纳川珠利原料药及其溶液剂的质量研究
摘要
纳川珠利系偏三嗪类药物，是本实验室在数十年抗球虫药物研究基础上优化、筛选出来的具有高效、低毒的新型药物。本研究的目的是完成纳川珠利对照品的研制及评价；建立一种HPLC方法测定纳川珠利原料药及其溶液剂的含量；利用建立的HPLC方法对原料药及其溶液剂的稳定性进行考察；然后对纳川珠利溶液剂进行了药效学评价，最终为纳川珠利的实际应用提供理论依据。结果如下：
将纳川珠利原料药采用不同方法精制，通过测定熔点和HPLC-UV面积归一化法测定含量跟踪比较几种方法，选择合适的精制方法制备纳川珠利化合物，然后采用HPLC-UV、HPLC-PDA和DSC检测该化合物的纯度，经检测，其含量不小于99.5%，熔点为170.0-170.5℃，熔程为0.5℃，符合研究用化学对照品的要求。
建立了一种HPLC方法用于纳川珠利原料药含量的测定及有关物质检查，结果表明，纳川珠利在0.08mg/ml-0.12mg/ml范围内线性良好（r=0.999）；不同浓度精密度好（RSD=0.12%,n=6；RSD=0.91%,n=6）；纳川珠利溶液稳定性良好（RSD=2.49%,n≥90d）；检测限、定量限分别为5ng和12.5ng,三批原料药有关物质含量均小于0.5%，故暂定有关物质限度为0.5%。此法简单、准确、专属性强、重现性好，可用作纳川珠利原料药和杂质控制的分析方法。
建立了一种HPLC法用于纳川珠利溶液剂含量的测定，结果表明，纳川珠利在0.04mg/mL-0.12mg/mL范围内线性良好（r=0.9999）；方法精密度好（RSD=0.57%,n=6）；溶液剂两种配方平均加样回收率分别是98.77%和98.76%,RSD分别为0.41%和0.78%；溶液剂两种配方稳定性良好(RSD=0.22%，RSD=0.34%,n≥12h)。本法简便快捷、结果准确，适用于纳川珠利溶液剂的含量测定。
对原料药进行了影响因素和加速试验，考察了稳定性试验要求的重点项目，影响因素试验表明原料药在光照条件下颜色略有加深，其他无明显变化，说明该药物对光照较敏感；故选用单层铝箔作为包装材料，6个月加速试验显示原料药含量和有关物质等重点考察项目均无明显变化。对溶液剂进行了加速试验、光加速试验，考察了稳定性试验要求的重点项目，光加速试验表明纳川珠利溶液剂在光照条件下颜色略有加深，均对光照有一定敏感性；加速试验显示配方1较配方2有更好的稳定性，最终确定配方1为纳川珠利溶液剂的较优配方。
纳川珠利溶液剂药效学研究是以抗球虫活性指数ACI为评价指标，以人工感染柔嫩艾美耳球虫病的鸡为模型，考察了纳川珠利溶液剂对鸡球虫病的防治效果。结果表明：纳川珠利溶液剂两种配方均具有良好的预防鸡柔嫩艾美耳球虫病的效果，给药剂量≥1mg/L时，ACI值已高于180，均属高效范围，初步推荐剂量为1-3mg/L；纳川珠利对鸡柔嫩艾美尔球虫病有治疗效果，给药剂量≥3mg/L时，ACI值属高效范围。
Nitromezuril belongs to Triazine drugs, the laboratory has spent several years on the anticoccidialdrugs in order to optimization, filtering efficiency, low toxicity of new drugs. The purpose of this studyis to complete the preparation and evaluation of the reference substance of Nitromezuril; created a kindof HPLC method for the content determination of the API and solution; HPLC method is used to detectthe stability of the API and the solution,the stability of the solution, agents provide a scientific basis forits packaging materials and transport conditions; Nitromezuril and solution agent pharmacodynamicevaluation, and ultimately provide a theoretical basis for practical application in the Nitromezuril.Theresults are as follows:
Using different methods refine the API, tracking content was determined by measuring themelting point and HPLC-UV area normalization method to compare several ways to select theappropriate purification methods of Nitromezuril compounds, and then detecting the content of them byHPLC-UV, HPLC-PDA and DSC (differential scanning calorimetry), initially identified in line withthe reference standard requirements, testing, its content is not less than99.5%.
To establish a HPLC method for the determination of Nitromezuril and its related substances in theAPI, The result showed the calibration curve was linear between0.08mg/ml-0.12mg/ml (r=0.999);the different precisions were good（RSD=0.12%,n=6；RSD=0.91%,n=6）; the good stability of thereference solution was RSD=2.49%(n≥90d); accurate, specific, reproducible and suitable fordetermination of the content of Nitromezuril and its related substances in the API
To establish a HPLC method for the determination of Nitromezuril solution, The result showed thecalibration curve was linear between0.04mg/ml-0.20mg/ml (r=0.9999); the method precision wasRSD=0.12%(n=6);the average recovery rate of two different recipes were98.77%and98.76%, andRSD were0.41%and0.78%; the good stability of two different recipes (RSD=0.22%，RSD=0.34%,n≥12h);.The method is simple, rapid, accurate and suitable for determination of thecontent of Nitromezuril solution.
The stability of API was tested by influencing testing and accelerated testing, the key projects ofstability test were examinated, influencing testing showed that the color became deeper underillumination,other did not changed obviously,indicating that the drug is more sensitive tolight.Therefore a single-layer aluminum foil is used in packaging material, accelerated testing showedthat the API and related substances did not changed significantly.The solution was treated withaccelerated testing for6months, accelerated testing (4500±500Lx) for10days,the key projects ofstability test were examinated that showed the color of two recipes were deeper under illumination,indicating that the solution is more sensitive to light; and meanwhile the recipe1was better than therecipe2through accelerated testing for6months.
Study on Pharmacodynamic of solution of Nitromezuril was assessed in light of anticoccidialindex(ACI),and artificially infected with Eimeria coccidiosis in chickens as a model,the preventive andtherapeutic effects of the solution were investigated.The preventive effect of Nitromezuril was good against E.tenella, ACI had a high efficiency when doses greater than or equal to1mg/L; the therapeuticeffect of Nitromezuril was good against E.tenella, ACI had a high efficiency when doses greater than orequal to3mg/L.
将纳川珠利原料药采用不同方法精制，通过测定熔点和HPLC-UV面积归一化法测定含量跟踪比较几种方法，选择合适的精制方法制备纳川珠利化合物，然后采用HPLC-UV、HPLC-PDA和DSC检测该化合物的纯度，经检测，其含量不小于99.5%，熔点为170.0-170.5℃，熔程为0.5℃，符合研究用化学对照品的要求。
建立了一种HPLC方法用于纳川珠利原料药含量的测定及有关物质检查，结果表明，纳川珠利在0.08mg/ml-0.12mg/ml范围内线性良好（r=0.999）；不同浓度精密度好（RSD=0.12%,n=6；RSD=0.91%,n=6）；纳川珠利溶液稳定性良好（RSD=2.49%,n≥90d）；检测限、定量限分别为5ng和12.5ng,三批原料药有关物质含量均小于0.5%，故暂定有关物质限度为0.5%。此法简单、准确、专属性强、重现性好，可用作纳川珠利原料药和杂质控制的分析方法。
建立了一种HPLC法用于纳川珠利溶液剂含量的测定，结果表明，纳川珠利在0.04mg/mL-0.12mg/mL范围内线性良好（r=0.9999）；方法精密度好（RSD=0.57%,n=6）；溶液剂两种配方平均加样回收率分别是98.77%和98.76%,RSD分别为0.41%和0.78%；溶液剂两种配方稳定性良好(RSD=0.22%，RSD=0.34%,n≥12h)。本法简便快捷、结果准确，适用于纳川珠利溶液剂的含量测定。
对原料药进行了影响因素和加速试验，考察了稳定性试验要求的重点项目，影响因素试验表明原料药在光照条件下颜色略有加深，其他无明显变化，说明该药物对光照较敏感；故选用单层铝箔作为包装材料，6个月加速试验显示原料药含量和有关物质等重点考察项目均无明显变化。对溶液剂进行了加速试验、光加速试验，考察了稳定性试验要求的重点项目，光加速试验表明纳川珠利溶液剂在光照条件下颜色略有加深，均对光照有一定敏感性；加速试验显示配方1较配方2有更好的稳定性，最终确定配方1为纳川珠利溶液剂的较优配方。
纳川珠利溶液剂药效学研究是以抗球虫活性指数ACI为评价指标，以人工感染柔嫩艾美耳球虫病的鸡为模型，考察了纳川珠利溶液剂对鸡球虫病的防治效果。结果表明：纳川珠利溶液剂两种配方均具有良好的预防鸡柔嫩艾美耳球虫病的效果，给药剂量≥1mg/L时，ACI值已高于180，均属高效范围，初步推荐剂量为1-3mg/L；纳川珠利对鸡柔嫩艾美尔球虫病有治疗效果，给药剂量≥3mg/L时，ACI值属高效范围。
Nitromezuril belongs to Triazine drugs, the laboratory has spent several years on the anticoccidialdrugs in order to optimization, filtering efficiency, low toxicity of new drugs. The purpose of this studyis to complete the preparation and evaluation of the reference substance of Nitromezuril; created a kindof HPLC method for the content determination of the API and solution; HPLC method is used to detectthe stability of the API and the solution,the stability of the solution, agents provide a scientific basis forits packaging materials and transport conditions; Nitromezuril and solution agent pharmacodynamicevaluation, and ultimately provide a theoretical basis for practical application in the Nitromezuril.Theresults are as follows:
Using different methods refine the API, tracking content was determined by measuring themelting point and HPLC-UV area normalization method to compare several ways to select theappropriate purification methods of Nitromezuril compounds, and then detecting the content of them byHPLC-UV, HPLC-PDA and DSC (differential scanning calorimetry), initially identified in line withthe reference standard requirements, testing, its content is not less than99.5%.
To establish a HPLC method for the determination of Nitromezuril and its related substances in theAPI, The result showed the calibration curve was linear between0.08mg/ml-0.12mg/ml (r=0.999);the different precisions were good（RSD=0.12%,n=6；RSD=0.91%,n=6）; the good stability of thereference solution was RSD=2.49%(n≥90d); accurate, specific, reproducible and suitable fordetermination of the content of Nitromezuril and its related substances in the API
To establish a HPLC method for the determination of Nitromezuril solution, The result showed thecalibration curve was linear between0.04mg/ml-0.20mg/ml (r=0.9999); the method precision wasRSD=0.12%(n=6);the average recovery rate of two different recipes were98.77%and98.76%, andRSD were0.41%and0.78%; the good stability of two different recipes (RSD=0.22%，RSD=0.34%,n≥12h);.The method is simple, rapid, accurate and suitable for determination of thecontent of Nitromezuril solution.
The stability of API was tested by influencing testing and accelerated testing, the key projects ofstability test were examinated, influencing testing showed that the color became deeper underillumination,other did not changed obviously,indicating that the drug is more sensitive tolight.Therefore a single-layer aluminum foil is used in packaging material, accelerated testing showedthat the API and related substances did not changed significantly.The solution was treated withaccelerated testing for6months, accelerated testing (4500±500Lx) for10days,the key projects ofstability test were examinated that showed the color of two recipes were deeper under illumination,indicating that the solution is more sensitive to light; and meanwhile the recipe1was better than therecipe2through accelerated testing for6months.
Study on Pharmacodynamic of solution of Nitromezuril was assessed in light of anticoccidialindex(ACI),and artificially infected with Eimeria coccidiosis in chickens as a model,the preventive andtherapeutic effects of the solution were investigated.The preventive effect of Nitromezuril was good against E.tenella, ACI had a high efficiency when doses greater than or equal to1mg/L; the therapeuticeffect of Nitromezuril was good against E.tenella, ACI had a high efficiency when doses greater than orequal to3mg/L.
引文
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49. Allen P C, Fetter R H. Recent advance in biology and immunobiology of Eimeria species and indiagnosis and control of infection with these coccidian parasites poulty [J]. Clin. Microbiol. Rev.2002,15(1):58-65.
50. Cuckler A C,Malanga C M. Studies on drug resistance in coccidian [J]. J Parasitol.1955,41:302-311
51. Conway, D.P. McKenzie, M.E., Poultry Coccidiosis Diagnostic and Testing Procedures [J]. PfizerInc.,New York,1991:32-35.
52. Gabriela Gómez-Verduzco1, Arturo Cortes-Cuevas1, Carlos López-Coello, et al. Dietarysupplementation of mannan-oligosaccharide enhances neonatal immune responses in chickensduring natural exposure to Eimeria spp [J]. Acta Veterinaria Scandinavica,2009,51(11):1-7.
53. G.F. Mathis, R. Froyman, T. Kennedy. Coccidiosis control by administering toltrazuril in thedrinking water for a2-day period [J]. Veterinary Parasitology,2004,121:1-9.
54. Grainger C, Auldist M J, Clarke T, et al. Use of Monensin Controlled-release Capsules to ReduceMethane Emission and Improve Milk Production of Dairy Cows Offered Pasture Supplementedwith Grain [J]. Dairy Sci,2008,91(3):1159-1165.
55. Hamet N. Resistance to Anticoccidial drugs in poultry farms in France from1975to1984[C]. ProcGeorgia Coccidosis Conf Univ Georgia Athens,1986,415-421
56. Hodgson J N, Ball SJ, Ryan KC, et al. The incidence of drug resistant strains of Eimeria in chickenin Great Britain [J]. Br.Vet, J.1969,125:31-35.
57. Jockel J, Wendt B. Loffler M. Structural and functional comparision of agents interfering withdihydroorotate succinate and NADH oxidation of rat liver mitochondrial [J]. Biochem Phannacol,1998,56(8):1053-1060.
58. Johnson J, Reid W M. Anticoccidial drugs leision scoring techniques in battery and floor-panexperiments with chickens [J]. Experimental parasitology,1970,28(1):30-36
59. Jurgen Krucken, Ralf J. Hosse, Aimdip N. Mouafo, et al. Excystation of Eimeria tenellaSporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22andGAM56[J].Eukaryotic Cell,2008,7(2):202-211.
60. Kawazon U, Fabio J D. Resistance to diclazuril in field isolates of Eimeria species obtained fromcommercial broiler flocks in Brazil [J]. Avian Pathology,1994,23:305-311.
61. Kohri N, Yamayoshi Y, Xie H, et al. Improving the Oral Bioavailability of Albendazole in Rabbitsby the Solid Dispersion Technique [J]. European Journal of Pharmacology,1999,51(2):159-162.
62. Lillehoj HS, Dalloul RA, Min W. Enhancing intestinal immunity to coccidiosis [J]. World Poult.2003,19(4):18-21.
63. Lusina M, Cindric T, Tomaic J,et al. Stability study of losartan/hydrochlorthiazide tablets [J]. Int JPharm,2005,291:127-137.
64. McDougald L R. A survey of sensitivity to anticoccidial drugs in60isolates of Coccidial frombroiler chickens in Brazil and Argertinal [J]. Avain diseases,1987,31(2):287-292.
65. Mehlhorn H. Schmahl G. Haberkorn A. Toltrazuril effective against a broad spectrum of protozoanparasites [J].Parasitol Research,1988,75(1):64-66.
66. M.G. Michels, L.C.T. Bertolini, A.F. Esteves, et al. Anticoccidial effects of coumestans fromclipta alba for sustainable control of Eimeria tenella parasitosis in poultry production [J].Veterinary Parasitology,2011,177:55-60.
67. Mian Muhammad Awais, Masood Akhtar, Faqir Muhammad, et al. Immunotherapeutic effects ofsome sugar cane (Saccharum officinarum L.) extracts against coccidiosis in industrial broilerchickens [J].Experimental Parasitology,2011,128:104-110.
68. Min W, Lillehoj HS, Ashwell CM et al. EST analysis of Eimeria-stimulated intestinalintraepithelial lymphocytes in chickens [J]. Mol.Biotechnol,2005,30(2):143-150.
69. N F. Morehouse, R R. Baron. Coccidiosis evaluation of coccidiostats of mortality, weight gainsand fecal scores [J]. Experimental parasitololy,1970,28(1):25-29
70. Oikawa H. Survey on drug resistance of coccidian in broilers [J]. Jap J Vet Sci,1975,37:357-362
71. Rehbein S, Barth D, Visser M, et al. Efficacy of an Ivermectin Controlled-release Capsule againstSome Rarer Nematode Parasites of Sheep [J]. Veterinary Parasitology,2000,88(3):293-298.
72. Rotibi A, McDougald L R, Solis J. Response of21Canadian field isolates of chicken coccidian tocommercial anticoccidial drugs [J]. Avain disease.1989,33:365-367
73. Seung I. Jang, Hyun S. Lillehoj, Sung Hyen Lee, et al. Eimeria maxima recombinant Gam82gametocyte antigen vaccine protects against coccidiosis and augments humoral and cell-mediatedimmunity [J].Vaccine,2010,28:2980-2985.
74. Shirley MW, Ivens A, Gruber A et al. The Eimeria genome projects: a sequence of events [J].Trends Parasitol.2004，20(5)：199-201.
75. Vjollca Konjufca, Soo-Young Wanda, Mark C. Jenkins, et al. A Recombinant AttenuatedSalmonella enterica Serovar Typhimurium Vaccine Encoding Eimeria acervulina Antigen OffersProtection against E. acervulina Challenge [J]. Infection and immunity,2006,74(12):6785-6796.
76. V. Naidoo, L.J. McGaw, S.P.R. Bisschop, et al The value of plant extracts with antioxidant activityin attenuating coccidiosis in broiler chickens [J].Veterinary Parasitology,2008,153:214-219.
77. Waletzky E. A field strain of E.tenella resistant to sulfon amide(abstr)[J]. J Parasitol.1954,4(a):24、
78. Williams RB. A compartmentalized model for the estimation of the cost of coccidiosis to theworld’s chicken production industry [J]. Parasitol,1999,29(8)：1209-1229.
79. Williams R B. Anticocidial vaccines for broil chicken:pathways to success [J]. Avian Pathol,2002,31(4):317-353.
80. Williams R B. Epidemiological aspects of the use of live anticoccidial vaccines for chickens[J]. Int.J.Parasitol,1998,28:1089-1098.
81. Xicheng Ding, Hyun S. Lillehoj, Marco A. Quiroz, et al. Protective Immunity against Eimeriaacervulina following In Ovo Immunization with a Recombinant Subunit Vaccine and CytokineGenes [J]. Infection and immunity,2004,72(12):6939-6944.