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

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。

The combined effect of isoniazid and rifampicin on the activities of CYP4501A2 and CYP4503A4 in primary hepatocytes from healthy human adults

OBJECTIVE: To study the combined effect of isoniazid and rifampicin on the activities of CYP1A2 and 3A4 in primary hepatocytes from healthy human adults. METHODS: The primary hepatocytes were isolated from adult healthy human livers, and cultured for 3 days. Then the cells were divided into 15 groups including two negative control groups (culture media alone) and 13 drug intervention groups, to which the following drugs were added: isoniazid (25 micromol/L, 50 micromol/L), rifampicin (12.5 micromol/L, 25 micromol/L) or both of them with different concentrations (CYP 1A2: rifampicin 12.5 micromol/L + isoniazid 50 micromol/L, rifampicin 25 micromol/L + isoniazid 50 micromol/L; CYP3A4: rifampicin 12.5 micromol/L + soniazid 50 micromol/L, rifampicin 25 micromol/L + isoniazid 25 micromol/L, rifampicin 25 micromol/L + isoniazid 50 micromol/L) respectively. All the concentrations were consistent with the range of maximum clinical blood concentrations. After culture for 2 days, substrates (phenacetin for CYP1A2 , testosterone for CYP3A4) were added, and then the peak area (unit: mAU. min) of their metabolites was measured with high performance liquid chromatography (HPLC) to assess the activities of CYP450 1A2 and 3A4. RESULTS: (1) The activity of CYP1A2 in isoniazid groups with concentrations of 25 micromol/L and 50 micromol/L was (3.33 +/- 0.65), (3.03 +/- 0.38) mAU.min respectively, significantly different compared with that in the negative control group (5.23 +/- 0.31) mAU.min, P < 0.01]. The activity of CYP450 1A2 in rifampicin groups with a concentration of 12.5 micromol/L was (6.07 +/- 0.55) mAU.min, which had significant difference compared with that in the negative control group (P < 0.05). There was no statistical difference of CYP1A2 activity between rifampicin with 25 micromol/L (4.93 +/- 0.57) mAU.min] and the negative control group (P > 0.05). The activity of CYP1A2 of groups with two kinds of different concentrations of isoniazid and rifampicin combined groups was (3.27 +/- 0.96), (3.97 +/- 0.25) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.05). (2) The activity of CYP3A4 in isoniazid groups with concentrations of 25 micromol/L and 50 micromol/L was (5.40 +/- 1.35), (2.63 +/- 0.06) mAU.min respectively, which had significant difference compared with that in the negative control group (12.53 +/- 0.51) mAU.min, P < 0.01]. The activity of CYP3A4 in rifampicin groups with concentrations of 12.5 micromol/L and 25 micromol/L was (165.17 +/- 11.47), (120.20 +/- 15.73) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.01). The activity of CYP3A4 in the three isoniazid and rifampicin combined groups with three kinds of different concentrations was (118.37 +/- 8.90), (77.53 +/- 6.91), (68.73 +/- 4.72) mAU.min respectively, which had significant difference compared with that in the negative control group (P < 0.01), but they were lower than those in rifampicin groups with corresponding concentrations (P < 0.05). CONCLUSIONS: Isoniazid and rifampicin in the range of maximum clinical blood concentration have no significant inducing or inhibiting effect on the activity of CYP1A2 of healthy adult human primary hepatocytes. Isoniazid in the range of maximum clinical blood concentration can inhibit the activity of CYP3A4, while rifampicin can induce the activity of CYP3A4; the combined effect of isoniazid and rifampicin being the induction of CYP3A4 activity, but the inducing effect was less than that of rifampicin alone with the same concentration.