Drug Metabolism Letters - Volume 3, Issue 3, 2009

A LC/MS-based metabolomic assay was utilized to investigate a drug's excretion kinetic profile in urine so that the drug toxicity information could be obtained. Groups of 10 male Sprague-Dawley rats per dose were orally gavaged with a single dose of 0.2% carboxymethylcellulose, 400 mg acetaminophen (APAP)/kg body weight or 1600 mg APAP/kg. UPLC/MS and NMR were used to evaluate the excretion kinetics of major drug metabolites. N-acetyl-Lcysteine acetaminophen (APAP-NAC) had statistically significant correlations with clinical chemistry data, endogenous metabolite concentrations and histopathology data. The potential toxicity of a drug can be assessed through the study of the drug's metabolite profiles.

Tylosin concentrations and its disposition kinetics in serum, urine, and milk of lactating goats following a single intravenous (i.v.) or intramuscular (i.m.) injection (10 mg kg-1 b.wt.) were carried out using high performance liquid chromatography (HPLC).The concentration-time curve of tylosin after i.v. injection could be described by a twocompartment open model. Tylosin was rapidly distributed and eliminated from goat's bodies with t1/2(β) value of 1.25 h. The V (d) was less than one litre/kg and the MRT was 1.40 h. Concomitant administration with albendazole decreased tylosin concentrations in serum after its i.v. injection and the MRT was 1.17 h. The AUC and AUMC showed a significant decrease in goats given albendazole prior to injection as compared with those given tylosin only. Following i.m. administration, the absorption half-life and the corresponding tmax revealed rapid absorption rate with systemic bioavailability (F%) of 76.2 %. Albendazole when given concurrently with tylosin decreased its serum concentrations due to lower bioavailability (43.25 %). Following i.v. or i.m. injection, tylosin was excreted rapidly in urine in concentration much higher than those determined in serum and milk. Tylosin administered in goats pretreated with albendazole was excreted at lower concentration in urine, with a significant decrease from 1st to 10th hours as compared with animals given tylosin only. Following i.v. or i.m. administration of tylosin, the drug was excreted in high concentrations in milk. A significant decrease in milk concentrations was reported in goats pretreated with albendazole.

The chemiluminescence intensity of luminol-dissolved oxygen was decreased when cephalosporins were mixed with luminol. The decrease chemiluminescence intensity was linear with the logarithm of cephalosporins concentration over the range from nanogram to microgramme level, with the limits of detection at nanogram level. The sensitivities of determination for cephalosporins were in the order of cefoperazone > ceftriaxone > cefuroxime > cefaclor > cefalexin > cefradine. The proposed method was applied to monitor the excretion of cefradine in human urine after taken cefradine capsules. The possible chemiluminescence mechanism and relationship between the determination sensitivities and generations of cephalosporins were also discussed.

To elucidate the differences in the cyclosporine A (CyA) PK between cadaveric and living-related renal transplantation (CRT and LRT, respectively) recipients, a retrospective cohort study of clinical PK was conducted. Data from 80 patients who received LRT (n=75) and CRT (n=5) over 4 years were included. The incidence of acute rejection in CRT recipients was over 5 times higher than that in LRT recipients. On day 14 after transplantation, the area under the blood concentration versus time curve (AUC) per dose up to 4 h in CRT recipients was 65.3 % that of LRT recipients, however, there was no difference in the blood trough levels. Unlike LRT, renal failure derived from long ischemia time was observed in CRT recipients, and it is speculated that renal failure affects the PK of CyA. Moreover, we performed intravenous. (i.v.) and intraduodenal (i.d.) PK studies of CyA using renal failure model rats prepared by renal ischemiareperfusion (RIR rats). There were no differences in PK profiles after i.v. administration of CyA between RIR and control rats; however, AUC up to infinity (1.81±0.18 μg·h/ml) in RIR rats after i.d. administration was significantly lower than in control rats (5.01±1.78 μg·h/ml). In addition, the absorption of CyA and midazolam, an ideal probe for CYP3A, from the intestinal loop in RIR rats was significantly less (69.8% and 42.8 %, respectively) than in control rats. These results suggest that the contribution of intestinal metabolism by CYP3A to decreasing CyA absorption in RIR rats is significant, namely, there is a possibility that the reason for poor absorption of CyA in CRT recipients is increasing intestinal CYP3A activity caused by renal injury derived from long renal ischemia. The results of this study provide a useful information for therapeutic drug monitoring of CyA in CRT recipients.

The present study demonstrated that in addition to CYP3A4 and CYP2D6, the metabolism of loratadine is also catalyzed by CYP1A1, CYP2C19, and to a lesser extent by CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP3A5. The biotransformation of loratadine was associated with the formation of desloratadine (DL) and further hydroxylation of both DL and the parent drug (loratadine). Based on the inhibition and correlation studies contribution of CYP2C19 in the formation of the major circulating metabolite DL seems to be minor. Reported clinical results suggest that the steady state mean (%CV) plasma Cmax and AUC24hr of loratadine were 4.73 ng/ml (119%) and 24.1 ng.hr/ml (157%), respectively, after dosing with 10 mg loratadine tablets for 10 days. High inter-subject variability in loratadine steady-state data is probably due to the phenotypical characteristics of CYP2D6, CYP2C19, and CYP3A4. The relative abundance of CYP3A4 in the human liver exceeds that of CYP2C19 and CYP2D6 and therefore the contribution of CYP3A4 in the metabolism of loratadine should be major (∼70%).

Cytochrome P450 (CYP) induction in rodents and humans is considered a liability for new chemical entities (NCEs) in drug discovery. In particular, CYP1A1 and CYP2B1/2 have been associated with the induction of liver tumors in oncogenicity studies during safety evaluation studies of potential drugs. In our laboratory, real time PCR (Taqman®) has been used to quantify the induction of rat hepatic CYP1A1 and CYP2B1/2 in precision-cut rat liver slices. A novel technology that does not require m-RNA isolation or RT-PCR, (developed by NanoString Technologies®) has been investigated to quantify CYP1A1 and CYP2B1/2 induction in rat liver slices. Seventeen commercially available compounds were evaluated using both Taqman® and NanoString® technologies. Precision-cut rat liver slices were incubated with individual compounds for 24 hr at 37°C in a humidified CO2 incubator and CYP1A1 and CYP2B1/2 m-RNA molecules were quantified. The results from the NanoString® technology were similar to those of the Taqman® with a high degree of correlation for both CYP isoforms (r2>0.85). Therefore, NanoString provides an additional new technology to evaluate the induction of CYP1A1 and 2B1/2, as well as potentially other enzymes or transporters in rat liver slices.

A 43 year old man with HIV and HCV infection and liver cirrhosis developed fatal lactic acidosis within five days from starting nifedipine for arterial hypertension. Multiple drug interactions, current and accumulated drug toxicities and the reduced liver function, might in combination have led to the acute lactic acidosis.

During LC-MS/MS quantification of a small molecule in human urine samples from a clinical study, an unexpected peak was observed to nearly co-elute with the analyte of interest in many study samples. Improved chromatographic resolution revealed the presence of at least 3 non-analyte peaks, which were identified as cysteine metabolites and N-acetyl (mercapturic acid) derivatives thereof. These metabolites produced artifact responses in the parent compound MRM channel due to decomposition in the ionization source of the mass spectrometer. Quantitative comparison of the analyte concentrations in study samples using the original chromatographic method and the improved chromatographic separation method demonstrated that the original method substantially over-estimated the analyte concentration in many cases. The substitution of electrospray ionization (ESI) for atmospheric pressure chemical ionization (APCI) nearly eliminated the source instability of these metabolites, which would have mitigated their interference in the quantification of the analyte, even without chromatographic separation. These results 1) demonstrate the potential for thiol metabolite interferences during the quantification of small molecules in pharmacokinetic samples, and 2) underscore the need to carefully evaluate LC-MS/MS methods for molecules that can undergo metabolism to thiol adducts to ensure that they are not susceptible to such interferences during quantification.

The microsomal metabolism of ketoconazole is revisited using accurate mass LC/MSn and deuterium labelling. Structures for sixteen metabolites are proposed from rat and human microsomal metabolism of commercial ketoconazole. Thirteen of the proposed structures are well determined and consistent with all data; five of the proposed structures are less certain. Ten of the metabolites are described for the first time. Reaction phenotyping shows that most of the metabolites arise from CYP3A4, the enzyme known to be well inhibited by ketoconazole.

UDP-glucuronosyltransferase 1A6 (UGT1A6) is involved in metabolizing non-steroidal anti-inflammatory drugs (NSAIDs). Genotype variation in UGT1A6 may influence the metabolism of NSAIDs and we studied whether this might modulate the gastrointestinal toxicity of NSAIDs. UGT1A6 genotypes of 114 patients with peptic ulcer haemorrhage were compared with those of two subsets of controls: 158 cardiology patients using similar amounts of NSAIDs and 140 healthy controls, hardly using NSAIDs. Risk factors for peptic ulcer bleeding were male gender (Odds ratio (OR) 2.66, 95% confidence interval (CI) 1.7-4.2), age above 60 years (OR 2.15, 95% CI 1.4-3.4) and use of NSAIDs/aspirin (OR 4.50, 95% CI 2.8-7.3). UGT1A6 genotype frequencies did not differ between patients with peptic ulcer and the two control groups (p=0.76). We conclude that polymorphic UGT1A6 is not implicated in the pathogenesis of NSAIDs-related peptic ulcer disease.