五种抗结核新药在小鼠体内的药代动力学/药效学初步研究

doi:10.3969/j.issn.1000-6621.2021.10.015

摘要/Abstract

摘要：

目的在BALB/c小鼠结核病模型中,研究普托马尼(pretomanid,PA-824)、德拉马尼(delamanid,Dlm)、氯法齐明(clofazimine,Cfz)、贝达喹啉(bedaquiline,Bdq)、PBTZ-169共5种抗结核药物在不同给药剂量下的药代动力学/药效学(pharmacokinetic/pharmacodynamics,PK/PD)。方法 BALB/c小鼠经气溶胶感染结核分枝杆菌标准株H37Rv,5种药物分为高中低3个剂量组灌胃给药。在给药4周和8周时,解剖小鼠,取脾、肺脏进行菌落单位(colony forming unit,CFU)计数以评价药物疗效,同时在4倍测定最低抑菌浓度(minimum inhibitory concentration,MIC)的含药培养板上,挑取单菌落并测定MIC值。在各给药组给予最后一个剂量3h后,通过眼眶采血,用液相色谱-质谱法测定血浆药物浓度。结果以各药物引起小鼠肺部CFU计数下降且与对照组差异有统计学意义为标准,得到最低有效剂量分别为PA-824 30mg/kg,Dlm 5mg/kg,Cfz 5mg/kg,Bdq 6.25mg/kg,PBTZ-169 20mg/kg。自最低有效剂量起,各药物均使得小鼠肺CFU计数下降,其中Bdq与PA-824高剂量组小鼠肺CFU计数分别下降2.99和2.66 log₁₀值。5种药物给药8周时,在最低有效剂量下的血药浓度(C_3h)分别为PA-824(7.60±1.28)μg/ml,Dlm(0.20±0.05)μg/ml,Cfz(0.26±0.02)μg/ml,Bdq(0.22±0.07)μg/ml,PBTZ-169(74.56±17.80)ng/ml。各药物高剂量时,C_max/MIC大于特定数值,即PA-824 100mg/kg时,C_max/MIC>147;Dlm 10mg/kg时,C_max/MIC>100;Cfz 20mg/kg时,C_max/MIC>2;Bdq 25mg/kg时,C_max/MIC>13.6;PBTZ-169 20mg/kg时,C_max/MIC>150,不易产生药物耐受性/表型抗性。结论 5种抗结核药物在有效剂量下,血药浓度随药物剂量增加而上升,CFU计数与给药剂量呈负相关,C_max/MIC是与疗效呈良好相关性的PK/PD参数,可为这些药物的临床使用提供指导。

关键词: 结核, 药代动力学, 抗药性, 研究

Abstract:

Objective To evaluate the pharmacokinetic/pharmacodynamics (PK/PD) of five anti-tuberculosis drugs including pretomanid (PA-824), delamanid (Dlm), clofazimine (Cfz), bedaquiline (Bdq) and PBTZ-169 in BALB/c mice tuberculosis model at different doses. Methods BALB/c mice were infected with Mycobacterium tuberculosis (H37Rv) by aerosol. Five drugs were divided into high, middle and low dose groups. After 4 and 8 weeks of treatment, the mice were dissected and the colony forming unit (CFU) of spleens and lungs were counted. At the same time, the lungs and spleens homogenate were inoculated on the plate containing 4×minimum inhibitory concentration (MIC) to observe the colony growth. Three hours after the last dose was given to each group, the orbital blood was collected and the drug concentration were determined by HPLC-MS/MS. Results The lowest effective dose of each drug was PA-824 30 mg/kg, Dlm 5 mg/kg, Cfz 5 mg/kg, Bdq 6.25 mg/kg, PBTZ-169 20 mg/kg, which obtained according to the significant difference of lung CFU count between treatment and control groups. From the lowest effective dose, the CFU counts in the lungs of mice were decreased in all treatment groups. At the high-dose of Bdq and PA-824, the CFU counts in the lungs of mice decreased by 2.99 and 2.66 log₁₀ values respectively. At 8 weeks, the plasma concentrations (C_3h) of each drug at the lowest effective dose were PA-824 (7.60±1.28) μg/ml, Dlm (0.20±0.05) μg/ml, Cfz (0.26±0.02) μg/ml, Bdq (0.22±0.07) μg/ml, PBTZ-169 (74.56±17.80) ng/ml, respectively. At high dose, when C_max/MIC was higher than the specific value (PA-824 100 mg/kg, C_max/MIC>147; Dlm 10 mg/kg, C_max/MIC>100; Cfz 20 mg/kg, C_max/MIC>2; Bdq 25 mg/kg, C_max/MIC>13.6; PBTZ-169 20 mg/kg, C_max/MIC>150), drug tolerance/phenotypic resistance was not easy to occur. Conclusion At the effective dose of the five anti-tuberculosis durgs, the plasma concentration increased with the increase of the dosage, but the CFU count was negatively correlated with the dosage. Our study showed that C_max/MIC was a good PK/PD parameter correlation with treatment effect, which could provide guidance for the clinical use of these drugs.

Key words: Tuberculosis, Pharmacokinetics, Drug resistance, Interdisciplinary studies

朱慧, 付雷, 张炜焱, 王彬, 陈曦, 陆宇. 五种抗结核新药在小鼠体内的药代动力学/药效学初步研究[J]. 中国防痨杂志, 2021, 43(10): 1056-1065. doi: 10.3969/j.issn.1000-6621.2021.10.015

ZHU Hui, FU Lei, ZHANG Wei-yan, WANG Bin, CHEN Xi, LU Yu. Preliminary study on pharmacokinetics/pharmacodynamics of five new anti-tuberculosis drugs in mice[J]. Chinese Journal of Antituberculosis, 2021, 43(10): 1056-1065. doi: 10.3969/j.issn.1000-6621.2021.10.015

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参考文献 19

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组别	剂量(mg/kg)	4周(g,$\bar{x}\pm s$)	t值	P值	8周(g,$\bar{x}\pm s$)	t值	P值
对照组		21.44±1.07			21.86±0.85
PA-824
L组	10	20.87±0.98	-0.883	0.403	22.56±1.05	1.169	0.276
M组	30	21.97±0.57	0.972	0.359	23.04±0.81	2.261	0.054
H组	100	22.49±0.71	1.829	0.105	22.35±1.01	0.838	0.426
Dlm
L组	2.5	21.56±0.37	0.241	0.816	22.56±0.85	1.306	0.228
M组	5	21.63±1.33	0.244	0.814	23.30±1.06	2.389	0.044
H组	10	21.39±1.05	-0.078	0.940	21.51±1.03	-0.577	0.580
Cfz
L组	5	22.01±1.04	0.846	0.422	23.20±1.20	2.047	0.075
M组	10	21.08±1.82	-0.385	0.710	22.41±1.18	0.854	0.418
H组	20	20.76±0.79	-1.152	0.283	22.82±0.67	1.988	0.082
Bdq
L组	6.25	22.14±1.12	1.016	0.339	22.10±0.48	0.556	0.593
M组	12.5	21.68±0.66	0.417	0.688	23.52±0.35	4.064	0.004
H组	25	21.32±1.05	-0.188	0.855	22.78±0.66	1.923	0.091
PBTZ-169
L组	5	21.30±0.48	-0.275	0.790	22.45±0.88	1.095	0.305
M组	10	21.37±0.38	-0.138	0.894	23.66±0.69	3.690	0.006
H组	20	20.84±0.75	-1.032	0.332	23.45±0.41	3.798	0.005

组别	剂量(mg/kg)	4周时浓度(μg/ml,$\bar{x}\pm s$)	8周时浓度(μg/ml,$\bar{x}\pm s$)	参考MIC^a(μg/ml)
PA-824				0.07
L组	10	3.75±0.92	2.84±0.61
M组	30	9.05±1.70	7.60±1.28
H组	100	17.95±2.87	14.68±1.02
Dlm				0.004
L组	2.5	0.02±0.01	0.20±0.06
M组	5	0.24±0.12	0.20±0.05
H组	10	0.43±0.17	0.40±0.07
Cfz				0.24
L组	5	0.22±0.01	0.26±0.02
M组	10	0.23±0.04	0.43±0.05
H组	20	0.36±0.08	0.47±0.07
Bdq				0.022~0.05
L组	6.25	0.31±0.10	0.22±0.07
M组	12.5	0.32±0.14	0.42±0.07
H组	25	0.68±0.25	0.68±0.26
PBTZ-169				0.4~0.5^b
L组	5	7.25±1.69^b	6.91±1.52^b
M组	10	18.70±6.69^b	21.23±4.45^b
H组	20	51.54±20.35^b	74.56±17.80^b

组别	剂量(mg/kg)		肺(log₁₀CFU,$\bar{x}\pm s$)		t值^a		P值^a		脾(log₁₀CFU,$\bar{x}\pm s$)		t值^a		P值^a
对照组
治疗前14d			4.70±0.11						1.48±0.00
治疗当天			5.21±0.08						3.60±0.18
治疗8周			4.90±0.16						3.08±0.44
PA-824
L组	10		4.72±0.19		-1.542		0.162		4/5为0,1只为2.08		-5.775		<0.001
M组	30		4.00±0.17		-8.654		<0.001		0		-15.475		<0.001
H组	100		2.24±0.22		-21.951		<0.001		0		-15.475		<0.001
Dlm
L组	2.5		4.75±0.20		-1.255		0.245		4/5为0,1只为2.08		-5.775		<0.001
M组	5		4.55±0.14		-3.627		0.007		4/5为0,1只为1.48		-7.803		<0.001
H组	10		3.54±0.16		-13.574		<0.001		0		-15.475		<0.001
Cfz
L组	5		4.07±0.12		-9.405		<0.001		2.47±0.19		-2.799		0.023
M组	10		4.02±0.27		-6.231		<0.001		2.40±0.80		-1.657		0.136
H组	20		3.41±0.28		-10.397		<0.001		2.06±0.23		-4.538		0.002
Bdq
L组		6.25		4.61±0.15		-2.918		0.019		2.90±0.33		-0.731	0.485
M组		12.5		3.85±0.51		-4.405		0.008		4/5为0,1只为2.38		-5.045	0.001
H组		25		1.91±1.08		-6.109		<0.001		0		-15.475	<0.001
PBTZ-169
L组		5		5.54±0.22		5.306		0.001		3.16±0.17		0.381	0.713
M组		10		4.71±0.23		-1.505		0.171		0		-15.475	<0.001
H组		20		3.36±0.21		-12.999		<0.001		4/5为0,1只为1.48		-7.803	<0.001

培养板脏器	L组(10mg/kg)		M组(30mg/kg)		H组(100mg/kg)
培养板脏器	不含药培养板	含药培养板	不含药培养板	含药培养板	不含药培养板	含药培养板
不含活性炭培养板
肺脏	+++	1只为 1CFU	74~171CFU	未生长	3~10CFU	未生长
脾脏	4只未生长, 1只为 4CFU	未生长	未生长	未生长	未生长	未生长
活性炭含药培养板
肺脏	-	100~300CFU	-	44~150CFU	-	0~73CFU
脾脏	-	4只未生长, 1只为 4CFU	-	未生长	-	未生长

培养板脏器	L组(2.5mg/kg)		M组(5mg/kg)		H组(10mg/kg)
培养板脏器	不含药培养板	含药培养板	不含药培养板	含药培养板	不含药培养板	含药培养板
不含活性炭培养板
肺脏	+++	未生长	+++	未生长	85~210CFU	未生长
脾脏	4只未生长, 1只为 4CFU	未生长	4只未生长, 1只为 1CFU	未生长	未生长	未生长
活性炭含药培养板
肺脏	-	1~200CFU	-	3~150CFU	-	1~16CFU
脾脏	-	4只未生长, 1只为 1CFU	-	4只未生长, 1只为1CFU	-	未生长