmax and AUC(0-12 h) calculated. On pill days 10 and 21, only 6-hour post-intake samples were obtained. Samples were analyzed for levels of ascorbic acid, free and sulfated ethinyl estradiol (and a number of other parameters). Cmax and AUC values for EE2 and EE2-sulfate in cycles with and without ascorbic acid were evaluated statistically by the Grizzle model for days 1 and 15 and the ratios of day 15/day 1 for each of the substances. No effect of ascorbic acid was observed (alpha = 0.05, 1-beta = 0.9). Only on day 15 was there a significantly lower AUC for EE2-sulfate in the presence of ascorbic acid intake. Thus, the competition between ascorbic acid and EE2 for sulfation does not lead to an increased systemic availability of EE2 and is, therefore, unlikely to be of any clinical importance. Ascorbic acid can, therefore, be removed from the list of drugs interfering with the pharmacokinetics of ethinyl estradiol.

Adolescent↗
PubMed1993

Metabolism of the oral contraceptive steroids ethynylestradiol, norgestimate and 3-ketodesogestrel by a human endometrial cancer cell line (HEC-1A) and endometrial tissue in vitro.

Human endometrial cancer cells and human endometrial tissue have been extensively used to study steroid hormone action and metabolism. The natural estrogens estradial (E2) and estrone (E1) are known to be metabolized by both cells and tissue with the interconversion of the two steroids and the formation of sulphate conjugates. The aim of the present work was to see if the commonly used oral contraceptive steroids ethynylestradiol (EE2), norgestimate (Ngmate) and 3-ketodesogestrel (3-KDG) were metabolized by a human endometrial cancer cell line (HEC-1A) and human endometrial tissue in vitro. Metabolites were analysed by on-line radiometric HPLC. Endometrial tissue was obtained from women undergoing dilation and curettage or hysterectomy operations. In preliminary studies with endogenous estrogens, HEC-1A cells were able to interconvert E1 and E2; the equilibrium favouring the formation of E2. Normal endometrial tissue extensively converted E2 to E1, tumour tissue appeared to catalyse this reaction much less avidly. In addition sulphate conjugates were formed by normal tissue from some patients. Cell line and endometrial tissue was able to hydrolyse estrone 3-sulphate. With EE2 as substrate there was no evidence of phase I metabolism by cell line or tissue. However, conversion to the presumed 3-sulphate conjugate was observed following incubation with normal tissue from some women. Deacetylation of the progestogen Ngmate to norgestrel oxime (NgOx) was complete within 24 h. There was also some loss of the oxime moiety to give norgestrel (Ng) following incubation with HEC-1A cells. Metabolism of Ngmate was also complete within 24 h following incubation with endometrial tissue. There were both qualitative and quantitative differences in metabolite formation between tissue obtained from different women. In contrast, 3-KDG was relatively resistant to metabolism by cell line and tissue. The major metabolite formed by HEC-1A cells accounted for only 3.3 +/- 0.4% of total added radiolabelled steroid and co-chromatographed with 3 alpha-hydroxydesogestrel. Smaller amounts of other radiometabolites were formed. No phase I metabolites of 3-KDG were formed by normal endometrial tissue, however small amounts of radiometabolites appeared to be formed by malignant tissue. These studies have provided evidence to suggest that the oral contraceptives EE2, Ngmate and 3-KDG are metabolized in the human endometrium. Knowledge of the metabolism of these in target tissues such as the endometrium may be pertinent considering the possibility that metabolites may exert specific effects.

Chromatography, High Pressure Liquid↗
PubMed1993

Cortisol metabolism in vitro--II. Species difference.

It has been suggested that cortisol 6 beta-hydroxylase activity specifically reflects cytochrome P4503A (CYP3A) levels in the liver. However, we have previously reported that the metabolism of cortisol in human liver fractions in vitro is extremely complex and variable, and therefore complete metabolite analysis must be undertaken if 6 beta-hydroxycortisol is to be used as a marker of CYP3A activity. In the present study, the metabolism of [3H]cortisol by hepatic microsomes from various animal species, and by cytosol from male and female rats, has been defined and compared with metabolites formed by human liver microsomes. Metabolites were characterized by co-chromatography with authentic standards, mass spectrometry, and quantified by radiometric HPLC. The results show that all microsomes prepared from animal species studied (male and female rat, male and female guinea-pig, male hamsters and mice) can metabolize cortisol, although the metabolic profiles are both quantitatively and qualitatively different from that obtained with human microsomes. In general the metabolic profiles for animal microsomes are simpler: hamster, mouse and guinea pig show only 6 beta-hydroxylase and 11 beta-dehydrogenase activity, although male rat shows both of these and 20 beta-reductase activity while the female rat possesses all of the above as well as the ability to reduce the A-ring (delta 4-reductase and 3-oxidoreductase activities). The female rat also produces two metabolites undetected in humans. Incubations with male rat cytosol generated 20 beta-dihydrocortisone as the major metabolite, and several unidentified minor polar metabolites, whereas female cytosolic products were identical to those generated by human cytosol, the major metabolite being 3 alpha,5 beta-tetrahydrocortisol. In conclusion, our studies have shown that hepatic cortisol metabolism is extremely variable amongst the species investigated and that the hamster provides the simplest model with which to explore cortisol 6 beta-hydroxylase activity.

Animals↗
PubMed1993

Metabolism of gestodene in human liver cytosol and microsomes in vitro.

The metabolism of the progestogen gestodene has been studied in human liver cytosol and microsomal incubations. Extraction with diethyl ether was followed by radiometric HPLC analysis. Metabolites were identified by co-chromatography with authentic standards and mass spectrometry (electron impact and chemical ionization). All the cytosolic incubations (n = 4 livers) produced dihydrogestodene as the major metabolite, with lesser amounts of a tetrahydro derivative. It was not possible to separate the 5 alpha- and 5 beta-isomers of dihydrogestodene on the chromatographic system used. Values of Km and V(max) for the delta 4 reductase were determined. Androstenedione (Ki = 2.85 +/- 1.5 microM; n = 4) and cortisol (ki = 24.1 +/- 8.9 microM; n = 4) both inhibited the delta 4-reductase. In contrast desogestrel showed virtually no inhibition at concentrations up to 200 microM. The major microsomal metabolite of gestodene was a hydroxylated derivative although mass spectral analysis was unable to determine the position of insertion of the hydroxyl moiety. The hydroxylation of gestodene (1 microM) was markedly inhibited by ketoconazole (IC50 < 0.1 microM), and also by cyclosporin. This suggests that the cytochrome P450 isozyme CYP3A4 is important in gestodene metabolism. Theophylline and tolbutamide (substrates of CYPIA and CYP2C, respectively) did not affect gestodene metabolism at concentrations up to 100 microM. In conclusion, the major biotransformation of gestodene (A-ring reduction) occurs in the cytosolic fraction of human liver. Microsomal hydroxylation appears to be catalysed by CYP3A4.

Adolescent↗
PubMed1993

Cortisol metabolism in vitro--III. Inhibition of microsomal 6 beta-hydroxylase and cytosolic 4-ene-reductase.

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3 alpha,5 beta-tetrahydrocortisol; 3 alpha,5 beta-THF), while microsomal incubations generate upto 7 metabolites, including 6 beta-hydroxycortisol (6 beta-OHF), and 6 beta-hydroxycortisone (6 beta-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6 beta-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [3H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The Km value for 6 beta-OHF and 6 beta-OHE formation was 15.2 +/- 2.1 microM (mean +/- SD; n = 4) and the Vmax value 6.43 +/- 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6 beta-hydroxylase was ketoconazole (Ki = 0.9 +/- 0.4 microM; n = 4), followed by gestodene (Ki = 5.6 +/- 0.6 microM) and cyclosporine (Ki = 6.8 +/- 1.4 microM). Both betamethasone and dexamethasone produced some inhibition (Ki = 31.3 and 54.5 microM, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The Km value for cortisol 4-ene-reductase was 26.5 +/- 11.2 microM (n = 4) and the Vmax value 107.7 +/- 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (Ki = 17.8 +/- 3.3 microM) and gestodene (Ki = 23.8 +/- 3.8 microM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1993

Cortisol metabolism by human liver in vitro--IV. Metabolism of 9 alpha-fluorocortisol by human liver microsomes and cytosol.

The oxidative and reductive biotransformations of 9 alpha-fluorocortisol (fluorocortisol) by human liver microsomes and cytosol have been characterized. 9 alpha-Fluorination greatly simplified cortisol metabolism in microsomes: dehydrogenation of the 11 beta-hydroxyl group and A-ring (4-ene-5 beta and 3 alpha-keto) reduction, the principle pathways, were completely blocked. Fluorocortisol was essentially metabolized by the remaining pathways, 20 beta-reduction and 6 beta-hydroxylation. In cytosol, 20 beta-reduction replaced the A-ring reduction of cortisol as the sole biotransformation. The major structure-metabolism relationships of fluorocortisol in man, i.e. complete and extensive inhibition of 11 beta-dehydrogenation and 4-ene-5 beta-reduction, respectively, were attributed to hepatic enzyme systems. Their mechanistic basis is discussed with reference to the electronic and conformational changes induced by 9 alpha-fluorination.

Adult↗
PubMed1993

Effect of etretinate on cyclosporin metabolism in vitro.

Cyclosporin (CyA) is an effective treatment for psoriasis, including cases unresponsive to other therapies. The major side-effect of CyA treatment is dose-related nephrotoxicity. Combinations of CyA and etretinate (Et) have been tested with a view to reducing CyA dose requirements, and therefore minimizing adverse effects. We have studied the effect of Et on the cytochrome P-450-mediated metabolism of CyA. Microsomes prepared from histologically normal human (obtained from four cadaver kidney transplant donors; all male; age range 21-56) were incubated with CyA and various concentrations of Et. Metabolism was quantified by high-performance liquid chromatography with radiometric detection, and metabolites tentatively identified from the retention times of authentic standards. After 30 min incubation of CyA and microsomal protein at 37 degrees C, 10.1 +/- 3.0% (mean +/- SD) 3H-CyA was converted to the monohydroxylated metabolites M1 and M17, and 3.3 +/- 0.8% to the N-demethylated metabolite M21. At an Et concentration of 100 microM inhibition of CyA hydroxylase and N-demethylase was < 20%. This study indicates that there is no metabolic interaction between CyA and Et in vitro; it is likely that the two drugs are metabolized by different P-450 isoenzymes.

Adult↗
PubMed1993

Radioimmunological analysis of ethinylestradiol in human serum. Validation of the method and comparison with a gas chromatographic/mass spectrometric assay.

The majority of combination oral contraceptives contain ethinylestradiol (EE2, CAS 57-63-6) as estrogenic component at doses between 50 and 20 micrograms/unit. Since the concentrations of EE2 in the serum of women under oral contraceptive (OC) therapy are in the lower pg-range, highly sensitive and specific analytical methods are required. Radioimmunoassay (RIA) has been the method of choice, but evidence of specificity in the presence of the coadministered progestogens and their metabolites has not always been provided. The present study compares two radioimmunological methods, which use the same antiserum but different sample volumes and standard curves (extracted vs. non-extracted), with a newly developed gas chromatographic/mass spectrometric (GC/MS) method, in order to cross-validate the methods. For that purpose, 51 serum samples obtained from women who had been taking two different combination oral contraceptives were analysed independently by all three methods. The specificity of the antiserum was further examined by submitting ex vivo serum samples obtained from OC-users to a combination of high pressure liquid chromatography (HPLC) and RIA. The results obtained by the three methods were very similar and correlation coefficients (r) obtained from linear regression analysis were about 0.7. There was no interference from the coadministered progestins on the analysis of EE2 as carried out by the three methods. There were no metabolites in the extracts of ex vivo serum samples which showed cross-reactivity with the antiserum used. Modifications of the radioimmunoassay procedure did not markedly affect the results of EE2 determination. If properly validated, radioimmunoassay can be used as an alternative to GC/MS in pharmacokinetic studies.

Chromatography, High Pressure Liquid↗
PubMed1992

The effect of malaria infection on 3'-azido-3'-deoxythymidine and paracetamol glucuronidation in rat liver microsomes.

The effect of malaria infection on UDP-glucuronosyltransferase (UDPGT) activity was investigated in rat liver microsomes using 3'-azido-3'-deoxythymidine and paracetamol. The Michaelis-Menten parameters, Km and Vmax were calculated and intrinsic clearance values were estimated for normal and infected livers. The results show that malaria infection alters the activity of UDPGT.

Acetaminophen↗
PubMed1992

Primaquine metabolism by human liver microsomes: effect of other antimalarial drugs.

A number of drugs have been studied for their effect on the metabolism of the antimalarial drug primaquine by human liver microsomes (N = 4) in vitro. The only metabolite generated was identified as carboxyprimaquine by co-chromatography with the authentic standard. Ketoconazole, a known inhibitor of cytochrome P450 isozymes, caused marked inhibition of carboxyprimaquine formation with IC50 and K(i) values of 15 and 6.7 microM, respectively. This finding and the dependency of metabolite formation on NADPH indicates that cytochrome P450 isozyme(s) catalysed metabolite production. Of compounds actually or likely to be coadministered with primaquine to malaria patients, only mefloquine produced any inhibition (K(i) = 52.5 microM). Quinine, artemether, artesunate, halofantrine and chloroquine did not significantly inhibit metabolite formation. It seems unlikely that the concurrent administration of mefloquine, or other antimalarials, with primaquine will lead to appreciably altered disposition.

Antimalarials↗
PubMed1992

Mefloquine metabolism by human liver microsomes. Effect of other antimalarial drugs.

A number of drugs have been studied for their effect on the metabolism of the antimalarial drug mefloquine by human liver microsomes (N = 6) in vitro. The only metabolite generated was identified as carboxymefloquine by co-chromatography with the authentic standard. Ketoconazole caused marked inhibition of carboxymefloquine formation with IC50 and Ki values of 7.5 and 11.2 microM, respectively. The inhibition of ketoconazole, a known inhibitor of cytochrome P450 isozymes, and the dependency of metabolite formation on the presence of NADPH indicated that cytochrome P450 isozyme(s) catalysed metabolite production. Of compounds actually or likely to be coadministered with mefloquine to malaria patients only primaquine and quinine produced marked inhibition (IC50, 17.5 and 122 microM; Ki, 8.6 and 28.5 microM, respectively). However, despite these in vitro data with primaquine, clinical studies have failed to show any significant effect of single dose primaquine on the pharmacokinetics of mefloquine. With quinine, because peak plasma concentrations are very close to the Ki value, there is likely to be inhibition of mefloquine metabolism in patients receiving both drugs. Sulfadoxine, artemether, artesunate and tetracycline did not significantly inhibit carboxymefloquine formation.

Biotransformation↗
PubMed1992

Relative expression of cytochrome P450 isoenzymes in human liver and association with the metabolism of drugs and xenobiotics.

Cytochrome P450s play a central role in the metabolism and disposition of an extremely wide range of drugs and chemical carcinogens. Individual differences in the expression of these enzymes may be an important determinant in susceptibility to adverse drug reactions, chemical toxins and mutagens. In this paper, we have measured the relative levels of expression of cytochrome P450 isoenzymes from eight gene families or subfamilies in a panel of twelve human liver samples in order to determine the individuality in their expression and whether any forms are co-regulated. Isoenzymes were identified in most cases on Western blots based on the mobility of authentic recombinant human cytochrome P450 standards. The levels of the following P450 proteins correlated with each other: CYP2A6, CYP2B6 and a protein from the CYP2C gene subfamily, CYP2E1 and a member of the CYP2A gene subfamily, CYP2C8, CYP3A3/A4 and total cytochrome P450 content. Also, the levels of two proteins in the CYP4A gene subfamily were highly correlated. These correlations are consistent with the relative regulation of members of these gene families in rats or mice. In addition, the level of expression of specific isoenzymes has also been compared with the rate of metabolism of a panel of drugs, carcinogens and model P450 substrates. These latter studies demonstrate and confirm that the correlations obtained in this manner represent a powerful approach towards the assignment of the metabolism of substrates by specific human P450 isoenzymes.

Antibodies↗
PubMed1992

Effect of tetracycline on mefloquine pharmacokinetics in Thai males.

The kinetics of a single oral dose of mefloquine given either alone or with tetracycline has been studied in 20 healthy Thai male volunteers. There was a significantly higher maximum whole blood mefloquine concentration after coadministration with tetracycline (1600 vs 1160 ng.ml-1), as well as a significantly reduced terminal half-life (14.4 vs 19.3 days), mean residence time (11.9 vs 16.0 days) and volume of distribution at steady state (13.3 vs 19.9 1.kg-1). Although there was no significant change in the AUC from zero time to infinity, the AUC from zero time to 7 days was significantly increased by tetracycline (6.18 vs 4.76 micrograms.ml-1.day). The changes in mefloquine disposition after tetracycline treatment are probably due to a reduction in enterohepatic recycling. The initial increase in mefloquine AUC without an apparent increase in side-effects suggests that this combination may have a place in the treatment of multi-drug resistant falciparum malaria.

Adult↗