Novel approaches to develop a treatment for cryptosporidiosis

描述（由申请人提供）：隐孢子虫病，由细胞内寄生虫小隐孢子虫或人原隐孢子虫引起的肠道感染，是美国水传播暴发导致腹泻的主要原因，也是发展中国家儿童感染性腹泻的主要原因。这种感染是慢性的，对免疫功能低下的患者来说往往是致命的，隐孢子虫病导致艾滋病患者多达50%的慢性腹泻。不幸的是，唯一已知有效的药物nitazoxanide对免疫功能正常的人只有中等疗效，对严重免疫功能低下的人相当于安慰剂。隐孢子虫病主要影响发展中国家的人们，新药物开发的高成本是开发有效治疗方法的主要障碍。利用药物重新利用的方法来解决这一经济障碍，该项目的最终目标是开发一种有效的隐孢子虫病治疗方法。目标产品要求治疗方法安全，成本低，在小肠和胆道内相关浓度下对隐孢子虫有选择性毒性，并且能够在缺乏有效宿主免疫的情况下从患者体内清除寄生虫。为此，提出了初步数据文件：1)开发和验证一个强大的，基于细胞的，高通量筛选（HTS）测定；2) fda批准的具有选择性抗隐孢子虫活性药物的鉴定；3)通过体外实验，比较不同处理方法从细胞组织培养系统中清除小弧菌的能力。

DESCRIPTION (provided by applicant): Cryptosporidiosis, due to intestinal infection with the intracellular parasites Cryptosporidium parvum or C. hominis, is the leading cause of diarrhea resulting from waterborne outbreaks in the United States, and it is a major cause of pediatric infectious diarrhea in the developing world. The infection is chronic and often fatal for immunocompromised patients, and cryptosporidiosis causes as much as 50% of chronic diarrhea in AIDS patients. Unfortunately, nitazoxanide, the only drug with known efficacy, is only moderately effective for treatment of immunocompetent people, and it is equivalent to placebo in severely immunocompromised people. Cryptosporidiosis predominantly affects people in the developing world, and the high cost of de novo drug development is a major barrier to developing an effective treatment. Using a drug repurposing approach to address this economic barrier, the ultimate goal of this project is to develop an effective therapy for cryptosporidiosis The target product profile requires that the treatment be safe, low in cost, selectively toxic for Cryptosporidium at concentrations relevant within the small intestine and biliary tract, and capable of eliminating the parasite from patients in the absence of effective host immunity. Towards this end, preliminary data are presented documenting: 1) development and validation of a robust, cell-based, high-throughput screening (HTS) assay; 2) identification of FDA-approved drugs with selective anti-Cryptosporidium activity; and 3) an in vitro assay to compare the ability of different treatments to eliminate C. parvum from a cell-based tissue culture system. In aim 1, the abilities of the identified anti-Cryptosporidium drug leads to eliminate C. parvum will be determined in vitro, and then the drug leads will be tested in vivo using a model of cryptosporidiosis in severely immunocompromised mice. Synergistic drug combinations sometimes possess cidal antimicrobial activity (i.e., kill microbes rather than merely inhibit microbial growth) at achievable tissue concentrations despite static activity of each drug used alone, and, in some cases, drug combinations enable successful treatment of immunocompromised patients where either drug alone is ineffective. Therefore, in aim 2, combinatorial drug screens will be undertaken to identify drugs with synergistic anti-Cryptosporidium activity when used with nitazoxanide or the primary screening hits. As proof of concept, a screen was conducted in the presence of a sub-inhibitory concentration of nitazoxanide, which yielded five approved drugs with no anti-cryptosporidial activity when used alone that were strong C. parvum growth inhibitors in combination with low dose nitazoxanide. Synergy of promising drug combinations will be confirmed with checkerboard assays, and the most promising will be followed up in vitro and in vivo as in aim 1. Successful completion of these specific aims will result in identification of at least one FDA-approved drug or a combination of drugs that cures immunocompromised mice infected with C. parvum and is suitable for testing in human clinical trials.