| Preclinical metabolism and pharmacokinetics study is important in drug discovery program and the basis of the study is a specific, sensitive method with good accuracy and precision. A new LC/MS/MS method for determination of adefovir in animal plasma was developed and validated and successfully used in the preclinical metabolism and pharmacokinetics study of adefovir dipivoxil.1. The development of a LC/MS/MS method for determination of adefovir in plasmaA simple, specific and sensitive LC/MS/MS method was established to determine adefovir in animal plasma. Adefovir and internal standard 9-(3-phosphonylmethoxypropyl)adenine were isolated from plasma using protein precipitation with methanol, then chromatographed using Diamonsil C18 column. The mobile phase consisted of methanol-water-formic acid (20: 80: 1, v/v/v). Elecrrospray ionization (ESI) source was applied and operated in the positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 274 →m/z 162 and m/z 288 → m/z 176 were used to quantify adefovir and the internal standard, respectively. The linear calibration curve was obtained in the concentration range of 20-4000 ng.mL-1. The lower limit of quantitation of adefovir was 20 ng.mL-1. The recovery of adefovir in sample preparation was more than 75%. The inter- and intra-day precision (RSD) was less than 9%, and accuracy (relative error) was within ±4.5%.2. The preclinical metabolism and pharmacokinetics of adefovir dipivoxil studied by LC/MS/MS methodThe preclinical metabolism and pharmacokinetics of adefovir dipivoxil was systematically studied by the validated LC/MS/MS method.Investigation of absorption of adefovir dipivoxilFollowing an oral administration of adefovir dipivoxil to rats, monkeys and dogs in three doses, respectively, the plasma concentration of adefovir was determined by the validated LC/MS/MS method. The pharmacokinetic parameters were assessed by non-compartmental method using TOPFIT. Cmax and AUC0-24 h were all dose proportional (r>0.6231, p<0.05). The concentration in plasma-time curves after an i.v. administration to rats, monkeys and dogs were all fitted to two-compartment models. Comparison between AUC0-24h after an oral administration and an i.v. administration indicated that the absolute bioavailabilities of adefovir dipivoxil in rats, monkeys and dogs were 63.7%, 19.6% and 37.3%, respectively.Investigation of the distribution of adefovir dipivoxilThe tissue distribution result of adefovir dipivoxil showed that high concentration presented in stomach, stomach content, intestine and intestine content; accumulation occurred in kidney and bladder indicating the major metabolic pathway of the drug; relative high concentration presented in liver, the target tissue and the lowest concentration existed in brain showing a poor penetration into central nervous system.Investigation of the biotransformation of adefovir dipivoxilThe biotransformation result of adefovir dipivoxil showed that adefovir dipivoxil was gradually transformed to adefovir in rats' stomach and the concentration of adefovir was higher than that of adefovir dipivoxil after 2 h of an oral administration. There was almost no adefovir dipivoxil in rats' intestine and plasma after an oral administration.Investigation of major metabolic pathway of adefovir dipivoxilAfter an i.v. administration of 2 mg.kg-1 adefovir dipivoxil to monkeys, 81.6% of the dose was recovered in urine and after an oral administration of 20mg.kg-1 adefovir dipivoxil to rats, the biliary excretion of adefovir dipivoxil was less than 1%.Investigation of the plasma protein binding of adefovirAt the human plasma concentrations of 500 ng . mL-1, 1000 ng . mL-1 and 5000 ng.mL-1 adefovir, the plasma protein bindings of adefovir were 32.9%, 21.2% and 20.0%, respectively.2209 samples of the appropriate biological matrix were determined by the LC/MS/MS method (164 samples for method validation, 540 samples for calibration curves and quality control and 1505 samples... |
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