Alexander Jetter

Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the cocktail approach

Phenotyping for drug metabolizing enzymes and transporters is used to assess quantitatively the effect of an intervention (e.g., drug therapy, diet) or a condition (e.g., genetic polymorphism, disease) on their activity. Appropriate selection of test drug and metric is essential to obtain results applicable for other substrates of the respective enzyme/transporter. The following phenotyping metrics are recommended based on the level of validation and on practicability: CYP1A2, paraxanthine/caffeine in plasma 6 h after 150 mg caffeine; CYP2C9, tolbutamide plasma concentration 24 h after 125 mg tolbutamide; CYP2C19, urinary excretion of 4′‐OH‐mephenytoin 0–12 h after 50 mg mephenytoin; CYP2D6, urinary molar ratio debrisoquine/4‐OH‐debrisoquine 0–8 h after 10 mg debrisoquine; and CYP3A4, plasma clearance of midazolam after 2 mg midazolam (all drugs given orally).

Impact of CYP2B6 983T>C polymorphism on non-nucleoside reverse transcriptase inhibitor plasma concentrations in HIV-infected patients

OBJECTIVES The aim of this study was to investigate the frequency of CYP2B6 polymorphisms (according to ethnicity) and the influence of heterozygosity and homozygosity on plasma concentrations of efavirenz and nevirapine. METHODS Following written informed consent, 225 Caucasians and 146 Blacks were recruited from the German Competence Network for HIV/AIDS. Plasma concentrations of efavirenz and nevirapine were assessed by HPLC, and genotyping for 516G>T, 983T>C and 1459T>C polymorphisms in CYP2B6 was conducted by real-time PCR-based allelic discrimination. RESULTS The minor allele frequency for 516G>T, 983T>C and 1459T>C was 0.29, 0 and 0.08 in Caucasians and 0.34, 0.07 and 0.02 in Blacks, respectively. Two Black patients with the 983C allele receiving efavirenz were identified and both were withdrawn from therapy within 1 week of sampling due to toxicity. In multivariate analyses, efavirenz and nevirapine plasma concentrations were significantly associated with 983T>C (P < 0.0001 and P = 0.02, respectively), 516G>T (P < 0.0001 and P = 0.002, respectively) and time of drug analysis post-dose (P < 0.0001 for both). Body mass index was independently related to efavirenz (P = 0.04) but not nevirapine concentrations, and age was related to nevirapine (P = 0.05) but not efavirenz concentrations. Consistent with other studies, 1459C>T was not associated with plasma concentrations of either drug (P > 0.05 for both drugs). CONCLUSIONS This is the first report that the 983T>C genotype (part of the CYP2B6*18 haplotype) impacts on nevirapine plasma concentrations and the first study to assess the impact of 983C homozygosity on efavirenz concentrations. These data have implications for administration of non-nucleoside reverse transcriptase inhibitors to Black patients.

Should We Use N-Acetyltransferase Type 2 Genotyping To Personalize Isoniazid Doses?

ABSTRACT Isoniazid is metabolized by the genetically polymorphic arylamine N-acetyltransferase type 2 (NAT2). A greater number of high-activity alleles are related to increased acetylation capacity and in some reports to low efficacy and toxicity of isoniazid. The objective of this study was to assess individual isoniazid exposure based on NAT2 genotype to predict a personalized therapeutic dose. Isoniazid was administered to 18 healthy Caucasians (age 30 ± 6 years, body weight 74 ± 10 kg, five women) in random order as a 200-mg infusion, a 100-mg oral, and a 300-mg oral single dose. For the assessment of NAT2 genotype, common single nucleotide polymorphisms identifying 99.9% of variant alleles were characterized. Noncompartmental pharmacokinetics and compartmental population pharmacokinetics were estimated from isoniazid plasma concentrations until 24 h postdose by high-pressure liquid chromatography. The influence of NAT2 genotype, drug formulation, body weight, and sex on dose-normalized isoniazid pharmacokinetics was assessed by analysis of variance from noncompartmental data and confirmed by population pharmacokinetics. Eight high-activity NAT2*4 alleles were identified. Sex had no effect; the other factors explained 93% of the variability in apparent isoniazid clearance (analysis of variance). NAT2 genotype alone accounted for 88% of variability. Individual isoniazid clearance could be predicted as clearance (liters/hour) = 10 + 9 × (number of NAT2*4 alleles). To achieve similar isoniazid exposure, current standard doses presumably appropriate for patients with one high-activity NAT2 allele may be decreased or increased by approximately 50% for patients with no or two such alleles, respectively. Prospective clinical trials are required to assess the merits of this approach.

Pharmacokinetics and pharmacodynamics of rosiglitazone in relation to CYP2C8 genotype.

Rosiglitazone is metabolically inactivated predominantly via the cytochrome P450 (CYP) enzyme CYP2C8. The functional impact of the CYP2C8*3 allele coding for the Arg139Lys and Lys399Arg amino acid substitutions is controversial. The purpose of this was to clarify the role of this polymorphism with regard to the pharmacokinetics and clinical effects of rosiglitazone.

Oral supplementation with 25(OH)D3 versus vitamin D3: effects on 25(OH)D levels, lower extremity function, blood pressure, and markers of innate immunity.

To test the effect of 25(OH)D3 (HyD) compared to vitamin D3 on serum 25‐hydroxyvitamin D levels (25(OH)D), lower extremity function, blood pressure, and markers of innate immunity. Twenty healthy postmenopausal women with an average 25(OH)D level of 13.2 ± 3.9 ng/mL (mean ± SD) and a mean age of 61.5 ± 7.2 years were randomized to either 20 µg of HyD or 20 µg (800 IU) of vitamin D3 per day in a double‐blind manner. We measured on 14 visits over 4 months, 25(OH)D serum levels, blood pressure, and seven markers of innate immunity (eotaxin, interleukin [IL]‐8, IL‐12, interferon gamma‐induced protein 10 kDa [IP‐10], monocyte chemotactic protein‐1 [MCP‐1], macrophage inflammatory protein beta [MIP‐1β], and “Regulated upon Activation, Normal T‐cell Expressed, and Secreted” [RANTES]). At baseline and at 4 months, a test battery for lower extremity function (knee extensor and flexor strength, timed up and go, repeated sit‐to‐stand) was assessed. All analyses were adjusted for baseline measurement, age, and body mass index. Mean 25(OH)D levels increased to 69.5 ng/mL in the HyD group. This rise was immediate and sustained. Mean 25(OH)D levels increased to 31.0 ng/mL with a slow increase in the vitamin D3 group. Women on HyD compared with vitamin D3 had a 2.8‐fold increased odds of maintained or improved lower extremity function (odds ratio [OR] = 2.79; 95% confidence interval [CI], 1.18–6.58), and a 5.7‐mmHg decrease in systolic blood pressure (p = 0.0002). Both types of vitamin D contributed to a decrease in five out of seven markers of innate immunity, significantly more pronounced with HyD for eotaxin, IL‐12, MCP‐1, and MIP‐1 β. There were no cases of hypercalcemia at any time point. Twenty micrograms (20 µg) of HyD per day resulted in a safe, immediate, and sustained increase in 25(OH)D serum levels in all participants, which may explain its significant benefit on lower extremity function, systolic blood pressure, and innate immune response compared with vitamin D3.

Toxicokinetics of Acrylamide in Humans after Ingestion of a Defined Dose in a Test Meal to Improve Risk Assessment for Acrylamide Carcinogenicity

High amounts of acrylamide in some foods result in an estimated daily mean intake of 50 μg for a western style diet. Animal studies have shown the carcinogenicity of acrylamide upon oral exposure. However, only sparse human toxicokinetic data is available for acrylamide, which is needed for the extrapolation of human cancer risk from animal data. We evaluated the toxicokinetics of acrylamide in six young healthy volunteers after the consumption of a meal containing 0.94 mg of acrylamide. Urine was collected up to 72 hours thereafter. Unchanged acrylamide, its mercapturic acid metabolite N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), its epoxy derivative glycidamide, and the respective metabolite of glycidamide, N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA), were quantified in the urine by liquid chromatography-mass spectrometry. Toxicokinetic variables were obtained by noncompartmental methods. Overall, 60.3 ± 11.2% of the dose was recovered in the urine. Although no glycidamide was found, unchanged acrylamide, AAMA, and GAMA accounted for urinary excretion of (mean ± SD) 4.4 ± 1.5%, 50.0 ± 9.4%, and 5.9 ± 1.2% of the dose, respectively. Apparent terminal elimination half-lives for the substances were 2.4 ± 0.4, 17.4 ± 3.9, and 25.1 ± 6.4 hours. The ratio of GAMA/AAMA amounts excreted was 0.12 ± 0.02. In conclusion, most of the acrylamide ingested with food is absorbed in humans. Conjugation with glutathione exceeds the formation of the reactive metabolite glycidamide. The data suggests an at least 2-fold and 4-fold lower relative internal exposure for glycidamide from dietary acrylamide in humans compared with rats or mice, respectively. This should be considered for quantitative cancer risk assessment. (Cancer Epidemiol Biomarkers Prev 2006;15(2):266–71)

Cytochrome P450 2B6 (CYP2B6) and constitutive androstane receptor (CAR) polymorphisms are associated with early discontinuation of efavirenz-containing regimens

OBJECTIVES Cytochrome P450 2B6 (CYP2B6) is responsible for the metabolic clearance of efavirenz and single nucleotide polymorphisms (SNPs) in the CYP2B6 gene are associated with efavirenz pharmacokinetics. Since the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) correlate with CYP2B6 in liver, and a CAR polymorphism (rs2307424) and smoking correlate with efavirenz plasma concentrations, we investigated their association with early (<3 months) discontinuation of efavirenz therapy. METHODS Three hundred and seventy-three patients initiating therapy with an efavirenz-based regimen were included (278 white patients and 95 black patients; 293 male). DNA was extracted from whole blood and genotyping for CYP2B6 (516G → T, rs3745274), CAR (540C → T, rs2307424) and PXR (44477T → C, rs1523130; 63396C → T, rs2472677; and 69789A → G, rs763645) was conducted. Binary logistic regression using the backwards method was employed to assess the influence of SNPs and demographics on early discontinuation. RESULTS Of the 373 patients, 131 withdrew from therapy within the first 3 months. Black ethnicity [odds ratio (OR) = 0.27; P = 0.0001], CYP2B6 516TT (OR = 2.81; P = 0.006), CAR rs2307424 CC (OR = 1.92; P = 0.007) and smoking status (OR = 0.45; P = 0.002) were associated with discontinuation within 3 months. CONCLUSIONS These data indicate that genetic variability in CYP2B6 and CAR contributes to early treatment discontinuation for efavirenz-based antiretroviral regimens. Further studies are now required to define the clinical utility of these associations.

Effects of grapefruit juice on the pharmacokinetics of sildenafil

Because of extensive first‐pass metabolism, oral bioavailability of sildenafil reaches only 40%. Formation of the primary metabolite, N‐desmethylsildenafil, is mainly mediated by the cytochrome P450 enzyme CYP3A4. In this study we investigated the influence of grapefruit juice, containing inhibitors of intestinal CYP3A4, on the pharmacokinetics of sildenafil and N‐desmethylsildenafil.

Clinical pharmacokinetics of trospium chloride.

Trospium chloride, a quaternary amine with anticholinergic properties, is used for the treatment of overactive bladder with symptoms of urge urinary incontinence, urgency and urinary frequency. The pharmacokinetics of trospium chloride have been investigated in healthy volunteers, in patients with renal and hepatic impairment, and in those with symptoms of overactive bladder, after oral, intravenous and intravesical administration.After oral administration, absorption of the hydrophilic trospium chloride is slow and incomplete. Peak plasma concentrations (Cmax) of approximately 4 ng/mL are reached 4–5 hours after administration of a 20mg immediate-release preparation. The mean bioavailability is approximately 10% and decreases by concomitant food intake (to a mean of 26% of the fasting area under the plasma concentration-time curve [AUC]). Trospium chloride displays dose proportional increases in AUC and Cmax after a single dose within the clinically relevant dose range (20–60mg). The mean volume of distribution is approximately 350–800L. The drug is minimally (mean approximately 10%) metabolised to spiroalcohol by hydrolysis, is 50% plasma protein bound and does not cross the blood-brain barrier. Urinary excretion of the parent compound plays a major role in the disposition of the drug, with a mean renal clearance of 29 L/h (accounting for approximately 70% of total clearance) and a mean elimination half-life ranging from 10 to 20 hours. Elimination of the drug is slowed in patients with renal insufficiency, and population pharmacokinetic modelling has demonstrated that drug clearance is correlated with serum creatinine concentration. Thus, dose reduction is needed in patients with severe renal impairment (i.e. creatinine clearance <30 mL/min).To date, no clinically relevant pharmacokinetic drug-drug interactions have been identified; the drug does not bind to any of the drug metabolising cytochrome P450 enzymes.The pharmacokinetics of the drug are compatible with twice-daily administration. A once-daily schedule may also be appropriate, but this regimen needs formal clinical evaluation.

Pharmacokinetics of oral vitamin D3 and calcifediol

AIM Long-term pharmacokinetics after supplementation with vitamin D3 or calcifediol (the 25-hydroxyvitamin D3 metabolite) are not well studied. Additionally, it is unclear whether bolus doses of vitamin D3 or calcifediol lead to 25(OH)D3 plasma concentrations considered desirable for fracture prevention (30ng/mL). We therefore investigated plasma pharmacokinetics of 25(OH)D3 during different vitamin D3 and calcifediol supplementation regimens. METHODS In this seven-arm, randomised, double-blind, controlled parallel-group study, 35 healthy females aged 50 - 70years (5 per group) received 20μg calcifediol or vitaminD3 daily, 140μg calcifediol or vitaminD3 weekly, for 15weeks, or a single bolus of either 140μg calcifediol, or vitaminD3, or both. 25(OH)D3 plasma concentrations were quantified using LC-MS/MS in 14 clinical visits among all participants. RESULTS For daily (weekly) dosing, the area under the concentration-time curve (AUC0-24h), which is the measure for exposure, was 28% (67%) higher after the first dose of calcifediol than after the first dose of vitaminD3. After 15weeks, this difference was 123% (178%). All women in the daily and weekly calcifediol groups achieved 25(OH)D3 concentrations >30ng/mL (mean, 16.8days), but only 70% in the vitaminD3 daily or weekly groups reached this concentration (mean, 68.4days). A single dose of 140μg calcifediol led to 117% higher 25(OH)D3 AUC0-96h values than 140μg vitaminD3, while the simultaneous intake of both did not further increase exposure. CONCLUSIONS Calcifediol given daily, weekly, or as a single bolus is about 2-3 times more potent in increasing plasma 25(OH)D3 concentrations than vitamin D3. Plasma 25(OH)D3 concentrations of 30ng/mL were reached more rapidly and reliably with calcifediol.