An improved LC-MS/MS method for determination of cinacalcet in human plasma and its application to the evaluation of food intake effect on the pharmacokinetics of cinacalcet in healthy volunteers.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of cinacalcet in human plasma was developed and validated. This assay was based on liquid-liquid extraction and cinacalcet-d4 was used as an internal standard (IS). Separation was achieved on a C18 column by the mobile phase A of water (containing 0.1% formic acid) and the mobile phase B of acetonitrile-water (95:5, v/v) (containing 0.2% formic acid) with gradient elution. Quantification was done using multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 358.2 → m/z 155.2 for cinacalcet and m/z 362.3 → m/z 155.0 for IS at positive ionization mode. The calibration curve was established over the range 0.05-20.0 ng/mL and the correlation coefficient was >0.99. The intra- and inter-day relative standard deviations were <5.8%. Accuracy determined at four concentrations ranged between 96.0 and 106.0%. This method was successfully applied to a pharmacokinetic description of oral dose of cinacalcet and the significant effect of food intake on the pharmacokinetics of cinacalcet was first demonstrated in Chinese healthy volunteers.

Prediction of tacrolimus metabolism and dosage requirements based on CYP3A4 phenotype and CYP3A5(*)3 genotype in Chinese renal transplant recipients.

AIM: To examine how the endogenous CYP3A4 phenotype and CYP3A5(*)3 genotype of Chinese renal transplant recipients influenced the dose-corrected trough concentration (C0/D) and weight-corrected daily dose (D/W) of tacrolimus. METHODS: A total of 101 medically stable kidney transplant recipients were enrolled, and their blood and urine samples were gathered. The endogenous CYP3A4 phenotype was assessed by the ratio of 6ß-hydroxycortisol and 6ß-hydroxycortisone to cortisol and cortisone in urine. CYP3A5(*)3 genotype was determined using PCR-RELP. RESULTS: In overall renal transplant recipients, a multiple regression analysis including the endogenous CYP3A4 phenotype, CYP3A5(*)3 genotype and post-operative period accounted for 60.1% of the variability in C0/D ratio; a regression equation consisting of the endogenous CYP3A4 phenotype, post-operative period, body mass index, CYP3A5(*)3 genotype, gender, total bilirubin and age explained 61.0% of the variability in D/W ratio. In CYP3A5(*)3/(*)3 subjects, a combination of the endogenous CYP3A4 phenotype, post-operative period and age was responsible for 65.3% of the variability in C0/D ratio; a predictive equation including the endogenous CYP3A4 phenotype, post-operative period, body mass index, gender and age explained 61.2% of the variability in the D/W ratio. Base on desired target range of tacrolimus trough concentrations, individual daily dosage regimen was calculated, and all the observed daily doses were within the predicted range. CONCLUSION: This study provides the equations to predict tacrolimus metabolism and dosage requirements based on the endogenous CYP3A4 phenotype, CYP3A5(*)3 genotype and other non-genetic variables.

Pharmacodynamic analysis of intravenous recombinant urate oxidase using an indirect pharmacological response model in healthy subjects.

AIM: Pharmacodynamic analysis of intravenous recombinant urate oxidase produced by Escherichia coli was performed in healthy subjects using a pharmacokinetic/pharmacodynamic (PK/PD) model. METHODS: A randomized, single-blind, placebo-controlled study was performed in 40 healthy Chinese subjects (4 groups of 10 subjects each, placebo 4:1 ratio) who received infusions of uricase (single doses of 0.1, 0.2, and 0.3 mg/kg; multiple doses of 0.2 mg·kg(-1)·d(-1) for 7 d). PK profiles were determined through plasma uricase activity, and PD profiles were established using uric acid levels in plasma and urine. The plasma PD parameter was estimated as changes in plasma uric acid levels as the effect in the indirect response model. Adverse events were also monitored. RESULTS: A two-compartment PK model with constant iv input and first-order output was used to describe the kinetic process of plasma uricase. The low value (2.8 U/L) of drug concentration that achieved 50% of maximum effect (EC50) indicated that low plasma uricase concentrations were sufficient to produce pharmacological effects. A strong relationship (r(2)=0.9991) between the mean uric acid concentration in blood and the mean uric acid excretion rate in urine in the range of 11 to 30 h after single dosing was found. Infusions of uricase were well tolerated in all subjects. CONCLUSION: The PK/PD model predicted the effective dose to be 0.1 mg/kg in healthy subjects. The excretion rate of uric acid in urine may be used as a new index for pharmacological effects in further clinical trials.

Development of a new LC-MS/MS based enzyme activity assay for recombinant urate oxidase in plasma and its application to pharmacokinetics in human.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of recombinant urate oxidase in human plasma. This assay was based on the determination of enzyme reaction product, (15)N-allantoin, and phenacetin was used as an internal standard (IS). Separation was achieved on a C18 column by the mobile phase of 30% water (containing 0.5% formic acid) and 70% methanol. Quantification was done using multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 161 → m/z 118 for (15)N-allantoin and m/z 180 → m/z 110.1 for IS at positive ionization mode. The calibration curve was established over the range of 2.077-42.06 U/l and the correlation coefficient was larger than 0.99. The intra-day and inter-day relative standard deviations were less than 10.6%. Accuracy determined at three concentrations ranged between 98.6% and 109.2%. This method was successfully applied to a pharmacokinetic study of intravenous recombinant urate oxidase produced from Escherichia coli in Chinese healthy volunteers.