Non-clinical Pharmacokinetics Characteristics Of Carbonic Anhydrase Inhibitor Tolsultazolamide

Backgrounds:Acute mountain sickness （AMS） is a most common disease occurring at highaltitudes. It is caused by hypoxia and is characterized by headache, weakness, fatigue,nausea, insomnia, and depressed appetite. Although AMS does not seem serious, if itis untreated, this symptom complex can progress to the life-threatening conditions ofhigh-altitude pulmonary edema and high-altitude cerebral edema. For our country hasthe largest plateau areas, the highest elevation and the most populations in the wordand as acute exposure to high altitudes is becoming increasingly common for bothwork-and travel-related reasons, AMS is becoming an outstanding problem affectingpeoples’ health.In recent years, acetazolamide, a typical carbonic anhydrase inhibitor, has beenwidely accepted as a critical drug for the treatment of AMS, but it is prone to causehigh chloride metabolic acidosis, limbs numb, tinnitus, decreased appetite, dysgeusia,gastrointestinal discomfort, ambiguity, and other side effects. The incidence ofgeneral adverse reactions was64.1%, which seriously affected human work capacityand limited its use as a preventive drug. Therefore, it is important to find a new kindof safe and effective medicine to prevent and treat AMS.Tolsultazolamide, a new derivative of acetazolamide, is a novel carbonicanhydrase inhibitor discovered by our laboratory. The results of animal experimenthave indicated that it could improve the endurance of mice under hypoxic conditionsand may function more effectively than acetazolamide (In the hypoxia experiment, theminimum effective dosage of anti-hypoxia for tolsultazolamide and acetazolamidewere6.25mg·kg^-1·d^-1and100mg·kg^-1·d^-1, respectively, which indicated that thepotency of tolsultazolamide was16times higher than acetazolamide).Pharmacokinetics studys play a very important role in drug discovery anddevelopment, not only to support toxicity and clinical studies but also to optimizecandidate drugs. There is no related study of its pharmacokinetic properties at homeand abroad, for tolsultazolamide is a national1.1kind of innovative drug. Therefore,the objective of the current study was to examine the pharmacokinetics, tissue distribution, excretion and plasma protein binding characteristics of tolsultazolamidein rats. This study will reveal the dynamic variation of tolsultazolamide in rats, andobtain the basic pharmacokinetic parameters of tolsultazolamide, and clarify theprocesses and characteristics of absorption, distribution and excretion oftolsultazolamide, which will provide information for future drug research anddevelopment, and provide a basis for the design of clinical trials.Methods:A high-performance liquid chromatography method, with liquid-liquid extraction,was established for the quantitative analysis of tolsultazolamide in various biologicalsamples, and it was validated via examining the specificity, linearity, precision,accuracy, extraction recovery and stability. In the pharmacokinetic study, wemeasured the concentrations of tolsultazolamide in rat plasma at different time pointsafter oral administration and intravenous administration, respectively, and calculatedthe pharmacokinetic parameters with non-compartment model （Winonlinpharmacokinetic software）, and thus obtained the oral absolute bioavailability anddynamic changes of tolsultazolamide in rats in vivo. In tissue distribution study, weexplored the changes of tolsultazolamide at different time points after oraladministration in the various tissues of rats in vivo. In excretion study, we estimatedthe unchanged fraction of tolsultazolamide from urine, feces and bile at different timeintervals in rats after oral administration. In the plasma protein binding study, weinvestigated the protein binding characteristics of tolsultazolamide with rat and humanplasmas by the equilibrium dialysis.Results:The first part: Under the chromatographic conditions used, tolsultazolamide wasresolved well and was free from endogenous interference. The standard curvesshowed excellent linearity, with correlation coefficient （r） greater than0.99. The inter-and intra-day precision （RSD） were below15%, and the accuracy was in the range of85%¹15%. The extraction recoveries of tolsultazolamide in all biosamples weregreater than70%. Tolsultazolamide was stable under the experimental conditions.These results indicated that the established method was sensitive and reliable,according with the requirements of the analysis of biological samples.The second part: After its oral administration of pharmacological doses (20,40and80mg·kg^-1) to rats, Tmaxand T1/2of tolsultazolamide were approximately2h and5⁷ h, respectively, and the concentration of tolsultazolamide in plasma was almostdecreased to less than1/10of the Cmaxat24h post-dosing. The oral absolutebioavailability of tolsultazolamide was about60%, which demonstrates thattolsultazolamide is absorbed well in rats. For low, middle and high three doses, Cmaxand AUC0-were523±213,710±245,1626±266ng·mL^-1and2730±1290,5580±1839,11438±1637h·ng·mL^-1, respectively. Cmaxand dosage, as well as AUC0-tand dosageshowed preferable linear relationships, with correlation coefficients of0.985and1,respectively. The ratio of AUC0-tunder three doses was1:2.0:4.2. These resultsdemonstrated that, after its oral administration, tolsultazolamide may dispay linearpharmacokinetic characteristics in rats within the dose range studied. However, whenoral dose up to400mg·kg^-1, the absorption of tolsultazolamide decreased, showingsaturated tendency. In addition, we found significant sex-based differences in thepharmacokinetics of orally administered tolsultazolamide, in that the concentrationsof tolsultazolamide were almost higher in female rats than in male rats at each timepoint. The Cmaxand AUC values for female rats were both about twofold higher thanthose for male rats, whereas the Vd/F and CL/F values for female rats weresignificantly lower than those for male rats.The third part: Tolsultazolamide distributed rapidly and extensivelyly to rats in vivo,for it was determined in all the tissues examined30min after its administration. Inmost tissues, tolsultazolamide reached a peak concentration at2h and was stillelevated8h after drug administration. By24h, less than50%of the maximumtolsultazolamide remained in most tissues. In addition to stomach and intestine, higherlevels of tolsultazolamide were detected in spleen, lung and kidney, implicating thatthese organizations were likely to be the target organs for the pharmacological effectsof tolsultazolamide. Moreover, gender also exerted a significant influence on thedistribution of tolsultazolamide in the kidney of rats, and the concentrations in femalerats were higher than that in male rats.The fourth part: After its oral administration, the cumulative excretion oftolsultazolamide was0.016±0.019%in the urine and1.115±0.506%in the feceswithin72h, and0.0148±0.0072%in the bile within24h. Only a small proportion（Less than2%） of tolsultazolamide was excreted intact, demonstrating the extensivemetabolism of this drug after its oral administration in rats.The fifth part: The average protein binding rates of tolsultazolamide with rat plasma and human plasma were96.03%±0.10%and94.24%±0.52%, respectively. There wereno significant differences in different concentration groups of rat plasma proteinbinding rates, as well as human plasma protein binding rates （P>0.05）, demonstratingthat the binding of tolsultazolamide with plasma protein was dose-independent.Conclusions:The is hitherto the first report to establish a method for the analysis oftolsultazolamide in biological samples, and this is also the first study to evaluate thepharmacokinetics, tissue distribution, excretion and plasma protein bindingcharacteristics of tolsultazolamide in rats. Following its oral administration,tolsultazolamide was rapidly absorbed showed relatively high bioavailability, about60%. The tissue distribution of tolsultazolamide was established rapidly and waswidespread. The excretion of unchanged tolsultazolamide from urine, feces and bilewere all very low, and the total recovery was less than2%. The protein bindingcapability of tolsultazolamide with rat plasma and human plasma were both verystrong, higher than90%. Furthermore, there were significant gender differences in thepharmacokinetics of tolsultazolamide after oral administration in rats, and the Cmaxand AUC values of female rats were higher than that of male rats, while the Vd/F andCL/F values in female rats were significantly lower than that in male rats. Theseresults provide reliable scientific data with which to design safe and effectivetreatment regimens for the clinical administration of the drug.