CYP2C-dependent drug metabolism in vivo; influence of genetics and drug interactions

Cytochrome P450 enzymes (CYPs) are responsible for the metabolism of the majority of therapeutic drugs. This thesis focuses on one of the CYP subfamilies, CYP2C, especially CYP2C9 and CYP2C19, which are responsible for the metabolism of 15– 20% of all drugs. All CYP2C enzymes are polymorphic, i.e. there are genetic variants, which have functional consequences for drug metabolism. Individuals can be classified according to their CYP2C metabolic capacity in extensive (EMs), intermediate (IMs) and poor metabolisers (PMs). Recently, a novel variant of the CYP2C19 gene was described in individuals with high metabolic capacity. This allele, CYP2C19*17, has been claimed to cause ultrarapid metabolism (UM) of CYP2C19 substrates. The aim of this thesis is to explore some of the aspects underlying varying metabolic capacity between, and within, individuals with the focus on genetics and drug–drug interactions. The thesis is based on five published papers. In Paper I we explored the influence of the genetic variant CYP2C9*3 on the CYP2C9 dependent metabolism of the anti-inflammatory and analgesic drug celecoxib. We found a seven-fold higher median exposure at steady-state in homozygous carriers of the CYP2C9*3 allele compared to homozygous wild-type subjects. This might be one factor behind the increased risk of cardiovascular events that has been observed in long-term users of celecoxib in a dose-dependent fashion. Paper II and III focused on the CYP2C19*17 allele that has been associated with extensive metabolism of CYP2C19 substrates. We showed a 52% lower exposure of omeprazole in homozygous *17 carriers compared to homozygous wild-type subjects after a single dose of 40 mg. Regarding steady-state levels of escitalopram (5 mg twice daily for a week), we noted a trend towards a 21% lower exposure in CYP2C19*17 homozygous individuals. However, this did not reach statistical significance in this study that was powered for a 40% difference. The clinical impact (or lack of impact) of this allele for various clinically important CYP2C19 substrates will be discussed in the thesis. A clinical consultation was the starting point for Paper IV in which we described eight cases of increased anticoagulant effect of warfarin in connection with concomitant use of noscapine; a cough medicine available over-the-counter. These cases were reported to the Swedish adverse drug reactions (ADR) register and we could show that they yielded a statistically significant signal worthy of further investigation. In vitro experiments were performed, showing that noscapine strongly inhibited CYP2C9 and CYP3A4, the key enzymes in warfarin metabolism. Besides noscapine, another OTC drug, glucosamine, has attracted interest for suspected interaction with warfarin. In Paper V we addressed the pharmacokinetic aspect of these interactions by giving a cocktail of four probe drugs before and during noscapine or glucosamine. Compared to baseline phenotyping, significant inhibition of both CYP2C9 (4.9-fold increase in the urinary losartan/E3174 ratio; 95% CI 2.8 - 8.4) and CYP2C19 (3.6-fold increase in the plasma omeprazole/5-hydroxyomeprazole ratio; 95% CI 2.6 - 4.8) was seen during noscapine treatment. This is likely to explain the observed interaction with warfarin. No enzyme inhibition was seen with glucosamine and a metabolic interaction between warfarin and glucosamine seems highly unlikely

Population pharmacokinetics of apramycin from first-in-human plasma and urine data to support prediction of efficacious dose

BACKGROUND: Apramycin is under development for human use as EBL-1003, a crystalline free base of apramycin, in face of increasing incidence of multidrug-resistant bacteria. Both toxicity and cross-resistance, commonly seen for other aminoglycosides, appear relatively low owing to its distinct chemical structure. OBJECTIVES: To perform a population pharmacokinetic (PPK) analysis and predict an efficacious dose based on data from a first-in-human Phase I trial. METHODS: The drug was administered intravenously over 30 min in five ascending-dose groups ranging from 0.3 to 30 mg/kg. Plasma and urine samples were collected from 30 healthy volunteers. PPK model development was performed stepwise and the final model was used for PTA analysis. RESULTS: A mammillary four-compartment PPK model, with linear elimination and a renal fractional excretion of 90%, described the data. Apramycin clearance was proportional to the absolute estimated glomerular filtration rate (eGFR). All fixed effect parameters were allometrically scaled to total body weight (TBW). Clearance and steady-state volume of distribution were estimated to 5.5 L/h and 16 L, respectively, for a typical individual with absolute eGFR of 124 mL/min and TBW of 70 kg. PTA analyses demonstrated that the anticipated efficacious dose (30 mg/kg daily, 30 min intravenous infusion) reaches a probability of 96.4% for a free AUC/MIC target of 40, given an MIC of 8 mg/L, in a virtual Phase II patient population with an absolute eGFR extrapolated to 80 mL/min. CONCLUSIONS: The results support further Phase II clinical trials with apramycin at an anticipated efficacious dose of 30 mg/kg once daily

Quantification of Tacrolimus and Three Demethylated Metabolites in Human Whole Blood Using LC-ESI-MS/MS

Background: A bioalanytical method for the quantification of tacrolimus (TAC) and 3 metabolites, 13-O, 15-O, and 31-O-demethylated TAC (M-I, M-III, and M-II) in human whole blood using liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS) was developed and validated. Method: The analytes were extracted from 85 mu L of blood by protein precipitation followed by solid-phase extraction and a concentration step. The analytes and the internal standard (IS, ascomycin) were separated on a C18 column using a slow gradient mobile phase elution, with an analysis time of 3.3 minutes. The ammonium-adduct ions with transitions of m/z 821.5 > 768.7 (TAC), 807.5 > 754.7 (M-I, M-III, M-II), and 809.4 > 756.7 (IS) were measured in selected reaction monitoring mode using electrospray ionization. Results: Measuring ranges were 0.1-50 ng/mL for M-II, M-III, and TAC and 0.15-39 ng/mL for M-I. Imprecision in quantification was 50% for all analytes. The sample's stability was proven for 1 month at -20 degrees C and 72 hours at room temperature. Three freeze-thaw cycles had no significant effect on the stability. The prepared samples were stable at least 16 hours at 8 degrees C. Analysis of 53 patient samples resulted in average concentrations of 7.2 for TAC, 0.8 for M-I, 0.4 for M-III, and 0.2 ng/mL for M-II. The total metabolite concentration was 17% (4%-52%) of the TAC concentration. The TAC concentration measured by LC-MS/MS was 36.1% +/- 27.1% lower than by immunochemical (enzyme multiplied immunoassay technique) analysis. When adding the metabolite crossreactivity in the presence of TAC, the difference between the 2 methods was still 29.8% +/- 28.3%, indicating that the overestimation of TAC concentration of enzyme multiplied immunoassay technique compared with liquid chromatography-tandem mass spectrometry cannot only be ascribed to the demethylated metabolites. Conclusions: An LC-ESI-MS/MS method for the quantitative analysis of TAC and 3 metabolites, using a 2-step sample preparation was successfully developed, validated, and applied on 53 patient samples

Population pharmacokinetics of apramycin from first-in-human plasma and urine data to support prediction of efficacious dose

Background Apramycin is under development for human use as EBL-1003, a crystalline free base of apramycin, in face of increasing incidence of multidrug-resistant bacteria. Both toxicity and cross-resistance, commonly seen for other aminoglycosides, appear relatively low owing to its distinct chemical structure. Objectives To perform a population pharmacokinetic (PPK) analysis and predict an efficacious dose based on data from a first-in-human Phase I trial. Methods The drug was administered intravenously over 30 min in five ascending-dose groups ranging from 0.3 to 30 mg/kg. Plasma and urine samples were collected from 30 healthy volunteers. PPK model development was performed stepwise and the final model was used for PTA analysis. Results A mammillary four-compartment PPK model, with linear elimination and a renal fractional excretion of 90%, described the data. Apramycin clearance was proportional to the absolute estimated glomerular filtration rate (eGFR). All fixed effect parameters were allometrically scaled to total body weight (TBW). Clearance and steady-state volume of distribution were estimated to 5.5 L/h and 16 L, respectively, for a typical individual with absolute eGFR of 124 mL/min and TBW of 70 kg. PTA analyses demonstrated that the anticipated efficacious dose (30 mg/kg daily, 30 min intravenous infusion) reaches a probability of 96.4% for a free AUC/MIC target of 40, given an MIC of 8 mg/L, in a virtual Phase II patient population with an absolute eGFR extrapolated to 80 mL/min. Conclusions The results support further Phase II clinical trials with apramycin at an anticipated efficacious dose of 30 mg/kg once daily