Determination Of Emtricitabine Concentration In Human Plasma, Urine And Their Pharmacokinetics Study In Healthy Volunteers

Emtricitabine (5-fluoro-1-(2R, 5S)-[2-(hydroxymethyl)-1, 3-oxathiolan-5-yl] cytosine, FTC) is a novel nucleoside reverse transcriptase inhibitor with activity against human immunodeficiency virus type-1, 2 (HIV-1, HIV-2) and hepatitis B virus (HBV). The chemical constitution and mechanism of action of FTC are similar to Lamivudine, but FTC is more effective with better tolerance and lower toxicity due to its reconstitution, and the activity in vitro of FTC against HIV-1 is approximately 4-10 fold more potent than Lamivudine. At present the studies about the treatments of HIV are in phase III clinical trials and HBV are in phase I/II clinical trials.Emtricitabine is rapidly and extensively absorbed following oral administration, then it is phosphorylated and actived to be an intracellular active 5'-triphosphate (TP) of FTC (FTC-TP). The FTC-TP competitively inhibits HIV-1 reverse transcriptase and HBV-DNA polymerase and can also be incorporated into viral genome, causing termination in DNA chain elongation. Recent data have shown that a higher level of intracellular FTC-TP was associated with a better suppression of plasma viral load in HIV infected individuals. The experiment is intend to assay Emtricitabine in plasma and urine of Chinese healthy volunteers and study the pharmacokinetics, which provides related reference to rational administration in clinic. Part1 The pharmacokinetics study of Emtricitabine in human plasmaObjective: To establish a SPE-HPLC method for the determination of Emtricitabine (FTC) in human plasma; To study the characteristics of pharmacokinetics in human plasma.Method: To use the SPE-HPLC method for the determination of Emtricitabine (FTC) in human plasma and study the characteristics of pharmacokinetics in Chinese healthy volunteers after oral administration (200mg).HPLC system: Separation was achieved on an InertisilC8 (150×4.6mm, 5μm) column at 30℃and detected at 280nm. The mobile phase consisted of acetonitrile and 0.02mol·mL^-1 potassium dihydrogen phosphate buffer salt (including 0.02% tetra-butyl ammonium hydroxide), (12:88, v/v) at a flow rate of 1.0 mL·min^-1.Pharmacokinetics: To draw blood from veins of upper extremity at rated time following oral administration (200mg); To use external standard method after solid phase extraction (SPE); To compute the pharmaceutical parameter by DSD pharmacokinetics software.Results: The retention time of Emtricitabine (FTC) in plasma is 3.5min and there are no interferences appeared at the peak position. The Emtricitabine(FTC) linear range is 0.05⁵.00μg·mL^-1, r=0.9997, the limit of detection is 0.021μg·mL^-1(S/N>3), n=5, the limit of quantification is 0.049μg·mL^-1(S/N>10), n=5, the extraction recovery is 91.5%⁹2.8% (n=6), the within-day precisions (RSD) is 1.02%².17% (n=6), the between-day precisions (RSD) is 1.49%².05% (n=4).The pharmacokinetics parameters of Emtricitabine(FTC) are: Cmax 2.17±0.62μg·mL^-1, t1/2 3.18±0.29min, AUC0¹4h 8.70±1.56μg·mL^-1·min^-1, AUC0^∝9.09±1.60μg·mL^-1·min^-1.Conclusion: The SPE-HPLC method to determine the concentration of Emtricitabine in plasma is convenient and sensitive, which is suitable to the clinical determination and pharmacokinetics study.Pharmacokinetics of Emtricitabine fits non-compartmental model, the pharmacokinetic parameters is slightly different from the records following the oral administration (200mg). Part2 The pharmacokinetics study of Emtricitabine and its metabolite in human urineObjective: To establish a fast, sensitive and accurate method for the determination of Emtricitabine(FTC) and its metabolite in human urine; To study the characteristics of pharmacokinetics in human urine.Method: To use the RE-HPLC method for the determination of Emtricitabine(FTC) and its metabolite in human urine and study the characteristics of pharmacokinetics in Chinese healthy volunteers after oral administration (200mg).HPLC system: Separation was achieved on a DiamonsilC18 (250×4.6mm, 5μm) column at 30℃and detected at 278nm. The mobile phase consisted of acetonitrile and 0.03% trifluoroacetic acid by gradient elution at a flow rate of 1.0 mL·min^-1.LC/MS condition: ion source: ESI; scanner mode: positive ionization mode; mass range: 100³00m/z; drying gas flow: N2, 1.5L·min^-1; drying gas pressure: 200Pa.Pharmacokinetics: To collect urine at rated time following oral administration (200mg); To determine the concentration of Emtricitabine and its main metabolite using internal standard method after centrifugalization; To inspect the pharmaceutical condition in urine in 48 hours.Results: The retention time of Emtricitabine(FTC) in urine is 21.2 min, the retention times of its metabolites: sulfoxide A and sulfoxide B, which are diastereoisomers, are 9.5min and 10.4min. The Emtricitabine(FTC) linear range is 1.00²50.0μg·mL^-1, r=0.9997, the limit of quantification is 0.98μg·mL^-1, the recovery is 100.0%¹02.2% (n=5), the within-day precisions (RSD) is 1.62%⁵.22% (n=5), the between-day precisions (RSD) is 1.85%⁶.20% (n=5).The retention time of main metabolite in urine is 10.4min, and the resolution between sulfoxide A and sulfoxide B is good, R>1.5. The sulfoxide B linear range is 1.00¹00.0μg·mL^-1, r=0.9993, the limit of quantification is 1.00μg·mL^-1, the recovery is 99.7%¹03.7% (n=5), the within-day precisions (RSD) is 1.80%⁵.58% (n=5), the between-day precisions (RSD) is 2.21%⁶.46% (n=5).The retention time of internal standard Floxuridine is 14.3min, and there is a good resolution during Floxuridine, Emtricitabine (FTC), sulfoxide A and sulfoxide B, and there are no interferences appeared at the peak position.During the 48 hours, the accumulative excretion of Emtricitabine (FTC) is 160.69±13.39mg, the accumulative excretory rate of Emtricitabine (FTC) is 80.35±6.70%; the accumulative excretion of sulfoxide B is 13.62±1.94mg, the accumulative excretory rate of sulfoxide B is 6.86±0.97%.Conclusion: The RE-HPLC method to determine the concentration of Emtricitabine(FTC) and its main metabolite sulfoxides in urine is convenient and sensitive, which is suitable to the clinical determination and pharmacokinetics study.There is no accumulation following the oral administration (200mg) in the healthy volunteers, and providing the administration to the patients with injury of kidney at clinic.