复方萘酚阿奇对延缓恶性疟原虫抗性的实验性研究
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摘要
1.研究目的
1.1观察对照组(对照组单一用萘酚喹)与实验组(萘酚喹与阿奇霉素联合用药)恶性疟原虫在体外药物刺激(压力)下,疟原虫对其敏感性的变化情况或产生速度。
1.2用WHO推荐的Rieckmann体外测定不同阶段对照组与实验组恶性疟原虫对萘酚喹、阿奇霉素、复方萘酚阿奇的敏感性,计算半数抑制量(IC50)。
1.3阐述两药物联用体外抗疟作用的特点；评价两药物联合有或无延缓抗性的作用或潜力。为疟疾的联合用药的临床治疗方案提供科学依据。
2.研究方法
2.1虫株与培养：恶性疟原虫FccSM/YN株,该株采自位于中老、中缅边境地区云南思茅,按Trager等蜡烛缸培养法,通过实验室驯化,建立体外传代培养,实验室长期保种虫株。
2.2药物作用观察：用体外剂量递增间隔接触法进行抗性培育
2.3体外测定：测试采用Rieckmann体外微量测定法。将同步化处理过的疟原虫稀释到20,000-60,000个/μl血,加5倍培养基稀释,混匀后加入测定板各药井,从低浓度到高浓度每井加50μl,每个浓度同时测定两组(2行)。37℃培养20-24h后收获1个对照井,视其疟原虫发育情况决定收获时间。用ICEstimator software计算IC50。
2.4判别标准接触药物后的恶性疟原虫的IC50较亲代上升5倍以上,可认为对该药的敏感性下降。在相同的抗性培育时间内,接触萘酚喹/阿奇霉素的恶性疟原虫的IC50分别较亲代上升5倍,可认为对该药的敏感性下降；萘酚喹/阿奇霉素对恶性疟原虫的IC50分别与亲代相比,无明显上升或者虽然有所上升,但明显低于对照组,可认为该联合用药有延缓抗性作用。
3.结果
3.1药物接触的次数、浓度及结果对照组恶性疟原虫接触萘酚喹120天,共接触药物15次,药物浓度分别为2.56nmol/L5次,160nmol/L2次,5、10、20、40、80、320、625、1280nmol/L各1次。接触药物前,敏感株(亲代系)的IC50为2.11nmol/L,药物刺激15次后,其IC50增至65.47nmol/L。为亲代原虫的31倍。实验组恶性疟原虫接触萘酚喹/阿奇霉素共120天,共接触药物19次,药物浓度分别为0.31/0.256nmol/L 8次,2.49/2.135 nmol/L 5次,0.625/0.53、4.98/4.27、9.735/8.34、19.47/16.685、38.94/33.375、77.88/66.755nmol/L各一次。接触药物前,敏感株(亲代系)的IC50为0.18 nmol/L、0.15nmol/L,药物刺激19次后,其IC50增至2.89 nmol/L、2.68 nmol/L,分别为亲代原虫的16.1倍、17.9倍。
3.2用药前恶性疟原虫的敏感性测定结果在恶性疟原虫未接触药物前,在萘酚喹测定板、阿奇霉素测定板及萘酚喹/阿奇霉素复合测定板中测定疟原虫的敏感性,在单一萘酚喹测定板中测得IC50为2.11nmol/L;在单一的阿奇霉素测定板中测得IC50为3.21 nmol/L;在萘酚喹/阿奇霉素复合测定板中测得萘酚喹与阿奇霉素的IC50分别为0.18nmol/L、0.15 nmol/L。单一测定板中萘酚喹的IC50是复合测定板的11.7(2.11/0.18)倍；阿奇霉素的IC50是复合测定板的21.4(3.21/0.15)倍。
3.3对照组恶性疟原虫接触萘酚喹前后(药物刺激15次,历时120天)的敏感性测定结果对照组恶性疟原虫在单一接触萘酚喹后的IC50为单一接触萘酚喹前的31.0(65.47/2.11)倍。对照组疟原虫在单一接触萘酚喹后对阿奇霉素的敏感性是单一接触萘酚喹前的21.4(68.58/3.21)倍。
3.4实验组恶性疟原虫接触萘酚喹/阿奇霉素前后(药物刺激19次,历时120天)的敏感性测定结果实验组疟原虫在接触萘酚喹/阿奇霉素后对萘酚喹的IC50为接触药物前的16.8(35.47/2.11)倍。实验组疟原虫在接触萘酚喹/阿奇霉素后对阿奇霉素的IC50为接触药物前的15.6(50.04/3.21)倍。
3.5萘酚喹/阿奇霉素联用与萘酚喹单用对恶性疟原虫作用的比较对照组在接触药物120天后在单一萘酚喹测定板中的IC50为65.47nmol/L,在单一阿奇霉素测定板中测得IC5。为68.58nmol/L,在萘酚喹/阿奇霉素复合测定板中的IC50分别为10.34nmol/L/8.46nmol/L。实验组在接触药物120天后在单一萘酚喹测定板中的IC50为35.47nmol/L,在单一阿奇霉素测定板中测得IC50为50.04nmol/L,在萘酚喹/阿奇霉素复合测定板中的IC50分别为2.89nmol/L、2.68 nmol/L。
4.结论
4.1恶性疟原虫在单一接触萘酚喹后一段时间敏感性有明显的下降,其IC50远远高于亲代的5倍以上,进一步证实用人工培养的方法可以培育出恶性疟原虫抗萘酚喹耐药株。
4.2萘酚喹与阿奇霉素配伍对恶性疟原虫的作用观察显示,无论对敏感株(对照组)还是抗药株在萘酚喹/阿奇霉素复合测定板中测的IC50显著低于单一的萘酚喹测定板和阿奇霉素测定板,提示萘酚喹与阿奇霉素配伍对恶性疟原虫的作用明显大于单一用药,两种药物联用有明显增效作用。
4.3实验组恶性疟原虫对药物耐受性的增长远远慢于对照组,前者在药物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分别上升16.8倍、15.6倍、16.1/17.9倍,后者在药物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分别上升31.0倍、21.4倍、57.4/56.4倍,提示萘酚喹与阿奇霉素两种药物联用有明显延缓抗性作用。
1. Objective
1.1 Observation of the experimental group (Naphthoquine combination with Azithromycin) and the control group (control group:single Naphthoquine use) of Plasmodium falciparum in vitro drug stimulation (pressure), in order to sensitivity and its changes in speed.
1.2 With the WHO recommended Rieckmann in vitro determination of the sensitivity of the different stages Plasmodium falciparum to Naphthoquine and Azithromycin, Naphthoquine/Azithromycin in control group and experimental group to calculate the amount of half inhibition (IC50).
1.3 Described the characteristics of the action for the two drugs combination in vitro and evaluated the two drugs combination whether or not delay resistance or potential. Combination therapy may provide a scientific basis for the clinical treatment malaria programs.
2. Methods
2.1 Strain and culture:Culture the Plasmodium falciparum strain selected from samples by Trager method in vitro
2.2 Observation the effect of drug:The resistant Plasmodium falciparum strains were stimulated by corresponding drugs with increasing concentrations disconnectedly.
2.3 In vitro assay:Rieckmann's in vitro micro-technique was used for assay. The concentration of in vitro cultured Plasmodium falciparum strain were diluted to 20,000-60,000/μ1 with culture medium, which was added to pre-prepared drug plates at 50μ/well after synchronization. Selected Plasmodium falciparum cultured in one of control wells after 20-24hours'culture in 37℃, the harvested time was determined by the growth of Plasmodium falciparum. Based on the number of schizonts in control well, counted the inhibitive ratio of schizonts in each well, calculated half inhibitive dose (IC50) of every drug to parasite by ICEstimator software.
2.4 Estimated standard:When IC50 of Plasmodium falciparmst to artesunat was 5 time than that of parent, then we consider corresponding P. falciparum as artesunate-resistant strain. During the same period of resistance culturing in vitro, if IC50 value of P. falciparumst to artesunat combined with clindamycin was 5 times than that of parent, which could be deemed that the P. falciparumst had resistance to the tow drugs; otherwise, the IC50 did not rise obviously or much more lower than that of parent, however, artesunate combined with clindamycin exhibit an apparent synergistic effect in vitro.
1. Results
3.1 Frequency, concentration and results of drug touching. The control group falciparum contact Naphthoquine 150 days, total contact drug 15 times, drug concentration tools 2.56 nmol/L five times,160nmol/L twice,5,10,20,40,80,320,625,1280nmol/L each time respectively. Contact drugs before, sensitive strains (parental department) of IC50 of 2.11 nmol/L, drug stimulates 15 times, after the IC50 to 65.47 nmol/L. Approximately dictated protozoon 31 times. Experimental falciparum contact Naphthoquine/Azithromycin 120 days, total contact drug nineteen times, drug concentration respectively for 0.31/0.256 nmol/L eight,2.49/2.135 nmol/L five times,0.625/0.53、4.98/4.2、 9.735/8.34、19.47/16.685、38.94/33.375、77.88/66.755nmol/L each time. Contact drugs before, sensitive strains (parental department) of IC50 for 0.18 nmol/L、0.15 nmol/L, drug stimulates nineteen times and its IC50 to 2.68 nmol/L、2.89nmol/L respectively, approximately dictated protozoon 16.1 times、17.9times.
3.2 Sensitivity of P. falciparum before adding drugs. In P. falciparum not contact with drugs, before Naphthoquine determination board, Azithromycin determination board and Naphthoquine/Azithromycin composite plate determination plasmodium P. falciparum determination sensitivity, in a single Naphthoquine determination plate IC50 of 2. llnmol/L, In a single Azithromycin determination plate measured IC50 for 3.21nmol/L, In Naphthoquine/Azithromycin composite determination boards measured with quetiapine alpha-naphthol of Azithromycin IC50 were detected 0.15 nmol/L. A single measurementboards Naphthoquine IC50) is the determination of the composite board 11.7 times (2.11/0.18). The determination of Azithromycin IC50 is composite plate 21.4 times (3.21/0.15).
3.3 Sensitivity of P. falciparum before and after adding Naphthoquine i.n contrast group The control group in a single contact Naphthoquine plasmodium falciparum IC50 for a single after contact with Naphthoquine before 31.0(65.47/2.11) times. The control group in a single contact Naphthoquine plasmodium falciparum to the sensitivity of the question of Azithromycin is single contact Naphthoquine 21.4 (65.58/3.21) times.
3.4 Sensitivity of P. falciparum before and after adding Naphthoquine/ Azithromycin in test group Experimental Plasmodium falciparumin contact Naphthoquine/Azithromycin after contact with drugs for IC50 before 16.8(35.47/2.11) times. Experimental plasmodium falciparum in contact Naphthoquine/Azithromycin after contact with drugs for IC50 before 15.6 (50.04/3.21) times.
3.5 Compare the effect of artesunate clindamycin combination and aretsunate single used to P. falciparum The control group in contact with drugs in a single Naphthoquine IC50 for the determination of board 65.47 nmol/L, in a single Azithromycin determination boards measured for IC50 68.58nmol/L, in Naphthoquine/Azithromycin compound respectively in the determination of board IC50 10.34nmol/L,8.46nmol/L. After the experimental drug in contact in a single Naphthoquine determination for the IC50 35.47nmol/L plate in a single/L, determination of Azithromycin plate measured for IC50 50.04nmol/L, in Naphthoquine/Azithromycin composite board of determination IC50 respectively 2.89 nmol/L,2.68nmol/L
4. Conclusions
4.1 Falciparum parasites in a single contact Naphthoquine after a period of time can produce resistance, and its IC50 far outclass dictated by more than 5 times, further confirmed that the artificial cultivation method can cultivate anti Naphthoquine resistant falciparum strains.
4.2 Drug tolerability growth on experimental falciparum far slower than control group, so it hints Naphthoquine and Azithromycin two drugs coupled have obvious synergy and delay the resistance and deserves further study.
4.3 Group of drug falciparum tolerated growth far slower than the control group, the former in drug stimulates 120 acquired Naphthoquine, Azithromycin, Naphthoquine/Azithromycin IC50 were up by 16.8 times of 15.5 times, and 16.1/17.9 times, the latter stimulate 120 days Naphthoquine, Azithromycin, Naphthoquine/Azithromycin IC50 were up by the victims times,31.0 times、21.4times、57.4/56.4 times, suggesting Naphthoquine and Azithromycin two drugs have obvious delay the resistance role.
1.1观察对照组(对照组单一用萘酚喹)与实验组(萘酚喹与阿奇霉素联合用药)恶性疟原虫在体外药物刺激(压力)下,疟原虫对其敏感性的变化情况或产生速度。
1.2用WHO推荐的Rieckmann体外测定不同阶段对照组与实验组恶性疟原虫对萘酚喹、阿奇霉素、复方萘酚阿奇的敏感性,计算半数抑制量(IC50)。
1.3阐述两药物联用体外抗疟作用的特点；评价两药物联合有或无延缓抗性的作用或潜力。为疟疾的联合用药的临床治疗方案提供科学依据。
2.研究方法
2.1虫株与培养：恶性疟原虫FccSM/YN株,该株采自位于中老、中缅边境地区云南思茅,按Trager等蜡烛缸培养法,通过实验室驯化,建立体外传代培养,实验室长期保种虫株。
2.2药物作用观察：用体外剂量递增间隔接触法进行抗性培育
2.3体外测定：测试采用Rieckmann体外微量测定法。将同步化处理过的疟原虫稀释到20,000-60,000个/μl血,加5倍培养基稀释,混匀后加入测定板各药井,从低浓度到高浓度每井加50μl,每个浓度同时测定两组(2行)。37℃培养20-24h后收获1个对照井,视其疟原虫发育情况决定收获时间。用ICEstimator software计算IC50。
2.4判别标准接触药物后的恶性疟原虫的IC50较亲代上升5倍以上,可认为对该药的敏感性下降。在相同的抗性培育时间内,接触萘酚喹/阿奇霉素的恶性疟原虫的IC50分别较亲代上升5倍,可认为对该药的敏感性下降；萘酚喹/阿奇霉素对恶性疟原虫的IC50分别与亲代相比,无明显上升或者虽然有所上升,但明显低于对照组,可认为该联合用药有延缓抗性作用。
3.结果
3.1药物接触的次数、浓度及结果对照组恶性疟原虫接触萘酚喹120天,共接触药物15次,药物浓度分别为2.56nmol/L5次,160nmol/L2次,5、10、20、40、80、320、625、1280nmol/L各1次。接触药物前,敏感株(亲代系)的IC50为2.11nmol/L,药物刺激15次后,其IC50增至65.47nmol/L。为亲代原虫的31倍。实验组恶性疟原虫接触萘酚喹/阿奇霉素共120天,共接触药物19次,药物浓度分别为0.31/0.256nmol/L 8次,2.49/2.135 nmol/L 5次,0.625/0.53、4.98/4.27、9.735/8.34、19.47/16.685、38.94/33.375、77.88/66.755nmol/L各一次。接触药物前,敏感株(亲代系)的IC50为0.18 nmol/L、0.15nmol/L,药物刺激19次后,其IC50增至2.89 nmol/L、2.68 nmol/L,分别为亲代原虫的16.1倍、17.9倍。
3.2用药前恶性疟原虫的敏感性测定结果在恶性疟原虫未接触药物前,在萘酚喹测定板、阿奇霉素测定板及萘酚喹/阿奇霉素复合测定板中测定疟原虫的敏感性,在单一萘酚喹测定板中测得IC50为2.11nmol/L;在单一的阿奇霉素测定板中测得IC50为3.21 nmol/L;在萘酚喹/阿奇霉素复合测定板中测得萘酚喹与阿奇霉素的IC50分别为0.18nmol/L、0.15 nmol/L。单一测定板中萘酚喹的IC50是复合测定板的11.7(2.11/0.18)倍；阿奇霉素的IC50是复合测定板的21.4(3.21/0.15)倍。
3.3对照组恶性疟原虫接触萘酚喹前后(药物刺激15次,历时120天)的敏感性测定结果对照组恶性疟原虫在单一接触萘酚喹后的IC50为单一接触萘酚喹前的31.0(65.47/2.11)倍。对照组疟原虫在单一接触萘酚喹后对阿奇霉素的敏感性是单一接触萘酚喹前的21.4(68.58/3.21)倍。
3.4实验组恶性疟原虫接触萘酚喹/阿奇霉素前后(药物刺激19次,历时120天)的敏感性测定结果实验组疟原虫在接触萘酚喹/阿奇霉素后对萘酚喹的IC50为接触药物前的16.8(35.47/2.11)倍。实验组疟原虫在接触萘酚喹/阿奇霉素后对阿奇霉素的IC50为接触药物前的15.6(50.04/3.21)倍。
3.5萘酚喹/阿奇霉素联用与萘酚喹单用对恶性疟原虫作用的比较对照组在接触药物120天后在单一萘酚喹测定板中的IC50为65.47nmol/L,在单一阿奇霉素测定板中测得IC5。为68.58nmol/L,在萘酚喹/阿奇霉素复合测定板中的IC50分别为10.34nmol/L/8.46nmol/L。实验组在接触药物120天后在单一萘酚喹测定板中的IC50为35.47nmol/L,在单一阿奇霉素测定板中测得IC50为50.04nmol/L,在萘酚喹/阿奇霉素复合测定板中的IC50分别为2.89nmol/L、2.68 nmol/L。
4.结论
4.1恶性疟原虫在单一接触萘酚喹后一段时间敏感性有明显的下降,其IC50远远高于亲代的5倍以上,进一步证实用人工培养的方法可以培育出恶性疟原虫抗萘酚喹耐药株。
4.2萘酚喹与阿奇霉素配伍对恶性疟原虫的作用观察显示,无论对敏感株(对照组)还是抗药株在萘酚喹/阿奇霉素复合测定板中测的IC50显著低于单一的萘酚喹测定板和阿奇霉素测定板,提示萘酚喹与阿奇霉素配伍对恶性疟原虫的作用明显大于单一用药,两种药物联用有明显增效作用。
4.3实验组恶性疟原虫对药物耐受性的增长远远慢于对照组,前者在药物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分别上升16.8倍、15.6倍、16.1/17.9倍,后者在药物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分别上升31.0倍、21.4倍、57.4/56.4倍,提示萘酚喹与阿奇霉素两种药物联用有明显延缓抗性作用。
1. Objective
1.1 Observation of the experimental group (Naphthoquine combination with Azithromycin) and the control group (control group:single Naphthoquine use) of Plasmodium falciparum in vitro drug stimulation (pressure), in order to sensitivity and its changes in speed.
1.2 With the WHO recommended Rieckmann in vitro determination of the sensitivity of the different stages Plasmodium falciparum to Naphthoquine and Azithromycin, Naphthoquine/Azithromycin in control group and experimental group to calculate the amount of half inhibition (IC50).
1.3 Described the characteristics of the action for the two drugs combination in vitro and evaluated the two drugs combination whether or not delay resistance or potential. Combination therapy may provide a scientific basis for the clinical treatment malaria programs.
2. Methods
2.1 Strain and culture:Culture the Plasmodium falciparum strain selected from samples by Trager method in vitro
2.2 Observation the effect of drug:The resistant Plasmodium falciparum strains were stimulated by corresponding drugs with increasing concentrations disconnectedly.
2.3 In vitro assay:Rieckmann's in vitro micro-technique was used for assay. The concentration of in vitro cultured Plasmodium falciparum strain were diluted to 20,000-60,000/μ1 with culture medium, which was added to pre-prepared drug plates at 50μ/well after synchronization. Selected Plasmodium falciparum cultured in one of control wells after 20-24hours'culture in 37℃, the harvested time was determined by the growth of Plasmodium falciparum. Based on the number of schizonts in control well, counted the inhibitive ratio of schizonts in each well, calculated half inhibitive dose (IC50) of every drug to parasite by ICEstimator software.
2.4 Estimated standard:When IC50 of Plasmodium falciparmst to artesunat was 5 time than that of parent, then we consider corresponding P. falciparum as artesunate-resistant strain. During the same period of resistance culturing in vitro, if IC50 value of P. falciparumst to artesunat combined with clindamycin was 5 times than that of parent, which could be deemed that the P. falciparumst had resistance to the tow drugs; otherwise, the IC50 did not rise obviously or much more lower than that of parent, however, artesunate combined with clindamycin exhibit an apparent synergistic effect in vitro.
1. Results
3.1 Frequency, concentration and results of drug touching. The control group falciparum contact Naphthoquine 150 days, total contact drug 15 times, drug concentration tools 2.56 nmol/L five times,160nmol/L twice,5,10,20,40,80,320,625,1280nmol/L each time respectively. Contact drugs before, sensitive strains (parental department) of IC50 of 2.11 nmol/L, drug stimulates 15 times, after the IC50 to 65.47 nmol/L. Approximately dictated protozoon 31 times. Experimental falciparum contact Naphthoquine/Azithromycin 120 days, total contact drug nineteen times, drug concentration respectively for 0.31/0.256 nmol/L eight,2.49/2.135 nmol/L five times,0.625/0.53、4.98/4.2、 9.735/8.34、19.47/16.685、38.94/33.375、77.88/66.755nmol/L each time. Contact drugs before, sensitive strains (parental department) of IC50 for 0.18 nmol/L、0.15 nmol/L, drug stimulates nineteen times and its IC50 to 2.68 nmol/L、2.89nmol/L respectively, approximately dictated protozoon 16.1 times、17.9times.
3.2 Sensitivity of P. falciparum before adding drugs. In P. falciparum not contact with drugs, before Naphthoquine determination board, Azithromycin determination board and Naphthoquine/Azithromycin composite plate determination plasmodium P. falciparum determination sensitivity, in a single Naphthoquine determination plate IC50 of 2. llnmol/L, In a single Azithromycin determination plate measured IC50 for 3.21nmol/L, In Naphthoquine/Azithromycin composite determination boards measured with quetiapine alpha-naphthol of Azithromycin IC50 were detected 0.15 nmol/L. A single measurementboards Naphthoquine IC50) is the determination of the composite board 11.7 times (2.11/0.18). The determination of Azithromycin IC50 is composite plate 21.4 times (3.21/0.15).
3.3 Sensitivity of P. falciparum before and after adding Naphthoquine i.n contrast group The control group in a single contact Naphthoquine plasmodium falciparum IC50 for a single after contact with Naphthoquine before 31.0(65.47/2.11) times. The control group in a single contact Naphthoquine plasmodium falciparum to the sensitivity of the question of Azithromycin is single contact Naphthoquine 21.4 (65.58/3.21) times.
3.4 Sensitivity of P. falciparum before and after adding Naphthoquine/ Azithromycin in test group Experimental Plasmodium falciparumin contact Naphthoquine/Azithromycin after contact with drugs for IC50 before 16.8(35.47/2.11) times. Experimental plasmodium falciparum in contact Naphthoquine/Azithromycin after contact with drugs for IC50 before 15.6 (50.04/3.21) times.
3.5 Compare the effect of artesunate clindamycin combination and aretsunate single used to P. falciparum The control group in contact with drugs in a single Naphthoquine IC50 for the determination of board 65.47 nmol/L, in a single Azithromycin determination boards measured for IC50 68.58nmol/L, in Naphthoquine/Azithromycin compound respectively in the determination of board IC50 10.34nmol/L,8.46nmol/L. After the experimental drug in contact in a single Naphthoquine determination for the IC50 35.47nmol/L plate in a single/L, determination of Azithromycin plate measured for IC50 50.04nmol/L, in Naphthoquine/Azithromycin composite board of determination IC50 respectively 2.89 nmol/L,2.68nmol/L
4. Conclusions
4.1 Falciparum parasites in a single contact Naphthoquine after a period of time can produce resistance, and its IC50 far outclass dictated by more than 5 times, further confirmed that the artificial cultivation method can cultivate anti Naphthoquine resistant falciparum strains.
4.2 Drug tolerability growth on experimental falciparum far slower than control group, so it hints Naphthoquine and Azithromycin two drugs coupled have obvious synergy and delay the resistance and deserves further study.
4.3 Group of drug falciparum tolerated growth far slower than the control group, the former in drug stimulates 120 acquired Naphthoquine, Azithromycin, Naphthoquine/Azithromycin IC50 were up by 16.8 times of 15.5 times, and 16.1/17.9 times, the latter stimulate 120 days Naphthoquine, Azithromycin, Naphthoquine/Azithromycin IC50 were up by the victims times,31.0 times、21.4times、57.4/56.4 times, suggesting Naphthoquine and Azithromycin two drugs have obvious delay the resistance role.
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