Optimal design for a study of butadiene toxicokinetics in humans.
The derivation of the optimal design for an upcoming toxicokinetic study of butadiene in humans is presented. The specific goal of the planned study is to obtain a precise estimate of butadiene metabolic clearance for each study subject, together with a good characterization of its population variance. We used a two-compartment toxicokinetic model, imbedded in a hierarchical population model of variability, in conjunction with a preliminary set of butadiene kinetic data in humans, as a basis for design optimization. Optimization was performed using Monte Carlo simulations. Candidate designs differed in the number and timing of exhaled air samples to be collected. Simulations indicated that only 10 air samples should be necessary to obtain a coefficient of variation of 15% for the estimated clearance rate, if the timing of those samples is properly chosen. Optimal sampling times were found to closely bracket the end of exposure. This efficient design will allow the recruitment of more subjects in the study, in particular to match prescribed levels of accuracy in the estimate of the population variance of the butadiene metabolic rate constant. The techniques presented here have general applicability to the design of human and animal toxicology studies. (+info)
Development of a transgenic mouse model for carcinogenesis bioassays: evaluation of chemically induced skin tumors in Tg.AC mice.
Transgenic rodent models have emerged as potentially useful tools in the assessment of drug and chemical safety. The transgenic Tg.AC mouse carries an inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin to these animals. The induction of epidermal papillomas in the area of topically applied chemical agents, for duration of not more than 26 weeks, acts as a reporter phenotype that defines the activity of the test article. We describe here the activity of six chemicals that have been previously characterized for activity in the standard 2-year bioassay conducted by the National Toxicology Program (NTP). Homozygous female Tg.AC mice were treated with benzene (BZ), benzethonium chloride (BZTC), o-benzyl-p-chlorophenol (BCP), 2-chloroethanol (2-CE), lauric acid diethanolamine (LADA) and triethanolamine (TEA). BZ and LADA induced skin papillomas in a dose-dependent manner, while BCP induced papillomas only at the highest dose. BZTC, 2-CE, and TEA exhibited no activity. The correspondence of chemical activity in Tg.AC mice with that in the 2-year bioassay was high. A comparison of responsiveness to BZ and LADA was made between hemizygous and homozygous female Tg.AC mice. Both genotypes appear to be equally sensitive to maximum doses of active compounds. The results reported here indicate that the Tg.AC transgenic mouse model can discriminate between carcinogens and noncarcinogens and that both mutagenic and nonmutagenic chemicals can be detected. These studies provide support for the adjunctive use of the Tg.AC transgenic mouse skin tumor model in drug and chemical safety assessment and for the prediction of the carcinogenic potential of chemicals. (+info)
A retrospective study of buprenorphine and norbuprenorphine in human hair after multiple doses.
The analysis of hair has been proposed as a tool for monitoring drug-treatment compliance. This study was performed to determine if buprenorphine (BPR) and norbuprenorphine (NBPR) could be detected in human hair after controlled administration of drug and to determine if segmental analysis of hair was an accurate record of the dosing history. Subjects with dark hair (six males, six females) received 8 mg sublingual BPR for a maximum of 180 days. Single hair collections were made once after BPR treatment and stored at -20 degrees C until analysis. Hair was aligned scalp-end to tip and then segmented in 3-cm sections. For this study, it was assumed that the mean hair growth rate was 1.0 cm/month. Deuterated internal standard was added to hair segments (2-20 mg of hair) and digested overnight at room temperature with 1 N NaOH. Specimens were extracted with a liquid-liquid procedure and analyzed by liquid chromatography-tandem mass spectrometry. The limits of quantitation for BPR and NBPR were 3 pg/mg and 5 pg/mg, respectively, for 20 mg of hair. BPR and NBPR concentrations were highest for all subjects in hair segments estimated to correspond to the subject's period of drug treatment. With one exception, NBPR was present in higher concentrations in hair than was the parent compound. BPR concentrations in hair segments ranged from 3.1 pg/mg to 123.8 pg/mg. NBPR concentrations ranged from 4.8 pg/mg to 1517.8 pg/mg. In one subject, BPR and NBPR were not detected in any hair segment. In some subjects, BPR and NBPR were detected in hair segments that did not correspond to the period of drug treatment, suggesting that drug movement may have occurred by diffusion in sweat and other mechanisms. The data from this study also indicate that there is a high degree of intersubject variability in measured concentration of BPR and NBPR in hair segments, even when subjects receive the same dose for an equivalent number of treatment days. Future prospective studies involving controlled drug administration will be necessary to evaluate whether hair can serve as an accurate historical record of variations in the pattern of drug use. (+info)
Inter-individual variability in drug response is a major clinical problem. Adverse drug reactions (ADRs) are common, are responsible for a number of debilitating side effects following drug therapy and are a significant cause of death. It is now clear that much of the observed variability in drug response has a genetic basis, arising as a result of genetically-determined differences in drug absorption, disposition, metabolism or excretion. The best characterised pharmacogenetic polymorphisms are those within the phase I cytochrome P450 family of drug metabolising enzymes. One of these enzymes, CYP2D6 (debrisoquine hydroxylase), metabolizes one-quarter of all prescribed drugs and is inactive in 6% of the Caucasian population. Individuals at risk of developing ADRs as a result of genetically-determined variation in genes such as CYP2D6 can now be identified using DNA-based tests. A detailed knowledge of the genetic basis of individual drug response is potentially of major clinical and economic importance and could provide the basis for a rational approach to drug prescription. This would have significant benefits for human health. (+info)
Pharmacogenomics: the genomics of drug response.
Pharmacogenomics is defined as the study of the association between genetics and drug response. This is a rapidly expanding field with the hope that, within a few years, prospective genotyping will lead to patients being prescribed drugs which are both safer and more effective ('the right drug for the right patient', or personalized medicine). There are many existing examples in the literature of strong associations between genetic variation and drug response, and some of these even form the basis of accepted clinical tests. The molecular basis for some of these associations is described, and includes examples of variation in genes responsible for absorption and metabolism of the drug, and in target and disease genes. However, there are many issues surrounding the legal, regulatory and ethical framework to these studies that remain unanswered, and a huge amount of education both for the public and healthcare professionals will be needed before the results of this new medicine can be widely accepted. (+info)
Impact of genomics on drug discovery and clinical medicine.
Genomics, particularly high-throughput sequencing and characterization of expressed human genes, has created new opportunities for drug discovery. Knowledge of all the human genes and their functions may allow effective preventive measures, and change drug research strategy and drug discovery development processes. Pharmacogenomics is the application of genomic technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. It applies the large-scale systematic approaches of genomics to speed the discovery of drug response markers, whether they act at the level of the drug target, drug metabolism, or disease pathways. The potential implication of genomics and pharmacogenomics in clinical research and clinical medicine is that disease could be treated according to genetic and specific individual markers, selecting medications and dosages that are optimized for individual patients. The possibility of defining patient populations genetically may improve outcomes by predicting individual responses to drugs, and could improve safety and efficacy in therapeutic areas such as neuropsychiatry, cardiovascular medicine, endocrinology (diabetes and obesity) and oncology. Ethical questions need to be addressed and guidelines established for the use of genomics in clinical research and clinical medicine. Significant achievements are possible with an interdisciplinary approach that includes genetic, technological and therapeutic measures. (+info)
Cytochrome P450 1B1 (CYP1B1) pharmacogenetics: association of polymorphisms with functional differences in estrogen hydroxylation activity.
Activation of 17beta-estradiol (E2) through the formation of catechol estrogen metabolites, 2-OH-E2 and 4-OH-E2, and the C-16alpha hydroxylation product, 16alpha-OH-E2, has been postulated to be a factor in mammary carcinogenesis. Cytochrome P450 1B1 (CYP1B1) exceeds other P450 enzymes in both estrogen hydroxylation activity and expression level in breast tissue. To determine whether inherited variants of CYP1B1 differ from wild-type CYP1B1 in estrogen hydroxylase activity, we expressed recombinant wild-type and five polymorphic variants of CYP1B1: variant 1 (codon 48Arg-->Gly), variant 2 (codon 119Ala-->Ser), variant 3 (codon 432Val-->Leu), variant 4 (codon453Asn-->Ser), variant 5 (48Gly, 119Ser, 432Leu, 453Ser). The His-tagged proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography and analyzed by electrophoresis and spectrophotometry. We performed assays of E2 hydroxylation activity and quantitated production of 2-OH-E2, 4-OH-E2, and 16alpha-OH-E2 by gas chromatography/mass spectrometry. Wild-type CYP1B1 formed 4-OH-E2 as main product (Km, 40+/-8 microM; k(cat) 4.4+/-0.4, min(-1); k(cat)/Km, 110 mM(-1) min(-1)), followed by 2-OH-E2 (Km, 34+/-4 microM; k(cat), 1.9+/-0.1 min(-1); k(cat)/Km, 55 mM(-1)min(-1)) and 16alpha-OH-E2 (Km, 39+/-5.7 microM; k(cat), 0.30+/-0.02 min(-1); k(cat)/Km, 7.6 mM(-1)min(-1)). The CYP1B1 variants also formed 4-OH-E2 as the main product but displayed 2.4- to 3.4-fold higher catalytic efficiencies k(cat)/Km than the wild-type enzyme, ranging from 270 mM(-1)min(-1) for variant 4, to 370 mM(-1)min(-1) for variant 2. The variant enzymes also exceeded wild-type CYP1B1 with respect to 2- and 16alpha-hydroxylation activity. Thus, inherited alterations in CYP1B1 estrogen hydroxylation activity may be associated with significant changes in estrogen metabolism and, thereby, may possibly explain interindividual differences in breast cancer risk associated with estrogen-mediated carcinogenicity. (+info)
Rabbit cytochrome P450 4B1: A novel prodrug activating gene for pharmacogene therapy of hepatocellular carcinoma.
Gene therapy using vector-mediated transfer of prodrug activating genes is a promising treatment approach for malignant tumors. As demonstrated recently, the novel prodrug activating gene coding for rabbit cytochrome P450 4B1 (CYP4B1) is able to induce tumor cell death at low micromolar concentrations in glioblastoma cells after treatment with the prodrug 4-ipomeanol (4-IM) in vitro and in vivo. The rabbit CYP4B1 converts this prodrug and other furane analogs and aromatic amines, such as 2-aminoanthracene, to highly toxic alkylating metabolites, whereas the human isoenzyme exhibits only minimal enzymatic activity. In the present study, the cDNA encoding rabbit CYP4B1 was used for pharmacogene therapy of hepatocellular carcinoma (HCC). Cell clones derived from the human HCC cell lines Hep3B, HuH-7, and HepG2 and stably expressing the chimeric protein CYP4B1-EGFP (the CYP4B1 coding sequence fused to the enhanced green fluorescent protein (EGFP) gene) were selected. HCC clones expressing EGFP served as controls. 4-IM rapidly induced tumor cell death in CYP4B1-EGFP-expressing clones at low concentrations (a 50% lethal dose of between 0.5 and 2 microg/mL). No signs of toxicity were found in control cells expressing EGFP even at high prodrug concentrations (20 microg/mL). Cell death occurred by apoptosis and was independent of functional p53. A pronounced direct bystander effect was observed in Hep3B cells, whereas bystander HepG2 and HuH-7 cells were highly resistant to toxic 4-IM metabolites. These results demonstrate that the CYP4B1/4-1M system efficiently and rapidly induces cell death in HCC cells, and that a cell line-specific mechanism may exist that limits the extent of the bystander effect of this novel prodrug activating system. (+info)