The Preclinical Pharmacokinetics And Drug-drug Interaction Study Of New Combinational Anti-tuberculosis Drugs

Tuberculosis (TB) is one kind of chronic infectious diseases caused by Mycobacterium tuberculosis due to a single infectious agent. Nowadays, it has been the second leading cause of death from an infectious disease worldwide, just after the HIV/AIDS. The latest WHO report estimated that there were 9 million new TB cases in 2013 and 1.5 million TB deaths. In order to prevent drug-resistant tuberculosis, the clinical treatment regimen for tuberculosis is fixed-dose combination. But the current regimen suffers from the disadvantages of large numbers of medicine, long term of treatment, high rate of adverse effect and cost consuming. Based on the screen and recombination of current anti-tuberculosis drugs by an original FSC technology, one new combinational anti-tuberculosis drug for TB containing of EMB, PZA, CLZ and PRO was obtained. We have carried out the pharmacokinetic and metabolic drug-drug interaction study of this new combinational anti-tuberculosis drug in SD rats and Beagle dogs, which can pave the way for its clinical use.1. Development and validation of LC-MS/MS method for simultaneous determination of standard four-drug combination and its pharmacokinetic study in animalsThe standard treatment regimen for tuberculosis contains four first-line anti-TB drugs of RFP, INH, EMB and PZA. LC-MS/MS method is the common tool for its pharmacokinetic study and therapeutic drug monitoring. The polarity of the four drugs is so different that it is difficult to fulfill the simultaneous determination of them under the same column and mobile phase condition. In this part, we compared different types of LC-MS/MS instruments and chromatographic columns, optimized gradient elution procedure and eliminated the matrix suppression of ethambutol and isoniazid in ESI detection. A sensitive, specific and repeatable LC-MS/MS method for simultaneous determination of the four first-line anti-TB drugs was developed. Acetonitrile was used to precipitate the protein in plasma samples. The four drugs were separated using a Waters Atlantis dC18 column. A rapid gradient elution procedure was used with the methanol:acetonitrile (1:1, v/v) as mobile phase A and water containing 0.1% formic acid as mobile phase B. The four drugs and internal standard performed good peak shape within 4 minutes. Per sample analysis time was 6 min. This method was fully validated both in SD rat plasma and Beagle dog plasma according to the requirements of the US Food and Drug Administration guidance. It has been successfully applied to the pharmacokinetic study of the standard four-drug combination in SD rats and Beagle dogs.2. Development and validation of LC-MS/MS method for simultaneous determination of new combinational anti-tuberculosis drug and its pharmacokinetic study in SD ratsIn this part, EMB, PZA, CLZ and PRO in the new combinational anti-tuberculosis drug were simultaneously determined using an Agilent ZORBAX SB-Aq column. Methanol and water (containing 0.1% formic acid and 5 mM ammonium acetate) were adopted as the mobile phase. Gradient elution was used and optimized to perform the separation of the analytes. The four drugs and internal standard were separated within 7 minutes and per sample analysis time was 10 min. A rapid and high-throughput sample preparation was applied by protein precipitation in Sirocco 96-well filtration plate with the use of only 20μL plasma. All data in the method validation met the acquirement of FDA guideline about preclinical pharmacokinetic study. The LC-MS/MS method was then used in the pharmacokinetic and drug-drug interaction study in SD rats.By comparing changes of the pharmacokinetic parameters after single dose of individual drug and the new four-drug combination, we found that the absorption of EMB may be postponed and PRO may has a promoted absorption in combination. But the t1/2 and MRT of the two drugs had no significant difference. This means that the combined administration had an effect on intestinal absorption and transport rather than elimination. CLZ and PZA had no obvious drug-drug interaction in combination. On the fifth day of multiple-dose test, the concentration of the four drugs in SD rats had reached to the steady state. According to the changes of pharmacokinetic parameters after single-dose and multiple-dose administration of both individual and combinational drugs, we found that all the four drugs have accumulated in SD rat after 7 days’ administration. However, there was no significant difference between tmax, t1/2, and MRT. This illustrated that the accumulation of the four drugs in vivo has no effect on the metabolic characteristics.3. Pharmacokinetic study of new combinational anti-tuberculosis drug in Beagle dogsIn order to know more about pharmacokinetic features of the new combinational anti-tuberculosis drug, further pharmacokinetic and drug-drug interaction study was carried out in Beagle dogs using the LC-MS/MS method and Sirocco 96-well filtration plate in Part 2. From the pharmacokinetic parameters after single dose of individual and combinational drugs, we found that the absorption of EMB, CLZ, and PRO was postponed, promoted and restrained after combinational administration. PZA had no obvious drug-drug interaction. On the fifth day of multiple-dose test, the concentration of EMB, PZA and CLZ in Beagle dogs had reached to the steady state. From the pharmacokinetic parameters after single-dose and multiple-dose administration of combinational drug, we found that there was no obvious accumulation of PZA and PRO in Beagle dogs after 7 days’ administration. This means that long-term administration of the combinational drug has no effect on pharmacokinetic features of PZA and PRO. However, EMB and CLZ have accumulated in Beagle dogs with no significant difference between tmax, t1/2, and MRT. This illustrated that the accumulation of the two drugs in vivo has no effect on the metabolic characteristics.4. Effect of the new combinational anti-tuberculosis drug on metabolic enzyme activity in SD rat liver microsomeIn this part, three fully validated LC-UV methods were used to assay the specific probe substrates phenacetin (CYP1A2), midazolam (CYP3A4), chlorzoxazone (CYP2E1) and their metabolites acetaminophen, 1’-hydroxymidazolam, and 6-hydroxychlorzoxazone, respectively in SD rat liver microsome. The induction or inhibition effect on the three enzymes after multiple doses of EMB, PZA, PRO, CLZ and the combination for 14 days was also evaluated.By comparing the amounts of metabolites in experimental groups with those in the control group, the following conclusions can be obtained:(1) No significant effect on CYP1A2 was detected after multiple doses of EMB; however, induction effect appeared on CYP1A2 after multiple doses of PZA, CLZ, PRO and the combination with no obvious gender difference. (2) No significant effect on CYP3A4 was detected after multiple doses of the four individual drugs and the combination for 14 days; but SD rats showed the gender difference. (3) No significant effect on CYP2E1 was detected after multiple doses of the four individual drugs; however, induction effect appeared on CYP2E1 after multiple doses of the combination for 14 days with no obvious gender difference. More than 20 drugs, such as caffeine and theophylline, are mainly metabolized by CYP1A2. Medical workers should keep an eye on efficacy and toxic effect of these drugs if they have to be used with the new combinational anti-tuberculosis drug. CYP2E1 is one of the critical enzymes in biotransformation of procarcinogens and has close relationship with hepatotoxicity. Special focus should be paid on the adverse effects of patients.