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目的明确异烟肼和利福平联合用药对健康成人原代肝细胞CYP450同工酶1A2和3A4活性的影响。方法从健康成人肝脏或肝叶中分离出肝细胞,分为CYP450同工酶1A2和3A4两部分,各分为阴性对照组及药物处理组共15组,放入24孔细胞培养板内,每组6个复孔,原代培养3 d,阴性对照组加入等量的细胞培养液,各药物处理组对应加入临床血药峰浓度范围内的异烟肼(25μmol/L,50μmol/L)、利福平(12.5μmol/L,25μmol/L)或两药联用(CYP1A2:利福平12.5μmol/L加异烟肼50μmol/L,利福平25μmol/L加异烟肼50μmol/L;CYP3A4:利福平12.5μmol/L加异烟肼25μmol/L,利福平25μmol/L加异烟肼25μmol/L,利福平25μmol/L加异烟肼50μmol/L),孵育2 d,再加入CYP450同工酶1A2和3A4的相应底物(非那西丁和睾酮),反应终止后用高效液相仪测量代谢产物(对乙酰氨基酚与6β-羟基睾酮)的峰面积(单位:mAU.m in)代表1A2和3A4的活性。结果(1)单用25μmol/L和50μmol/L浓度异烟肼肝细胞CYP450同工酶1A2的活性分别是(3.33±0.65)、(3.03±0.38)mAU.m in,与阴性对照组的(5.23±0.31)mAU.m in比较差异均有统计学意义(P均<0.01);单用12.5μmol/L浓度利福平肝细胞CYP450同工酶1A2的活性为(6.07±0.55)mAU.m in,与阴性对照组比较差异有统计学意义(P<0.05),单用25μmol/L浓度利福平肝细胞CYP450同工酶1A2的活性为(4.93±0.57)mAU.m in,与阴性对照组比较差异无统计学意义(P>0.05);异烟肼和利福平2种浓度联合配伍,肝细胞CYP450同工酶3A4的活性分别是(3.27±0.96)、(3.97±0.25)mAU.m in,与阴性对照组比较差异均有统计学意义(P均<0.05)。(2)单用25μmol/L和50μmol/L浓度异烟肼肝细胞CYP450同工酶1A2的活性分别是(5.40±1.35)、(2.63±0.06)mAU.m in,与阴性对照组的(12.53±0.51)mAU.m in比较差异均有统计学意义(P均<0.01);单用12.5和25μmol/L浓度利福平肝细胞CYP450同工酶3A4的活性分别为(165.17±11.47)、(120.20±15.73)mAU.m in,与阴性对照组比较差异均有统计学意义(P均<0.01);异烟肼和利福平3种浓度联合配伍,肝细胞CYP450同工酶3A4的活性分别是(118.37±8.90)、(77.53±6.91)、(68.73±4.72)mAU.m in,与阴性对照组比较差异均有统计学意义(P均<0.01),但低于相应浓度单用利福平组(P<0.05或0.01)。结论临床血药峰浓度范围的异烟肼与利福平单用或联用对CYP450同工酶1A2活性影响未达到抑制或诱导水平。临床血药峰浓度范围的异烟肼抑制CYP450同工酶3A4的活性,利福平诱导CYP450同工酶3A4的活性,两药联合仍呈诱导作用。
Objective To determine the effects of combination of isoniazid and rifampicin on the activities of CYP450 isoenzyme 1A2 and 3A4 in healthy adult primary hepatocytes. Methods Hepatocytes were isolated from healthy adult liver or liver lobe and divided into two groups: CYP450 isoenzyme 1A2 and 3A4. Each group was divided into 15 groups, negative control group and drug treatment group, and placed in 24-well cell culture plates. 6 replicate wells were cultured in primary culture for 3 days. The same amount of cell culture medium was added to the negative control group. Isoniazid (25μmol / L, 50μmol / L) Rifampicin (12.5μmol / L, 25μmol / L) or combination of the two drugs (CYP1A2: rifampin 12.5μmol / L plus isoniazid 50μmol / L, rifampicin 25μmol / L plus isoniazid 50μmol / L; Rifampicin 25μmol / L plus isoniazid 25μmol / L, Rifampicin 25μmol / L plus isoniazid 50μmol / L) for 2 days, Then, the corresponding substrates (phenacetin and testosterone) of CYP450 isoenzyme 1A2 and 3A4 were added. After termination of the reaction, the peak area of metabolites (paracetamol and 6β-hydroxytestosterone) (unit: mAU.m in) represents the activity of 1A2 and 3A4. Results (1) The activity of CYP450 isoenzyme 1A2 in isoniazid hepatocytes with the concentration of 25μmol / L and 50μmol / L alone was (3.33 ± 0.65), (3.03 ± 0.38) mAU.m in, respectively. Compared with the negative control group 5.23 ± 0.31) mAU.m in (P <0.01). The activity of CYP450 isoenzyme 1A2 in rifampin hepatocytes with 12.5 μmol / L alone was (6.07 ± 0.55) mAU.m (P <0.05). The activity of CYP450 isozyme 1A2 in rifampicin hepatocytes with 25 μmol / L alone was (4.93 ± 0.57) mAU.m in, which was significantly lower than that in the negative control (P> 0.05). The activity of CYP450 isoenzyme 3A4 in hepatocytes was (3.27 ± 0.96) and (3.97 ± 0.25) mAU, respectively, with the combination of isoniazid and rifampicin. m in and negative control group, the difference was statistically significant (P all <0.05). (2) The activities of CYP450 isoenzyme 1A2 in isoniazid hepatocytes with 25μmol / L and 50μmol / L alone were (5.40 ± 1.35) and (2.63 ± 0.06) mAU.m in, respectively, ± 0.51) mAU.m in (P <0.01). The activities of CYP450 isoenzyme 3A4 in rifampin hepatocytes treated with 12.5 and 25 μmol / L alone were (165.17 ± 11.47), 120.20 ± 15.73) mAU.m in, respectively, compared with the negative control group (all P <0.01). The activities of isoniazid and rifampin in combination with three concentrations and CYP450 isoenzyme 3A4 Were (118.37 ± 8.90), (77.53 ± 6.91) and (68.73 ± 4.72) mAU.m in, respectively, which were significantly different from those of the negative control group (all P <0.01) Flat group (P <0.05 or 0.01). Conclusions The effects of isoniazid and rifampicin alone or in combination on the activity of CYP450 isoenzyme 1A2 have not been inhibited or induced in the range of clinical peak plasma concentrations. Isoniazid in the concentration range of clinical blood serum inhibits the activity of CYP450 isoenzyme 3A4 and rifampicin induces the activity of CYP450 isozyme 3A4, and the combination of the two drugs still shows an induction effect.