Inhibition of human CYP3A catalyzed 1'-hydroxy midazolam formation by ketoconazole, nifedipine, erythromycin, cimetidine, and nizatidine.
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Chromatography, High Pressure Liquid↗
Biomedical subjects
B J Ring
Publications and source records attributed to B J Ring.
In vitro methods for assessing human hepatic drug metabolism: their use in drug development.
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Cytochrome P-450 Enzyme System↗
The influence of time of maternal exposure to 2,4,5,2',4',5'-hexachlorobiphenyl on its accumulation in their nursing offspring.
2,4,5,2',4',5'-Hexachlorobiphenyl (6-CB) is mobilized from rodent tissues during the lipid depletion associated with food restriction or lactation, the latter condition resulting in the substantial elimination of the maternal body burden of the chemical to nursing offspring. The present study was undertaken to determine whether the rate and/or magnitude of accumulation of 6-CB in nursing offspring differed with time following PCB administration to the maternal animal. Female ICR mice were administered two doses of 6-CB. Group I animals received [14C]-6-CB as weanlings (15-20 g) followed by unlabeled 6-CB 5 weeks later, after mating, on Day 1 of gestation. Group II received unlabeled 6-CB as weanlings and [14C]-6-CB on Day 1 of gestation. Thus, 14C identified the mobilization and elimination of either the first or the second dose of 6-CB in the treatment groups (I = [14C]-6-CB, 6-CB; II = 6-CB, [14C]-6-CB). Both groups of animals retained approximately 80% of the administered radiolabeled dose. The tissue distribution of [14C]-6-CB in group II as a percentage of the body burden was not different from that in group I as determined from maternal tissue concentrations on Day 14 of gestation. The percentage of the maternal body burden of [14C]-6-CB accumulated in suckling offspring of group II mothers was significantly greater than that in group I offspring on Day 1 (I, 2.2 +/- 0.5%; II, 3.5 +/- 0.4%), Day 3 (I, 14.8 +/- 1.9%; II, 24.6 +/- 2.7%), Day 5 (I, 16.8 +/- 1.4%; II, 24.8 +/- 0.8%), and Day 12 (I, 32.3 +/- 0.5%; II, 45.5 +/- 1.7%) postpartum. This differential elimination was reflected in the t1/2 of elimination of the radiolabeled dose from parametrial fat during lactation, which was significantly longer in group I (14 days) than group II maternal animals (9 days). The observations that the last dose of 6-CB administered was the first to be mobilized from the whole animal, and that this was reflected in 6-CB release from parametrial fat, suggest that this highly lipophilic chemical is not homogeneously distributed within storage depots.
Adipose Tissue↗
The effect of diet and litter size on the elimination of 2,4,5,2',4',5'-[14C]hexachlorobiphenyl from lactating mice.
It was shown that 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) administered to adult female mice accumulated in their nursing offspring more rapidly than a dose administered to weanling mice when treated animals were bred at equivalent ages. This suggested that the PCB was eliminated from the maternal animal relative to its time of sequestration into storage depots. Using a model which more closely approximates conditions during human lactation, the influence of a high-fat diet and decreased litter size on this phenomenon was examined. Female ICR mice were treated with 4 mg/kg [14C]-6-CB as 13-g weanlings (dW) at 3 weeks of age or as adults (dA) at 11 weeks of age. All animals were mated at 11 weeks of age. On Day 1 of pregnancy, mice were placed on a low-fat (11.5% of the total calories) or high-fat (43.8% of total calories) diet. At parturition, litters were adjusted to either two or eight within each diet group. Elimination of maternal 6-CB was determined by assessing radioactivity in offspring carcasses on Day 15 of gestation or Day 1, 3, 5, 10, or 15 postpartum. Consumption of a high-fat diet significantly extended the t1/2 of elimination of 6-CB from mothers nursing a litter of two in the dW group (low fat = 7.3 days; high fat = 12.4 days) and in both the dW and dA groups nursing litters of eight (dW: low fat = 4.6 days; high fat = 6.8 days; and dA: low fat = 1.8 days; high fat = 3.0 days). Within diet and group, reducing litter size to two also significantly decreased the rate of elimination of 6-CB from maternal animals. 6-CB was eliminated to offspring more rapidly from the dA group when compared to the dW group regardless of diet in animals nursing litters of eight. This relationship was not observed in maternal animals nursing litters of two. In general, exposure to a high-fat diet increased the t1/2 of elimination of 6-CB from maternal animals. Reducing litter size had a greater influence and also masked the ordered mobilization of 6-CB relative to its time of sequestration. Thus, under experimental conditions which more closely mimic the human situation, 6-CB was eliminated less rapidly in the rodent, and sequential 6-CB doses were equally available to nursing offspring.
Adipose Tissue↗
Computed temperature distributions around ectatic capillaries exposed to yellow (578 nm) laser light.
We have numerically modelled the thermal effects of yellow (577 or 578 nm) light illumination on the ectatic blood vessels of port wine stains. We investigated the effect of the laser treatment parameters of light irradiance and illumination time on the extent of coagulation (coagulation thickness) of the endothelial cells of the ectatic vessels. We assumed that this coagulation is dependent on heating the cells to a critical temperature (coagulation temperature). We iteratively adjusted the treatment parameters so that the model vessels had a maximum temperature that did not exceed the boiling point of blood. Given the likely range of variation of coagulation temperature, coagulation thickness and vessel size, coagulation temperature was found to have the greatest effect on the treatment parameters. Variations in coagulation thickness had less influence, and the diameter of the ectatic vessel (typically in the 30-80 microns diameter range) had the least effect. The treatment parameters that have been shown clinically to cause purpura (e.g. 6-7 cm-2 in 360 microseconds) were also studied. The purpura seen by some workers using pulsed dye lasers is most likely to be the result of vaporisation of blood leading to the rupture of the vessel. We conclude that, in order to achieve coagulation with these short laser pulses, the choice of irradiance is critical.
Capillaries↗
Thermal profiles of blood vessels heated by a laser.
Thermal profiles of ectatic capillaries, modelled on those found in port wine stain birthmarks, are calculated by a method of finite differences. Yellow 578nm light is assumed to illuminate these vessels. The coagulation of endothelial cells is assumed to occur when the cells are heated to at least 70 degrees C. We model this by asking that a point 6 microns above the top of the vessel lumen attains a temperature of 70 degrees C. We constrain the parameters to prevent heating of blood above 100 degrees C, so as to avoid vapourisation. The treatment parameters of dose and illumination time are varied until they produce thermal profiles that show the model's coagulation conditions. being treated and are rather longer than the thermal relaxation time of the vessel and also much longer than the pulses of pulsed dye lasers.
Body Temperature↗
Identification of a polymorphically expressed member of the human cytochrome P-450III family.
The human liver cytochrome P-450III family contains two highly related forms of cytochrome P-450 (P450), HLp and P450NF, that are expressed in the adult and a form, HLp2, that is expressed in the fetus. Immunoblot analyses of 46 liver specimens developed with an anti-HLp antibody demonstrated that, in addition to HLp, 11 specimens contained a previously undected higher molecular weight protein (termed HLp3). The expression of HLp3 did not correlate with the age, gender, smoking habits, or drug history of the patients. This protein was purified and found to be a P450 with a molecular weight of 52,000. Ouchterlony analyses using a polyclonal anti-HLp antibody yielded lines of partial identity between HLp3 and both HLp and HLp2. In addition, structural comparisons between these three proteins, including amino-terminal amino acid analyses and peptide mapping, indicated that HLp3, HLp2, and HLp are highly related but distinct proteins. In reconstituted systems, HLp and HLp3 were found to hydroxylate testosterone at the 6 beta and 2 beta positions. In microsomes, the rates of hydroxylation of testosterone at the 2 beta, 6 alpha, 6 beta, 15 beta, and 18 positions and the formation of an unknown were found to correlate with the levels of total HLp-related protein and these activities were inhibited by anti-HLp antibodies. In conclusion, our data demonstrate that HLp3 is a member of the human P450III family and is polymorphically expressed.
Amino Acid Sequence↗
Transfer of 2,4,5,2',4',5'-hexachlorobiphenyl across the in situ perfused guinea pig placenta.
The transplacental crossover of 14C-2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) from the maternal circulation to the fetal side of the placenta was examined in intact fetuses and following the in situ perfusion of the guinea pig placenta. Fetal, late pregnant, and nonpregnant female guinea pig lipoprotein profiles and the association of 6-CB with these plasma constituents were also determined in vivo. Very low density lipoprotein (VLDL) concentrations were 10-fold higher in fetal than in maternal plasma, and the great majority of 6-CB which was transferred to intact fetuses became associated with this plasma fraction. 6-CB was found primarily in association with low density lipoproteins (LDL) in nonpregnant animals. In the late pregnant guinea pig, 6-CB became primarily associated with plasma protein in spite of circulating protein concentrations lower than those seen in the nonpregnant state. No differences in the levels of the three plasma lipoprotein classes were observed between pregnant and nonpregnant animals. It was found that an amount of 6-CB similar to that found in intact litter mates crossed the perfused placenta over the same time period. Despite the much higher VLDL concentrations on the fetal side of the placenta and the association of 6-CB with VLDL in intact fetuses, addition of 1,000 mg/dl VLDL to the 5.4% bovine serum albumin perfusion medium failed to influence the magnitude of 6-CB crossover. 6-CB crossover was influenced by protein concentration in the perfusion media in a concentration-dependent fashion. It is hypothesized that 6-CB and free fatty acids traverse the placenta and are retained by the fetus via similar mechanisms.
Animals↗
Chronic effects of fractionated renal irradiation on the pharmacokinetics of intravenous methotrexate.
the chronic effects of renal irradiation on the pharmacology of methotrexate was studied in a rat model. Unanesthetized rats received 2 doses of bilateral fractionated kidney irradiation (16.2 Gy or 19.8 Gy in 9 fractions). Alterations in renal function were first seen at 3 months in the 19.8 Gy group and 12 months in the 16.2 Gy groups. Life table analysis showed a shift in the survival curve of about 3 months between the 2 radiation doses. The pharmacokinetics of i.v. methotrexate showed an increase in the area under the plasma curve beginning at 9 months in the 19.8 Gy group and at 15 months in the 16.2 Gy group. The volume of distribution of methotrexate was smaller in the irradiated rats than in unirradiated controls. Multiple linear regression models showed significant correlations between parameters of methotrexate clearance and certain renal function tests. Nevertheless, no set of renal function tests consistently predicted alteration in methotrexate clearance in the 2 radiation groups. Furthermore, time after irradiation remained a highly significant variable indicating that renal irradiation causes time dependent change in methotrexate pharmacokinetics that can not be accounted for by the usual tests of renal function.
Animals↗
Influence of lipolysis on the mobilization of 2,4,5,2'4',5'-hexachlorobiphenyl from adipocytes in vitro.
Epididymal adipocytes, isolated from rats pretreated with [14C]-2,4,5,2',4',5'-hexachlorobiphenyl (6-CB), were utilized to examine the relationship between the mobilization of lipid and 6-CB and to determine whether 6-CB was differentially associated with subcellular organelles over time as has been demonstrated for newly synthesized lipid. Lipolysis, induced by the presence of 8 X 10(-7) M isoproterenol (ISO) for 50 min, depleted approximately 1% of total cellular triacylglycerols (TG) regardless of time from treatment with 6-CB. The percentage of cellular 6-CB released from adipocytes to incubation buffer infranatants was not correlated with the magnitude of lipolysis produced over the 50-min incubation period; nor was the percentage of 6-CB released to the buffers correlated with the length of the incubation period, regardless of the presence of ISO. Although adipocytes responded similarly to lipolytic stimuli independent of time (days) since 6-CB treatment, significant decreases were found in the percentage of 6-CB released from adipocytes over time. The in vitro labeling of this newly synthesized TG in fat cells with [U-14C]glucose or [1-14C]palmitate demonstrated that TG was differentially distributed among adipocyte organelles. Newly synthesized TG was also the first to be mobilized under lipolytic stimulus. 6-CB was not released in a similar fashion, since radioactivity associated with the chemical levels of [14C]-6-CB and glucose-derived 14C in buffers were not correlated over time. 6-CB was found to redistribute to all available lipid pools during the subcellular fractionation procedure and thus did not resemble TG. However, it is possible that 6-CB may exist in equilibrium among organelle fractions and that it becomes sequestered within the nonsoluble lipid compartment with time, thus decreasing its appearance in the soluble buffer infranatants over the experimental time course.
Adipose Tissue↗
The lack of effects of alkylating agents on mammalian cell membranes.
The importance of cell membrane components as target sites for the action of 2,3,5-tris(ethyleneimino)-benzoquinone (Trenimon), mechlorethamine hydrochloride (HN2) and tris(2-chloroethyl) amine hydrochloride (HN3) was investigated. Uptake of 2-aminoisobutyric acid (AIBA) was studied under nonsaturating conditions where the transport system was rate-limiting for the uptake. Uptake of AIBA into L5178Y leukaemic cells was either inhibited or stimulated, depending on the type of the drug, the drug concentration and the length of incubation. Treatment of human erythrocytes with 10(-3) M HN3 produced new high-molecular-weight protein bands on SDS-polyacrylamide gel electrophoresis. Conversely, HN3 had no effect on L5178Y cell membrane proteins. Neither HN2 nor Trenimon produced any detectable changes in membrane proteins of L5178Y cells or human erythrocytes. None of the three drugs at concentrations and incubation conditions which inhibited cell replication changed the stoichiometry or dissociation constant of concanavalin A (Con A) binding sites on L5178Y cells. Trenimon at highly toxic concentrations had no effect on the fluidity of phospholipid membranes or of membranes of Ehrlich ascites tumour cells as analysed by ESR spin-label methods. The results presented here do not support the hypothesis that cell membranes are the primary target sites for alkylating drugs.
Alkylating Agents↗
Effect of asparaginase on cell membranes of sensitive and resistant mouse lymphoma cells.
High concentrations of Escherichia coli asparaginase (80 U/ml) altered the binding of concanavalin A (Con A) to L 5178Y murine lymphoma cells that are sensitive to the cytotoxic action of this enzyme. Incubation of the asparaginase sensitive line in asparagine-free media or media containing Acinetobacter glutaminase-asparaginase did not alter the Con A binding of these cells. Escherichia coli asparaginase had no effect on Con A binding of two asparaginase resistant L5178Y cell lines that were isolated and maintained in asparagine depleted or asparaginase containing medium. The E. coli asparaginase preparation inhibited protein and glycoprotein biosynthesis to comparable degrees. It did not have proteolytic or glycolytic activity. Escherichia coli asparaginase did not alter the binding of wheat germ, soybean or ricin agglutinins to any of these cell lines. These data suggest that high concentrations of E. coli asparaginase have a specific effect on the Con A receptor in the sensitive line.
Amino Acids↗
Effects of single-dose and fractionated cranial irradiation on rat brain accumulation of methotrexate.
The effects of single-dose and fractionated whole-brain irradiation on brain methotrexate (MTX) has been studied in a rat model. The amount of MTX present in the brain 24 hr after a single i.p. dose (100 mg/kg) was the same wether animals were sham irradiated or given a single dose of 2000 rads 6 or 48 hr prior to the drug (6.9, 8.3, and 6.8 pmol MTX/g, wet weight, respectively). Animals sham irradiated or given 2000 rads in 10 fractions over 11 days and treated with an average dose of 1.2 mg MTX/kg i.p. twice a week for 24 weeks did not differ significantly in their brain MTX concentration (7.9 and 8.3 pmol MTX/g, wet weight, respectively). Chronically MTX-treated animals became folate deficient whether they were irradiated or not (450 and 670 pmol folate/g, wet weight, brain in MTX-treated and control animals). Thus, MTX accumulates in the brain with acute or chronic administration, and this accumulation is not altered by this amount of brain irradiation.
Animals↗
Effect of hepatic irradiation on the toxicity and pharmacokinetics of adriamycin in children.
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Child↗
Phase I study of succinylated acinetobacter L-glutaminase-L-asparaginase.
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Acinetobacter↗
Human pharmacology and toxicology of succinylated Acinetobacter glutaminase-asparaginase.
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Acinetobacter↗
Present and future in vitro approaches for drug metabolism.
The 1980s through 1990s witnessed the widespread incorporation of in vitro absorption, distribution, metabolism, and excretion (ADME) approaches into drug development by drug companies. This has been exemplified by the integration of the basic science of cytochrome P450s (CYPs) into most drug metabolism departments so that information on the metabolic pathways of drugs and drug-drug interactions (DDIs) is no longer an academic exercise, but essential for regulatory submission. This has come about due to the application of a variety of new technologies and in vitro models. For example, subcellular fractions have been widely used in metabolism studies since the 1960s. The last two decades has seen the increased use of hepatocytes as the reproducibility of cell isolations improved. The 1990s saw the rejuvenation of liver slices (as new slicers were developed) and the utilization of cDNA expressed enzymes as these technologies matured. In addition, there has been considerable interest in extrapolating in vitro data to in vivo for parameters such as absorption, clearance and DDIs. The current philosophy of drug development is moving to a 'fail early--fail cheaply' paradigm. Therefore, in vitro ADME approaches are being applied to drug candidates earlier in development since they are essential for identifying compounds likely to present ADME challenges in the latter stages of drug development. These in vitro tools are also being used earlier in lead optimization biology, in parallel with approaches for optimizing target structure activity relationships, as well as identification of DDI and the involvement of metabolic pathways that demonstrate genetic polymorphisms. This would suggest that the line between discovery and development drug metabolism has blurred. In vitro approaches to ADME are increasingly being linked with high-throughput automation and analysis. Further, if we think of perhaps the fastest available way to screen for successful drugs with optimal ADME characteristics, then we arrive at predictive computational algorithms, which are only now being generated and validated in parallel with in vitro and in vivo methods. In addition, as we increase the number of ADME parameters determined early, the overall amount of data generated for both discovery and development will increase. This will present challenges for the efficient and fast interpretation of such data, as well as incorporation and communication to chemistry, biology, and clinical colleagues. This review will focus on and assess the nature of present in vitro metabolism approaches and indicate how they are likely to develop in the future.
Animals↗
A novel testosterone 6 beta-hydroxylase activity assay for the study of CYP3A-mediated metabolism, inhibition, and induction in vitro.
INTRODUCTION: In order to examine CYP3A-mediated metabolism in vitro, a unique analytical assay was developed to detect the formation of 63-hydroxytestosterone (63-OHT). This assay has been determined to be useful for the study of both inhibition- and induction-related drug-drug interactions in vitro and involves simple incubation and sample handling procedures. METHODS: A primary and three backup sets of analytical conditions were developed to detect interference between a test compound and either 6beta-OHT or the internal standard. RESULTS: The primary set of conditions was validated with a three-batch validation, and the remaining sets of conditions were validated with one-batch validations, all in human liver microsomes. The primary assay was also validated with a single batch for CYP3A induction studies in primary human hepatocytes. Enzyme kinetic parameters of 6beta-OHT formation (Km, Vmax) were determined to be reproducible in human liver microsomes. DISCUSSION: Utility of the assay in inhibition studies and induction studies, respectively, was confirmed with the test compounds ketoconazole and rifampicin. In addition, superiority to existing methods was demonstrated in three areas: ease of sample preparation, short run times, and low detection limits.
Cytochrome P-450 CYP3A↗