Study On The Pharmacokinetics Of Vonoprazan Pyroglutamate In Sprague-dawley Rats

Acid-related diseases?ARDs?are one class of gastrointestinal disorders closely related to the secretion of gastric acid.The pathological mechanism of ARDs is complicated and various.After several years study,it was accepted that inhibition of gastric acid secretion is the key to the treatment of ARDs and the development of H⁺-K⁺-ATPase inhibitors have become the target of the treatment.Vonoprazan Fumarate?TAK-438 F?was a new and effective potassium-competitive acid blocker being developed by Takeda for the treatment and prevention of acid-related diseases?ARDs?.Compared with the traditional drugs proton-pump inhibitors?PPIs?,TAK-438 F exhibited many advantages,such as a fast onset of action,high accumulation,slow clearance,better control of night-time acid secretion,does not require acid protection so they don't exhibit acid-dependent activation like PPIs,and show limited gene polymorphism and so on.Meanwhile,there were many literature reports for the superior efficacy of TAK-438 F as the significantly longer and higher antisecretory effect after comparing to the classical PPIs.So TAK-438 F rapidly became a hot worldwide topic since being approved in 2014 and the sales increased year after year.However,poorly soluble in water and low bioavailability of TAK-438 F limit their clinical applications.As tablets can not be used for the patients who lost consciousness or were fasted or had difficulty in swallowing.Hence,based on the structure modification of TAK-438 F,Vonoprazan pyroglutamate?TAK-438 P?was synthesized by our research team because of the high water solubility?1000 higher than TAK-438 F?and more potent antisecretory effect.But the changes of physicochemical property may influenced the pharmacokinetic characters.A high performance liquid chromatographic method was established and validated for the determination of TAK-438 P and TAK-438 F.And then applied it to the determination of bulk drugs.And a reliable and sensitive LC-MS/MS method was establish and validate for the determination of biological samples.Then the developed method was further applied to validating the clinic feasibility of TAK-438 P by investigating the bioequivalence between TAK-438 P and TAK-438 F,as well as the pharmacokinetics,tissue distribution and excretion of TAK-438 P from the perspective of pharmacokinetics.1. Determination of TAK-438 P and TAK-438 F by High Performance Liquid ChromatographyShimadzu liquid chromatography system?LC-2030,Japan?was applied as HPLC system.The column of Intertsil ODS-3 was used,5?m,4.6×150mm.The mobile phase was composed of methanol and a mixture of 0.15%phosphoric acid and 0.15%triethylamine solution?p H=3?.It was adopted gradient elution and the flow rate was set at1.0 m L?min^-1.The column temperature was 30?and the sample size was 10?L,the detection wave length was 230nm.The standard curve of TAK-438 P showed a good linearity over the range of 2.059¹31.8?g?m L^-1 with a correlation coefficient of 0.9995and the regression equation was Y=17448x-16018.The precision of TAK-438 P?Relative standard deviation?was 0.38%and the stability was good.The average spiked recovery of TAK-438 P was 99.40%.The standard curves of TAK-438 F was Y=16770X+8055.9 and the linear ranges were 1.844¹18.0?g?m L^-1.The precision of TAK-438 F?Relative standard deviation?was 0.34%and the stability was good.The average spiked recovery of TAK-438 F was 100.67%.Then bulk drugs of TAK-438 P and TAK-438 F were all determined,the content of three batches of TAK-438 P?1507111026?1507111027?1507111028?were 98.7%?99.0%?98.4%,respectively.The content of three batches?1510091431?1510091432?1510091433?of TAK-438 F were 98.5%?98.2%?98.9%,respectively.2.Determination and validation of TAK-438 P and TAK-438 F by LC-MS/MS method in biological samples.API 4000 tandem and Shimadzu liquid chromatography system LC-30 was applied as LC-MS/MS system.An Agelient C18 analytical column?4.6×150mm,3.5?m?was used by isocratic elution with phase A?10m M ammonium acetate and 0.1%formic acid?and phase B?Methanol?in the ratio of 15:85,at a flow rate of 0.6 m L?min^-1.The total run time was 5 min and the sample volume injected was 5?L.An Electrospray Ionization?ESI?source and multiple reaction monitoring?MRM?were selected with the transitions of m/z346.2 to 315.2 for TAK-438 P and TAK-438 F,and m/z 237.2 to 194 for IS.For the determination of TAK-438 P and TAK-438 F in rat plasma and tissues?heart,liver,spleen,liver,kidney,rain,stomach and small intestine?,selectivity,linearity,sensitivity,accuracy,precision,stability,recovery and matrix effect were all within the acceptable range described in guideline for bioanalytical method validation.For the determination of TAK-438 P and TAK-438 F in rat and excretion,phase A?10m M ammonium acetate and0.1%formic acid?and phase B?Methanol?were in the ratio of 40:60,the total run time was 6 min and the other condition was the same as mentioned above.All validation parameters were within the acceptable range.3.The bioequivalence between TAK-438 P and TAK-438 F after oral administrationSD rats were orally administered low,middle and high doses of TAK-438 P(2.16mg·kg^-1?4.32 mg·kg^-1?8.64 mg·kg^-1)and equimolar TAK-438 F(2.1 mg·kg^-1?4.2mg·kg^-1?8.4 mg·kg^-1)to analyzing the bioequivalence between TAK-438 P and TAK-438F.They were both rapidly absorbed?can be detected in only 5 min?,the T_max was about30⁴5min,and T_1/2 was 1.8².1h in rat plasma.Meanwhile,the bioavailability was increased as the administration doses increasing from low to high.It is worth noting that doublet peaks were shown in the concentration-time curves of TAK-438 F in about 1² h,but there was not obvious doublet peaks in the concentration-time curves of TAK-438 P.The AUC_?0-t?and C_max of TAK-438 P was within the range of 70%¹43%of TAK-438 F and the C_max of TAK-438 P was within the range of 80%¹25%of TAK-438 F.TAK-438 P and TAK-438 F were bioequivalent.4.Study on the pharmacokinetics of TAK-438 P after intravenous administrationSD rats were intravenous administered low and high doses(0.75mg·kg^-1 and2.25mg·kg^-1)of TAK-438 P.The C_max and AUC_?0-t?of TAK-438 P were obviously higher than that of the literature reported of TAK-438 F after intravenous administration to SD rats at the dose.The absolute bioavailability of TAK-438 F was only 10%(oral administration of 2mg·kg^-1 and intravenous administration of 0.75mg·kg^-1).The absolute bioavailability of TAK-438 P was 9.07%,13.91%,30.57%for low,middle,high doses respectively.The bioavailability of TAK-438 P and TAK-438 F was low because of the first pass effect.5.Tissue distribution of TAK-438 P in SD ratsHeart,liver,spleen,lung,kidney,brain,stomach,small intestine tissues were took out,blood was collected from abdomen at 0.25,0.75,2h,8h,12 h after oral administration of4.32 mg·kg^-1 TAK-438 P in rats.Each tissue was thawed,weighed and homogenized in0.9%physiological saline?1:1,g/m L?and then determined by the LC-MS/MS.It indicated that TAK-438 P distributed rapidly(can be detected in 15min and T_max was 45min).The C_max of TAK-438 P in most tissues was higher than that in plasm,which were ordered as below:stomach>liver>lung>kidney>spleen>small intestine>heart>plasma>brain.The concentration in stomach was extremely higher than other tissues,followed by liver.However,the concentration in brain was poor,which revealed that TAK-438 P could not cross the blood-brain barrier efficiently.There was no long-term accumulation in most tissues as the concentration of TAK-438 P was low or undetected in 12 h,except stomach.However,it was worth noting that there was still 2017.49 ng?m L^-1 TAK-438 P left in stomach 12 h after oral administration,which proved that stomach was likely the target organs of TAK-438 P.Overall,TAK-438 P had a high selectivity for stomach and own notable advantage of high accumulation,slow clearance.6.Excretion study of TAK-438 P and TAK-438 F in ratsAfter oral administration of 4.32 mg·kg^-1 TAK-438 P and equimolar TAK-438 F in rats,the accumulative urine excretion rate of prototype drug were 2.11%and 2.03%.The low excretion ratio indicated that metabolism might be the major clearance mechanism of TAK-438 P and TAK-438 F.The excretion rate of TAK-438 F was obvious lower than TAK-438 P in 2⁴h,in contrast with higher excretion rate of TAK-438 F in 4⁸ h.It was associated with the doublet peaks in the concentration-time curves of TAK-438 F,which may be related to hepato-enteric circulation of TAK-438 F.The concentration of TAK-438P and TAK-438 F in feces was below the detection limit.It was reported in the literature that metabolites?not prototype drug?were excreted into feces.