Clinical Pharmacology Study Of Tenofovir Dipivoxil Fumarate And Anti-HBV Study Of Total Alkaloids From Chickpea

Part Ⅰ: Clinical pharmacology study of tenofovir dipivoxilfumarateTenofovir dipivoxil fumarate is a new prodrug of tenofovir, exploited byJiangsu Chia-Tai Tianqing Pharmaceutical Co. Ltd., used for the treatment ofchronic hepatitis B. It’s registered for category1.1of chemical drugs accordingto Drug Registration Regulation. The clinical pharmacology study was designedstrictly following “Technical guidance for chemical drugs in clinicalpharmacokinetic studies” and “The appendix Ⅱ of Chinese pharmacopoeia” inorder to comprehensively and systematically clarify the laws of safety,absorption, metabolic, distribution, excretion of the new drug in human body,and to provide the evidence of dosage regimen for Phase II clinical trials. Thisstudy obtained scientific significance and application values in following fiveaspects:1. Establishment of drug analysis methods. Objective: To establish a rapid, specific and sensitive LC-MS/MS methodto detect tenofovir in plasma and urine and levocarnitine in plasma: Applicationto a clinical pharmacology study of tenofovir dipivoxil fumarate in human.Methods:1. Detection of tenofovir in plasma and urine: Massspectrometric detection was performed on a triple quad LC–MS/MS inelectrospray positive ionization using multiple reaction monitoring. The masstransition was m/z288.1�'176for tenofovir, and m/z278.1�'152.0forentecavir, respectively.2. Detection of levocarnitine in plasma: Mass spectrometric detection wasperformed on a triple quad LC–MS/MS in electrospray positive ionization usingmultiple reaction monitoring. The mass transition was m/z162�'84.65forlevocarnitine, and m/z326.2�'146.9for tolterodine, respectively.Result: The linear range of tenofovir in plasma is2^-1200ng/mL, inter andintra precision is in the range of1.1⁴.8%and8.4¹2.9%, respectively.Absolute recovery is greater than90%. The linear range of tenofovir in urine is800-64000ng/mL, inter and intra precision is in the range of0.9³.4%and0.8².6%, respectively. Absolute recovery is nearly100%. The linear range oflevocarnitine in plasma is2-20μg/mL, inter and intra precision is in the range of2.7³.0%and6.6⁸.9%. Absolute recovery is greater than90%.Conclusion: The simple, specific and rapid LC-MS/MS method foranalysis of tenofovir in human plasma and urine and levocarnitine in plasmahave been developed and validated.2. The study of pharmacokineticsObjective: To detect tenofovir in plasma and urine and evaluate the singleand multiple pharmacokinetic characteristics for absorption, distribution andeliminate of tenofovir dipivoxil fumarate in human.Methods:24healthy volunteers are divided into two groups randomly. Todetect tenofovir in plasma and urine by LC-MS/MS and calculate thepharmacokinetic parameters. Result: After administration of single150,300or600mg of tenofovirdipivoxil fumarate, elimination half-life (t_1/2) of tenofovir are14.06±1.83h,17.00±0.84h,16.32±2.29h, AUC0-721410.05±200.42ng·h·mL^-1,2872.94±335.99ng·h·mL^-1,5728.67±1056.59ng·h·mL^-1, T_max1.5±0.8h、0.8±0.3h and1.5±0.5h, C_max209.23±54.47ng·mL^-1,511.82±67.78ng·mL^-1and959.91±275.03ng·mL^-1, urine cumulative excretion rates of0～48h is29.58±2.84%,28.05±3.55%,30.81±7.35%. After administration of multiple oral doses of tenofovirdipivoxil fumarate, the steady average concentration （Cssav） is156.81±33.0ng·mL^-1, fluctuations degrees （DF） is3.94±0.5, cumulative constant （R） is1.59±0.15, t_1/2is19.58±2.11h.Conclusion: AUC（0-72）、C_maxwere found to be a linear relationship withdose in the range dose of150³00mg. Accumulation appears in human aftergiven tenofovir for7days.3. The study of food effectObjective: To investigate the effect of high fat and calorie diet on theabsorption, distribution, metabolism and excretion after administration ofmiddle doses of tenofovir dipivoxil fumarate300mg.Methods:12healthy volunteers were oral administered300mg of dipivoxilfumarate in fed or fast state in a single-dose, randomized,2-way, open-label,crossover study.Result: The main pharmacokinetic parameters of tenofovir in fed statewere AUC0-723270.37±290.52h·ng/ml, C_max432.62±85.02ng/ml, T_max1.5±0.5h,t_1/215.48±2.04h.Conclusion: The AUC, C_max, T_max, and t_1/2have the significantdifferences in fasted or fed, and the bioavailability increased14.7%after a highfat and calorie diet.4. The study of relative bioavailabilityObjective: To evaluate of the relative bioavailability between tenofovir dipivoxil fumarate and viread after administration a comparative same dose.Methods:18healthy male volunteers were administered test and reference,respectively, in a single-dose, randomized,2-way, open-label, crossover study.Result: The main pharmacokinetic parameters of tenofovir afteradministration test or reference were t1/215.74±1.68h,16.75±2.11h, AUC0-722695.62±483.42ng·h·mL^-1,2250.41±387.31ng·h·mL^-1, T_max1.1±0.5h,0.5±0.2hand C_max466.72±162.55ng·mL^-1,445.54±109.44ng·mL^-1, respectively.Conclusion: The relative bioavailability of tenofovir dipivoxil was20%higher compared with tenofovir disoproxil fumarate.5. The effect of tenofovir dipivoxil fumarate on the endogenouslevocarnitineObjective: To evaluate the effect of tenofovir dipivoxil fumarate on theendogenous levocarnitine.Methods: To determine levels of levocarnitine before and after the clinicaltrials, and to observe changes of levocarnitine before and after the clinical trials.Result: The process of absorption, distribution, metabolism andelimination of tenofovir dipivoxil fumarate could affect the levels of endogenouslevocarnitine in human. The level of levocarnitine could decrease whenconcentration of tenofovir rise to a certain degree. Levocarnitine will graduallyreturn to normal after drug elimination of tenofovir. After multiple doses,levocarnitine will keep in a low level.Conclusion: Levocarnitine was advised to be administered whenadministration of multiple doses of tenofovir dipivoxil fumarate. Objective: To extract and separate of chickpeas and conduct anti-HBVexperiments in vitro for total alkaloids separated from chickpeas. Methods: To separate the part of N-butyl alcohol systematically extractedfrom chickpea using silica gel column chromatography method, the chloroform-methanol-water was used for gradient elution. The sub-total alkaloids wereconducted in vitro anti-hepatitis pharmacology study.Results: For the first time, alkaloids were isolated from the chickpeas, andthe pharmacology study found that the total alkaloids had in vitro anti-hepatitisB virus activity, which could significantly inhibit HBsAg, HBeAg, HBV DNAin the supernatant of HepG2.2.15cells dependent on the dose and time. Furtherto isolate the alkaloid monomers from the total alkaloids, and identify theirstructure by physicochemical properties and the means of modern spectroscopic（UV, IR, MS,1H-NMR,13C-NMR and2D-NMR） methods. Crystal singlestructure of compound1was obtained by single crystal cultivation to clarify thestereochemistry of the compound1.Conclusion: Chickpeas alkaloids have in vitro anti-hepatitis B virus activity.