Nitazoxanide in Treatment of Helicobacterpylori : a Clinical and In VitroStudy

Rühle

et al. 2006

The thiazolides represent a novel class of anti-infective drugs, with the nitrothiazole nitazoxanide [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide] (NTZ) as the parent compound. NTZ exhibits a broad spectrum of activities against a wide variety of helminths, protozoa, and enteric bacteria infecting animals and humans. In vivo, NTZ is rapidly deacetylated to tizoxanide (TIZ), which exhibits similar activities. We have here comparatively investigated the in vitro effects of NTZ, TIZ, a number of other modified thiazolides, and metronidazole (MTZ) on Giardia lamblia trophozoites grown under axenic culture conditions and in coculture with the human cancer colon cell line Caco2. The modifications of the thiazolides included, on one hand, the replacement of the nitro group on the thiazole ring with a bromide, and, on the other hand, the differential positioning of methyl groups on the benzene ring. Of seven compounds with a bromo instead of a nitro group, only one, RM4820, showed moderate inhibition of Giardia proliferation in axenic culture, but not in coculture with Caco2 cells, with a 50% inhibitory concentration (IC 50 ) of 18.8 M; in comparison, NTZ and tizoxanide had IC 50 s of 2.4 M, and MTZ had an IC 50 of 7.8 M. Moreover, the methylation or carboxylation of the benzene ring at position 3 resulted in a significant decrease of activity, and methylation at position 5 completely abrogated the antiparasitic effect of the nitrothiazole compound. Trophozoites treated with NTZ showed distinct lesions on the ventral disk as soon as 2 to 3 h after treatment, whereas treatment with metronidazole resulted in severe damage to the dorsal surface membrane at later time points.Giardia lamblia (also known as G. duodenalis or G. intestinalis), a flagellated protozoan, is the most common causative agent of persistent diarrhea worldwide. The life cycle includes motile, flagellated trophozoites parasitizing the upper intestine and thick-walled cysts forming in the lower intestine, which are subsequently shed with the feces (29). Antigiardial chemotherapy is directed against the trophozoite stage. Metronidazole (MTZ) and other nitroimidazoles have been used against giardiasis since the late 1950s (12, 30). These compounds can cause, however, a number of side effects, including headache, vertigo, nausea, and pancreatitis, as well as central nervous system toxicity at high doses. Moreover, MTZ has been shown to be mutagenic in bacteria and carcinogenic in mice (2). Other treatments include quinolones (28), benzimidazoles such as albendazole (7), nitrofurans, paromomycin, and bacitracin zinc, all compounds that can exhibit notorious side effects (12).Originally developed as a veterinary antihelminthic (24), 2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide (NTZ), also known as Alinia, has been shown to exhibit a wide spectrum of in vivo activity against a broad spectrum of intestinal parasites, such as Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis (1, 5), the apicomplexan Cryptosporidium parvum (11), and enteric bact...

Section: Discussionsupporting

confidence: 87%

In Vitro Effects of Thiazolides on Giardia lamblia WB Clone C6 Cultured Axenically and in Coculture with Caco2 Cells

Rühle

et al. 2006

“…Despite the susceptibility of H. pylori strains to NTZ in vitro (MICs, 2 to 8 g/ml), the drug has little therapeutic efficacy (10). Based on our studies showing that only the anion exhibits biological activity, we suggest that NTZ in the stomach is inactivated (protonated) by gastric acid (pH ϳ3).…”

Section: Discussionmentioning

confidence: 89%

Antiparasitic Drug Nitazoxanide Inhibits the Pyruvate Oxidoreductases of Helicobacter pylori , Selected Anaerobic Bacteria and Parasites, and Campylobacter jejuni

Hoffman

Sisson²,

Croxen

et al. 2007

225

213

Nitazoxanide (NTZ) exhibits broad-spectrum activity against anaerobic bacteria and parasites and the ulcer-causing pathogen Helicobacter pylori. Here we show that NTZ is a noncompetitive inhibitor (K i , 2 to 10 M) of the pyruvate:ferredoxin/flavodoxin oxidoreductases (PFORs) of Trichomonas vaginalis, Entamoeba histolytica, Giardia intestinalis, Clostridium difficile, Clostridium perfringens, H. pylori, and Campylobacter jejuni and is weakly active against the pyruvate dehydrogenase of Escherichia coli. To further mechanistic studies, the PFOR operon of H. pylori was cloned and overexpressed in E. coli, and the multisubunit complex was purified by ion-exchange chromatography. Pyruvate-dependent PFOR activity with NTZ, as measured by a decrease in absorbance at 418 nm (spectral shift from 418 to 351 nm), unlike the reduction of viologen dyes, did not result in the accumulation of products (acetyl coenzyme A and CO 2 ) and pyruvate was not consumed in the reaction. NTZ did not displace the thiamine pyrophosphate (TPP) cofactor of PFOR, and the 351-nm absorbing form of NTZ was inactive. Optical scans and 1 H nuclear magnetic resonance analyses determined that the spectral shift (A 418 to A 351 ) of NTZ was due to protonation of the anion (NTZ ؊ ) of the 2-amino group of the thiazole ring which could be generated with the pure compound under acidic solutions (pK a ‫؍‬ 6.18). We propose that NTZ ؊ intercepts PFOR at an early step in the formation of the lactyl-TPP transition intermediate, resulting in the reversal of pyruvate binding prior to decarboxylation and in coordination with proton transfer to NTZ. Thus, NTZ might be the first example of an antimicrobial that targets the "activated cofactor" of an enzymatic reaction rather than its substrate or catalytic sites, a novel mechanism that may escape mutation-based drug resistance.Nitazoxanide [2-acetyloxy-N-(5-nitro-2-thiazolyl) benzamide] (NTZ) is a broad-spectrum drug that is efficacious for the treatment of infections caused by amitochondriate luminal parasites and helminths (8,15,21,26) and that shows promise as an alternative therapy for treating infections caused by Clostridium difficile (20). More generally, NTZ appears to be an effective treatment for persistent diarrhea (35) and even infections caused by rotavirus (26). NTZ shares some structural similarities with thiamine pyrophosphate (TPP) (Fig. 1) and exhibits a spectrum of activity similar to those of metronidazole (MTZ) and nitrofuran drugs (22), but several lines of evidence suggest that this drug is mechanistically different from the redox-active prodrugs (8,18,28). For example, NTZ does not increase the mutation frequency or produce DNA damage in Helicobacter pylori (28), and there is no cross-resistance with MTZ, as MTZ-resistant strains of Trichomonas vaginalis and H. pylori remain susceptible to NTZ (1,18,28,34). Unlike MTZ, resistance to NTZ has not been observed clinically or generated by in vitro methods commonly used to isolate MTZ r mutants of H. pylori (18). Despite the ever widening spe...

“…We had previously determined that amixicile and NTZ were inhibitory to H. pylori and C. jejuni in the 0.5-to 1-g/ml range by MIC tests (14,15). Moreover, we had tested NTZ for efficacy against H. pylori in a C57BL/6 mouse model and as in human studies, NTZ proved ineffective at eradicating the infection (35). In the mouse model, H. pylori resides in the gastric mucosa but does not appreciably invade gastric epithelial cells, as demonstrated by hematoxylinand-eosin staining of stomach tissue (27).…”

Section: Discussionmentioning

confidence: 99%

“…To test whether plasma protein binding might affect the bioavailability and therefore the therapeutic efficacy of amixicile, we used a mouse model of H. pylori infection where plasma drug levels above the MIC are required for eradication. Both human and animal studies have established that NTZ is not an effective therapy for H. pylori infections, despite excellent in vitro efficacy (15,35). In contrast, MTZ is an effective therapeutic for eradication of H. pylori infection in mice but not as a monotherapy in humans or when strains are resistant to MTZ (27,36).…”

Section: Drug Metabolismmentioning

confidence: 99%

Preclinical Studies of Amixicile, a Systemic Therapeutic Developed for Treatment of Clostridium difficile Infections That Also Shows Efficacy against Helicobacter pylori

Hoffman

Bruce

Olekhnovich

et al. 2014

dAmixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 M (NTZ was toxic above 10 M); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · g/ml (30 mg/kg dose) to 328 h · g/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 g/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.