Nitazoxanide (also known as NTZ; NSC 697855) is a synthetic nitrothiazolyl-salicylamide derivative and a broad spectrum antiprotozoal agent with IC50 for canine influenza virus ranges from 0.17 to 0.21 μM. It was approved for treating human protozoan infections. Nitazoxanide reduces parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity. Nitazoxanide is a new thiazolide antiparasitic agent that shows excellent in vitro activity against a wide variety of protozoa and helminths. Nitazoxanide and its metabolite tizoxanide are more active in vitro than metronidazole against G. intestinalis, E. histolytica and T. vaginalis.

Nitazoxanide is a new nitrothiazole compound with broad-spectrum activity against numerous intestinal protozoa, helminths, and anaerobic bacteria. It is presently approved to treat infections due to G. intestinalis in children and adults and infections due to Cryptosporidium species in children. Approval for use in adults with Cryptosporidium infection and the immunocompromised population is on the horizon. Nitazoxanide is an important new addition to the antiparasitic pharmacopeia. The drug has few side effects and requires a short course of treatment. Nevertheless, a need remains for further studies of its molecular mechanisms of action, bioavailability, and drug interactions to learn whether it can be safely used in a variety of patient groups. Because the majority of parasitic infections occur in the developing world, further data about drug potency and stability are also needed to support its widespread use in this context. Similarly, clinical and pharmacological data on absorption, dosage, and duration of therapy in patients with AIDS and chronic cryptosporidiosis are necessary. In view of its unique mechanism of action, nitazoxanide should be considered for further clinical evaluation in the treatment of parasitic infections (e.g., in combination with paromomycin or azithromycin for treatment of cryptosporidiosis and in combination with albendazole for treatment of intestinal helminth infections) and in reducing the emergence of metronidazole resistance, particularly with Giardia and H. pylori. Additional clinical trial data that would expand our knowledge of nitazoxanide's utility in these contexts is important. With these questions answered, nitazoxanide may represent a significant advance in the treatment of intestinal parasitic infections worldwide.

Physicochemical Properties

Molecular Formula	C12H9N3O5S
Molecular Weight	307.28
Exact Mass	307.026
Elemental Analysis	C, 46.90; H, 2.95; N, 13.67; O, 26.03; S, 10.44
CAS #	55981-09-4
Related CAS #	Nitazoxanide-d4;1246819-17-9
PubChem CID	41684
Appearance	Light yellow to yellow solid powder
Density	1.5±0.1 g/cm3
Melting Point	202ºC
Index of Refraction	1.673
LogP	1.79
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	4
Heavy Atom Count	21
Complexity	428
Defined Atom Stereocenter Count	0
SMILES	S1C(=C([H])N=C1N([H])C(C1=C([H])C([H])=C([H])C([H])=C1OC(C([H])([H])[H])=O)=O)[N+](=O)[O-]
InChi Key	YQNQNVDNTFHQSW-UHFFFAOYSA-N
InChi Code	InChI=1S/C12H9N3O5S/c1-7(16)20-9-5-3-2-4-8(9)11(17)14-12-13-6-10(21-12)15(18)19/h2-6H,1H3,(H,13,14,17)
Chemical Name	[2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl] acetate
Synonyms	NSC-697855; NTZ; NSC 697855;NSC697855; NITAZOXANIDE; 55981-09-4; Alinia; Nitazoxanida; 2-((5-nitrothiazol-2-yl)carbamoyl)phenyl acetate; Nitazoxamide; Nitazoxanidum; Daxon; Alinia, Colufase, Daxon, Nitazoxamide
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Broad spectrum anthelmintic
ln Vitro	Globally, the most frequent cause of persistent diarrhea is the flagellated protozoan Giardia lamblia[1]. With an IC50 of 2.4 μM, nitazoxanide has an impact on the growth of G. lamblia trophozoite in axenic culture[1]. NTZ/Nitazoxanide significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 ± 0.04 μg/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 ± 0.31 μg/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. [2] NTZ/Nitazoxanide derivatives containing a bromo instead of a nitro group (Table 1) were all inactive against Giardia trophozoites (IC50s of >50 μM), with the exception of RM4820, which showed a moderate inhibitory activity (IC50 of 18.8 μM) that was lower than the ones for RM4802 and RM4805. The efficacy of MTZ was lower than those of NTZ and TIZ but still higher than that of any other drug tested in this study. In order to visualize the morphological alterations of G. lamblia trophozoites induced by Nitazoxanide/NTZ and compare them with those induced by MTZ, confluent axenic trophozoite cultures were treated with 50 μM NTZ or MTZ or with DMSO as a control. Observations with the light microscope showed that after 3 h of treatment with NTZ, approximately 50% of trophozoites were immobile; at 5 h of treatment, over 95% of trophozoites were immobile and formed large multicellular aggregates; and at 24 h of NTZ treatment, no motile trophozoites were found (data not shown). They all exhibited aberrant vacuolar cytoplasmic structures. Inspection by TEM (Fig. 1) largely confirmed these findings and showed that at 1 h of NTZ treatment, a considerable number of trophozoites already exhibited relatively small, aberrant cytoplasmic inclusions within their cytoplasm (Fig. 1A and B). At 3 h of treatment, larger vacuoles containing membranous inclusions or membrane stacks could be observed in a large number of parasites (Fig. 1C), and at 24 h of treatment, these parasites were seriously damaged, as exhibited either by a dissociation of their cytoplasmic organization (Fig. 1D) or, in many instances, by large vacuoles containing membrane residues (Fig. 1E). Besides this, however, the cytoskeletal elements of trophozoites, such as filaments associated with the ventral disk or the flagella and basal bodies, were not notably altered. In control preparations incubated in DMSO alone, no obvious changes in parasite ultrastructure could be detected (Fig. 1F).[1] In a first series of experiments, Caco2 cells were incubated with increasing numbers of trophozoites (103 to 106 parasites per well) in the presence of DMSO as a solvent control or in the presence of 30 μM NTZ/Nitazoxanide. The parasites attaching to Caco2 cells were then quantified by real-time PCR (Fig. 4). In the absence of any drug and at an initial inoculum density of 105 parasites per well, 70 to 90% of the trophozoites remained attached to the Caco2 cells for a period of 24 to 48 h. At an inoculum density 10 times higher (106 parasites/well), this value was decreased to nearly 50%, indicating that binding to the Caco2 cell surface is saturable and dependent on the presence of suitable binding sites and/or host cell surface receptors. In the presence of 30 μM NTZ, with an inoculum density of 105 trophozoites, the number of parasites still attached to Caco2 cells after 24 h decreased to less than 20% of the control value. Based upon these findings, the effects of different thiazolides compared to those of MTZ were investigated. Confluent Caco2 cells were supplemented with fresh Caco2 growth medium. Trophozoites (105) were added to each well, and a number of thiazolides, MTZ, and DMSO were added (Fig. 5). Cells were harvested after 24 h, and the attached trophozoites were quantified by real-time PCR. Only those compounds that had exhibited strong inhibitory effects in axenic cultures (NTZ, TIZ, RM4802, RM4805, and MTZ) interfered with trophozoite attachment in the Caco2 coculture system. In contrast to what was seen with axenic culture, the efficacies of RM4802, RM4805, and MTZ in Caco2 coculture were comparable to those of NTZ and TIZ (Fig. 5). Concentrations lower than 15 μM did not show any significant inhibitory effects for any of the drugs tested.[1] Studies of protozoa and anaerobic bacteria have shown that Nitazoxanide inhibits pyruvate-ferredoxin oxidoreductase (PFOR), an enzyme essential to anaerobic energy metabolism. However, interference with the PFOR enzyme–dependent electron transfer reaction may not be the only pathway by which nitazoxanide exhibits antiprotozoal activity, and the mechanism of nitazoxanide's activity against helminths is unknown. Nitazoxanide has demonstrated in vitro activity against C. parvum and G. intestinalis. It has been shown to inhibit the growth of sporozoites of C. parvum on its own, and has also demonstrated combined in vitro activity with both azithromycin and rifampin, suppressing growth of C. parvum by 83.9% and 79.8%, respectively, compared with 56.1% when used alone. Similarly, in vitro studies of nitazoxanide and its derivative, tizoxanide, have shown greater efficacy than metronidazole against G. intestinalis. Specifically, tizoxanide was demonstrated to be 8 times more active than metronidazole against metronidazole-susceptible isolates of G. intestinalis and twice as active against resistant isolates. Nitazoxanide has also shown broad in vitro activity against numerous other parasitic and microbial pathogens, including E. intestinalis, V. corneae, E. histolytica, T. vaginalis, B. hominis, Echinococcus multilocularis, Echinococcus granulosus,, and F. hepatica. The antimicrobial properties of nitazoxanide and tizoxanide have been tested against 241 anaerobes, the majority of which were inhibited in vitro, with an MIC90 between 0.06 mg/L and 4 mg/L. Nitazoxanide has also shown in vitro and in vivo antimicrobial activity against Clostridium difficile and both metronidazole-susceptible and metronidazole-resistant strains of H. pylori [4].
ln Vivo	A variety of intestinal parasites, including Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis, the apicomplexan Cryptosporidium parvum, and enteric bacteria that infect both humans and animals, are resistant to nitazoxanide's broad range of in vivo activity[1]. Mice infected with the Japanese encephalitis virus (JEV) strain have a lower mortality rate when given nitazoxanide (50, 75, or 100 mg/kg/day; intragastric administration for up to 25 days) and are protected against a lethal dose challenge of JEV[2]. NTZ/Nitazoxanide reduces the mortality of mice challenged with a lethal dose of JEV [2] To evaluate the protective effect of NTZ on mice challenged with a lethal dose of JEV, NTZ was administered intragastrically at the indicated doses from 1 day post-infection, daily, for up to 25 days. The mice that were infected with JEV and received a placebo (DMSO) treatment (group JEV + DMSO) started to show the clinical signs of JE including limb paralysis, restriction of movements, piloerection, body stiffening and whole body tremors, from 5 days post-infection, and all mice (10/10 mice) died within 9 days post-infection. In contrast, the mice that were infected with JEV and received NTZ treatment (group JEV + NTZ, 100 mg/kg/day) showed the clinical signs of JE from 11 days post-infection, among these 10 mice, 1 died within 12 days post-infection and 9 survived the experimental period (25 days) (Figure 6A). The NTZ-mediated protection appeared to be dose dependent, as the infected mice receiving 50 mg/kg/day, 75 mg/kg/day and 100 mg/kg/day NTZ led to 30%, 70% and 90% mice survival, respectively (Figure 6A). These data suggested that NTZ treatment reduced the mortality of JEV-infected mice and protected mice from a lethal dose challenge of JEV. The mice that were mock-infected with JEV and received NTZ treatment (group Mock + NTZ) showed no detectable signs of abnormal behavior, similarly to the mice that were mock-infected and received DMSO treatment (group Mock + DMSO) (Figure 6A). Analysis of JEV titers in the brain samples from the experimental mice indicated that NTZ treatment significantly reduced the virus load in the brain from the group JEV + NTZ compared with that from group JEV + DMSO (Figure 6B). The brain samples from the experimental mice were examined for the presence of viral NS3 protein by immunohistochemistry. The viral NS3 protein was stained as brown deposits in the cytoplasm of neuronal cells (Additional file 1, arrowed cells). The brain sections from the JEV + DMSO group of mice showed remarkably higher numbers of NS3-stained positive cells than the sections from the JEV + NTZ group. No NS3-stained positive cells were detected in the sections from the Mock + NTZ or Mock + DMSO groups of mice. Studies with mice. [3] The efficacy of NTZ/Nitazoxanide, either alone or in combination with PRM, was tested in the anti-IFN-γ-conditioned SCID mouse model of acute cryptosporidiosis. While an initial study showed that a partial reduction in oocyst shedding (data not shown) was produced by NTZ, we were unable to replicate this result in several subsequent experiments. The following are the combined results of two independent trials that failed to show efficacy in this model. No difference in the log oocyst shedding between any of the groups treated with NTZ alone and the placebo control group was observed (Fig. 1). In contrast, all mice treated with PRM shed oocysts at significantly lower levels than mice treated with either NTZ alone or the placebo (P < 0.001). Coadministration of NTZ and PRM was no more effective at reducing the level of oocyst shedding than treatment with PRM alone. In general, no significant differences in mean body weight were observed between any of the groups of mice (data not shown). Student-Newman-Keuls analysis of variance revealed that the extent of mucosal infection was significantly greater in mice treated with either NTZ alone or the placebo than in animals treated with PRM (Fig. 2; P < 0.001). Coadministration of NTZ and PRM did not significantly alter the extent of mucosal infection compared to that in mice that received PRM alone. Studies with piglets. [3] The piglet model offers an added advantage in that the piglets develop diarrhea as a consequence of infection. In addition to the piglet euthanized before treatment began, 4 of the 25 animals challenged with C. parvum were euthanized due to poor health associated with diarrhea, including 1 piglet from the placebo group (on day 5 after challenge), 2 from the group receiving 250 mg of Nitazoxanide/NTZ/kg (on days 8 and 9 after challenge), and 1 from the PRM-treated group (on day 5 after challenge). Three additional piglets, 1 placebo-treated, 1 PRM-treated, and 1 uninfected control piglet, were euthanized on day 8 after challenge for a comparative analysis of the extent of mucosal infection. The analysis of both the level of oocyst shedding and extent of mucosal infection revealed that NTZ at 250 mg/kg/day significantly reduced the extent of mucosal infection in these piglets, but it was not as effective as PRM at 500 mg/kg/day (Fig. 3 and 4). With the exception of those in the PRM-treated group, all infected piglets manifested diarrhea of various degrees within 56 h after challenge. Diarrhea persisted until the end of the experiment, 13 days later (11 days after the start of treatment). Table 2 provides a cumulative analysis of the number of days of diarrhea observed for each of the treatment groups. A chi-square analysis of these data revealed very significant differences among the treatment groups (overall Pearson’s chi-square value of 88.096 with 5 df; P < 0.001). Uninfected piglets given Nitazoxanide/NTZ at a dose of 125 mg/kg/day did not have diarrhea (0 of 24 days of observation). In contrast, on 22 of 36 observation days, uninfected piglets given NTZ at a dose of 250 mg/kg/day had significant drug-induced diarrhea (P ≤ 0.001 for both Pearson’s chi-square and Fisher’s exact test [two-tailed]). Among the infected groups, only the PRM-treated group had less-frequent diarrhea than the infected placebo control group. The percentage of days of observation that the piglets in the PRM-treated group showed diarrhea was significantly lower than that in any of the other infected groups (chi-square P < 0.001, and Fisher’s exact two-tailed test, P < 0.001, for comparison with the infected placebo control group, the group receiving NTZ at 125 mg/kg/day, and the group receiving NTZ at 250 mg/kg/day).
Cell Assay	Cell Line: Giardia lamblia trophozoites were cultured in increasing numbers (103–106 parasites per well) in human cancer colon Caco2 cells. Concentration: 30 μM Incubation Time: 24 hours Result: When Nitazoxanide was not present and the initial inoculum density was 105 parasites per well, 70–90% of the trophozoites stayed adhered to the Caco2 cells for a duration of 24–48 hours.In the presence of 30 μM Nitazoxanide with an inoculum density of 105 trophozoites, the number of parasites still attached to Caco2 cells after 24 hours dropped to less than 20% of the control value. Virus, cells and Nitazoxanide/NTZ administration [2] JEV strain (SH-JEV01) was grown in 3-day-old BALB/c mice and titrated by a plaque assay using BHK-21 cells as described below. BHK-21 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in an atmosphere containing 5% CO2. NTZ/Nitazoxanide (purity ≥ 98%) was dissolved in culture-grade DMSO at a concentration of 50 μg/μl. NTZ solution was added immediately after a 1 h adsorption period and was kept in the culture medium for the duration of the experiment, unless specified otherwise. Controls received equal amounts of DMSO (final concentration ≤ 0.06%), which did not affect cell viability or virus replication. Cytotoxicity test[2] BHK-21 cells were seeded in a 96-well plate at a density of 5 × 103 cells per well. Following 24 h of incubation, the cells were treated with NTZ at various concentrations ranging from 0.1 to 32 μg/ml at 37°C for 48 h. Cells treated with DMSO alone were used as a control. The cellular toxicity of NTZ was assessed using MTT assay. Cell viability was calculated as a percentage of the total number of viable DMSO-treated control cells. The CC50, which is defined as the concentration that inhibits the proliferation of exponentially growing cells by 50%, was calculated as described. Analysis of the antiviral effect of NTZ/Nitazoxanide in BHK-21 cells [2] BHK-21 cells in six-well plates were infected with JEV at a MOI of 0.001. After a 1 h adsorption period, the cells were treated with NTZ at concentrations ranging from 0.01 to 10 μg/ml and incubated at 37°C for 48 h or the indicated times. The virus yield was determined by a plaque assay and qRT-PCR. The reduction in the virus titer was calculated as follows:% virus titer reduction = [1-(PFUJEV+NTZ/PFUJEV+DMSO)] × 100. The EC50 that is defined as the concentration offering 50% inhibition of viral yield in cells was calculated as described. NTZ/Nitazoxanide activity in cell culture. [3] MDBK cells selectively cloned for susceptibility to C. parvum infection were plated in 96-well microliter plates. To determine the dose response to NTZ, 3.0 × 104 C. parvum oocysts were added with or without drugs to each well 72 h later, when the cells were confluent. Paromomycin sulfate (PRM) was used as a positive control drug. All drug dilutions were made in Dulbecco’s minimum essential medium supplemented with 5% fetal bovine serum, 500 U of penicillin, 500 μg of streptomycin/ml, 1 mM sodium pyruvate, 2 mM l-glutamine, and 0.2% dimethyl sulfoxide (DMSO) (culture medium). Culture medium was added to wells containing MDBK cells infected with C. parvum oocysts as a negative control. All drug concentrations and controls were tested in quadruplicate. Following an incubation for 48 h (37°C, 8% CO2) the monolayers were methanol fixed and reacted in an indirect immunofluorescence assay to determine the intensity of infection. Fixed wells were rehydrated for 15 min with phosphate-buffered saline (PBS) containing 1% normal goat serum (NGS. Following rehydration, the parasite-reactive rabbit antiserum was diluted 1:1,000 in PBS containing 1% NGS, added to the wells, and incubated for 1 h at room temperature. All wells were washed three times with PBS, and bound antibody was detected with a fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody diluted 1:100 in PBS containing 1% NGS. Following a 1-h incubation at room temperature, the wells were washed three times with PBS and dried. The extent of C. parvum infection was quantitated, under UV light microscopy, with a microcomputer video imaging device specifically designed for this purpose. The percent inhibition of infection was calculated as follows: 1 − (mean number of parasites in wells with drug/mean number of parasites in control wells) × 100. All results were assigned inhibition scores of 0, 1, 2, 3, and 4 for inhibition of 0 to 30%, 31 to 55%, 56 to 70%, 71 to 90%, and 91 to 100%, respectively. Cytotoxicity assay. [3] The cytotoxicities of NTZ/Nitazoxanide and PRM to MDBK cells were determined by the CellTiter 96 AQueous non-radioactive cell proliferation assay. Controls for each cytotoxicity assay included (i) uninfected cells incubated in culture medium, (ii) infected cells incubated in culture medium, and (iii) cells exposed to a freeze-thaw lysate containing 3.0 × 104 oocyst equivalents in culture medium. The percent cytotoxicity was calculated from the optical density (OD) as follows: [(mean OD of uninfected cells − mean OD of infected cells)/mean OD of uninfected cells] × 100. All results were assigned cytotoxicity scores of 0, 1, 2, 3, and 4 for percent cytotoxicity of 0 to 5%, 6 to 25%, 26 to 50%, 51 to 75%, and 76 to 100%, respectively. Cytotoxicity scores of 0, 1, and 2 are considered to indicate nontoxicity, mild toxicity, and moderate toxicity, respectively, and scores of 3 and 4 are regarded as indicating severe toxicity for MDBK cells. A negative percent toxicity results when the OD of the infected cells is greater than the OD of the uninfected cells.
Animal Protocol	Animal Model: JEV was injected intraperitoneally into female Chinese Kunming mice that were three weeks old and weighed between 12 and 14 grams.[2] Dosage: 50, 75 or 100 mg/kg/day Administration: Administered intragastrically by gavage Result: 50 mg/kg/day, 75 mg/kg/day and 100 mg/kg/day led to 30%, 70% and 90% mice survival, respectively. NTZ/Nitazoxanide activity in SCID mice. [3] The anti-gamma-interferon (IFN-γ)-conditioned SCID mouse model has been described previously. Briefly, newly weaned (3- to 4-week-old) male inbred C.B-17 SCID mice were housed in microisolator cages in IACUC-approved facilities. Prior to the initiation of a drug trial, the animals were randomized into seven groups of seven mice each. Each mouse was primed with an intraperitoneal injection of 1 mg of XMG1.2, an IFN-γ-neutralizing monoclonal antibody. Two hours later, each mouse in six of the seven groups received an oral inoculation of 107 oocysts. Drug treatment was initiated on day 6 of infection, coinciding with the onset of oocyst excretion in the feces. Treatment schedules were as follows: group 1, 200 mg of NTZ/kg of body weight/day; group 2, 100 mg of NTZ/kg/day; group 3, 200 mg of NTZ/kg/day combined with 2,500 mg of PRM/kg/day; group 4, 100 mg of NTZ/kg/day combined with 2,500 mg of PRM/kg/day, and group 5, 2,500 mg of PRM/kg/day. NTZ was dissolved in 100% DMSO and administered orally in two divided doses of 30 μl each per day. PRM was dissolved in the drinking water to a concentration of 10 mg/ml (16.2 mM), resulting in a dose of 2,500 mg/kg/day based on the daily water consumption. Group 6 consisted of uninfected mice treated with 200 mg of NTZ/kg/day combined with 2,500 mg of PRM/kg/day (drug toxicity control group). Group 7 consisted of seven mice treated orally with 30 μl of DMSO twice per day (the placebo control group). All mice were treated for 10 days and maintained for an additional 5 days after the end of treatment. The level of oocyst shedding was determined three times per week throughout the study by microscopic observation of 30 high-power fields of a modified acid-fast stained fecal smear from each infected animal. Results are presented as the mean log oocysts shed per group ± the 95% confidence intervals. Body weights were determined one time per week throughout the study. Results are presented as the mean body weight per group ± the 95% confidence intervals. At necropsy, sections were taken from the pyloric region of the stomach, mid-small intestine, ileum, cecum, and proximal colon for histologic analysis to determine the extent of mucosal infection. Each site was assigned a score depending on the extent of infection, as follows: 0, no infection; 1, very difficult-to-find parasite forms; 2, sparse but easily found parasite forms; 3, abundant parasite forms but focally distributed; 4, extensive presence of parasite forms covering most mucosal surfaces; and 5, extensive presence of parasite forms covering the entire mucosal surface. The data are presented as the mean total score of the five sites ± 95% confidence intervals. NTZ/Nitazoxanide activity in the piglet diarrhea model. [3] Thirty-one gnotobiotic piglets derived by cesarean section from four litters were maintained inside sterile isolators for the duration of the experiment as described previously. Twenty-six of the 31 piglets were challenged with oocysts 24 h after derivation. Because these experiments were not performed simultaneously, the infecting dose of 5 × 106 excysting oocysts was calculated based on the percent oocyst excystation in vitro (rate of excystation). The rate of in vitro excystation was determined following incubation of the oocysts in 0.75% taurocholic acid for 45 min at 37°C. Once the in vitro excystation rate was determined, the inocula were adjusted accordingly so that 13 piglets received 2 × 107 oocysts and 13 piglets received 7 × 106 oocysts. Piglets were observed two or three times daily for signs of diarrhea, depression, and anorexia and for overall appearance. Diarrhea was defined as a twofold increase in the frequency, volume, and water content of the fecal discharge of a piglet compared to those in uninfected control piglets. Body weights and fecal samples were obtained daily. Within 3 days after challenge, piglets were assigned to groups based on a combination of body weight, onset of oocyst shedding, and diarrhea status. The piglets were then started on a daily treatment schedule of either 250 mg of Nitazoxanide/NTZ/kg (five piglets), 125 mg of NTZ/kg (six piglets), 500 mg of PRM/kg (five piglets), or a placebo (milk; nine piglets). Five uninfected control animals served as drug toxicity controls; three piglets received NTZ at 250 mg/kg/day, and two piglets received 125 mg/kg/day. One of the 26 infected piglets was euthanized because of severe illness and was excluded from the study. All drugs were administered via the milk diet in two divided doses daily for 11 days. The number of oocysts present in an entire modified-acid-fast-stained fecal smear was determined daily for each piglet. Since piglets develop diarrhea as a consequence of C. parvum infection, determination of the number of oocysts shed must account for any variability in fecal consistency that we observe. In particular, the presence of watery diarrhea will influence the number of oocysts detected in a fecal smear due to the effective dilution of the fecal material by the increased fluid content. Because of this, we have devised a scoring system that accounts for both the qualitative nature of the fecal material and the number of oocysts detected. Scores are assigned in this system as follows: 0, no oocysts detected; 1, ≤10 oocysts; 2, ≤25 oocysts; 3, ≤50 oocysts; 4, ≤100 oocysts; and 5, >100 oocysts. Results are presented as the mean oocyst shedding score for each treatment group ± standard error of the mean. Surviving piglets were euthanized 11 days after the onset of treatment, and six gut sections (the pyloric region of the stomach, three equally spaced small intestinal sites, the cecum, and the colon) were removed for histologic analysis of the extent of mucosal infection. Each site was assigned a score depending on the extent of infection by the system described above for the anti-IFN-γ-conditioned SCID mouse. Results are presented as the total score of the six sites for individual piglets. Analysis of the antiviral effect of NTZ/Nitazoxanide in a mouse model[2] Three-week old female Chinese Kunming mice (12–14 g body weight) were randomly divided into six groups (10 mice/group). Group JEV + NTZ was infected with JEV and received NTZ treatment (50, 75 or 100 mg/kg/day). Group JEV + DMSO was infected with JEV and received a placebo (DMSO) treatment. Group Mock + NTZ was mock-infected with JEV and received NTZ treatment. Group Mock + DMSO was mock-infected with JEV and received a placebo (DMSO) treatment. For infection, mice were infected intraperitoneally with 6×104 PFU of JEV (containing 50 × LD50 of JEV). For NTZ treatment, NTZ was dissolved in DMSO and administered intragastrically by gavage, in which a feeding needle was introduced into the esophagus and NTZ was delivered directly into the stomach. NTZ was tested at a total dose of 50, 75 or 100 mg/kg/day, and was consecutively administered from 1 day post-infection, daily, for up to 25 days. The mice were monitored daily for morbidity and mortality.
ADME/Pharmacokinetics	Absorption, Distribution and Excretion The relative bioavailability of the suspension compared to the tablet was 70%. When administered with food the AUC and Cmax increased by two-fold and 50%, respectively, for the tablet and 45 to 50% and ≤ 10%, respectively, for the oral suspension. Tizoxanide is excreted in the urine, bile and feces, and tizoxanide glucuronide is excreted in urine and bile. Approximately 2/3 of the oral dose of nitazoxanide is excreted in the faeces and 1/3 in the urine. Nitazoxanide is cleared in the urine and feces. The metabolite, tizoxanide, is also found in the urine, plasma, and breastmilk. The drug is not found unchanged in the urine. Metabolism / Metabolites The active metabolite of this drug is tizoxanide (desacetyl-nitazoxanide). The initial reaction in the metabolic pathway of Nitazoxanide is hydrolysis to tizoxanide, followed by conjugation, primarily by glucuronidation to tizoxanide glucuronide. The oral suspension bioavailability of this drug is not equivalent to that of the oral tablets. Compared to the to the tablet, the bioavailability of the suspension was 70%. When administered with food, the AUCt of tizoxanide and tizoxanide glucuronide in plasma is increased to almost two-fold and the maximum concentration is increased by almost 50% compared to when ingested without food. When the oral suspension was ingested with food, the AUC of tizoxanide and tizoxanide glucuronide increased by approximately 50% and the Cmax increased by less than 10%. Biological Half-Life 7.3h
Toxicity/Toxicokinetics	Hepatotoxicity Nitazoxanide therapy has not been associated with elevations in serum aminotransferase levels nor with clinically apparent acute liver injury. However, there have been few studies of long term therapy with nitazoxanide and most controlled trials of this agent used short term courses without serum aminotransferase monitoring. Nitazoxanide has been used as adjunctive therapy for chronic hepatitis C, usually in combination with peginterferon with or without ribavirin; in these studies, most patients had improvements in serum aminotransferase levels, and no instances of acute exacerbation of hepatitis or jaundice were reported. Likelihood score: E (unlikely cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Limited information indicates that a maternal dose of 500 mg of nitazoxanide produces low levels of an active metabolite, tizoxanide, in breastmilk and would not be expected to cause any adverse effects in breastfed infants, especially if the infant is older than 2 months. But until more data become available, an alternate drug may be preferred, especially while nursing a newborn or preterm infant. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding Very High (greater than 99%), bound to proteins in the plasma. Drug Interactions [4] At this time, no drug-drug interaction studies have been conducted with nitazoxanide in vivo. Because >99% of tizoxanide is bound to plasma proteins, caution should be used when administering nitazoxanide concurrently with other highly plasma protein—bound drugs with narrow therapeutic indices. It is recommended to monitor the prothrombin time for patients who are concurrently taking warfarin and nitazoxanide. Side Effects [4] Nitazoxanide is generally well tolerated, and no significant adverse events have been noted in human trials. Adverse events have been mild and transient and principally related to the gastrointestinal tract, such as abdominal pain, diarrhea, and nausea. Adverse events occurring in <1% of more than 2000 HIV-uninfected patients participating in clinical trials included anorexia, flatulence, increased appetite, enlarged salivary glands, fever, infection, malaise, elevated creatinine levels, elevated levels of alanine aminotransferase in serum, pruritus, sweat, pale yellow sclerae, rhinitis, dizziness, and discolored urine. In addition, there have been no significant changes in results of electrocardiography, vital signs, or hematologic, clinical chemistry, or urinalysis parameters in patients treated with nitazoxanide. Nitazoxanide has been well tolerated up to the maximum dose of 4 g when taken with or without food, but the frequency of gastrointestinal side effects increases significantly with the dose level.
References	[1]. J Biol Chem. 2009 Oct 23;284(43):29798-808. [2]. Virol J. 2014 Jan 23;11:10. [3]. Antimicrob Agents Chemother.1998 Aug;42(8):1959-65. [4]. Clin Infect Dis.2005 Apr 15;40(8):1173-80.
Additional Infomation	Pharmacodynamics The general effect of this medication is the prevention of microbe activity through disruption of important energy pathways for survival and proliferation. Nitazoxanide exhibits antiprotozoal activity by interfering with the pyruvate ferredoxin/flavodoxin oxidoreductase dependent electron transfer reaction, an essential reaction need for anaerobic energy metabolism of various microorganisms. Sporozoites of Cryptosporidium parvum and trophozoites of Giardia lamblia are therefore inhibited, relieving symptoms of diahrrea. Interference with the PFOR enzyme-dependent electron transfer reaction may only be one of the many pathways by which nitazoxanide exhibits antiprotozoal activity. Acetic acid [2-[[(5-nitro-2-thiazolyl)amino]-oxomethyl]phenyl] ester is a carboxylic ester and a member of benzamides. It is functionally related to a salicylamide. Nitazoxanide belongs to the class of drugs known as thiazolides. Nitazoxanide (NTZ) is a broad-spectrum anti-infective drug that markedly modulates the survival, growth, and proliferation of a range of extracellular and intracellular protozoa, helminths, anaerobic and microaerophilic bacteria, in addition to viruses. This drug is effective in the treatment of gastrointestinal infections including Cryptosporidium parvum or Giardia lamblia in healthy subjects. It is generally well tolerated. Nitazoxanide is a first-line, standard treatment for illness caused by C. parvum or G. lamblia infection in healthy (not immunosuppressed) adults and children and may also be considered in the treatment of illnesses caused by other protozoa or helminths. Recently, this drug has been studied as a broad-spectrum antiviral agent due to its ability to inhibit the replication of several RNA and DNA viruses. Nitazoxanide is an Antiprotozoal. Nitazoxanide is an antimicrobial with activity against several parasitic worms and protozoa that is used predominantly in the United States in treatment of giardiasis and cryptosporidiosis. Nitazoxanide therapy has not been reported to cause serum aminotransferase elevations during therapy or clinically apparent liver injury. Nitazoxanide is a synthetic benzamide with antiprotozoal activity. Nitazoxanide exerts its antiprotozoal activity by interfering with the pyruvate ferredoxin/flavodoxin oxidoreductase dependent electron transfer reaction, which is essential to anaerobic energy metabolism. PFOR enzyme reduces nitazoxanide, thereby impairing the energy metabolism. However, interference with the PFOR enzyme-dependent electron transfer reaction may not be the only pathway by which nitazoxanide exhibits antiprotozoal activity. Nitazoxanide is active against Giardia lamblia and Cryptosporidium parvum. NITAZOXANIDE is a small molecule drug with a maximum clinical trial phase of IV (across all indications) that was first approved in 2002 and is indicated for amebiasis and diarrhea and has 26 investigational indications. 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 (IC50) of 18.8 microM; in comparison, NTZ and tizoxanide had IC50s of 2.4 microM, and MTZ had an IC50 of 7.8 microM. 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. [1] Background: Japanese encephalitis virus (JEV) has a significant impact on public health. An estimated three billion people in 'at-risk' regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing. Consequently, there is an urgent need for the development of novel therapeutic agents against Japanese encephalitis. Nitazoxanide (NTZ) is a thiazolide anti-infective licensed for the treatment of parasitic gastroenteritis. Recently, NTZ has been demonstrated to have antiviral properties. In this study, the anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model. Methods: JEV-infected cells were treated with NTZ at different concentrations. The replication of JEV in the mock- and NTZ-treated cells was examined by virus titration. NTZ was administered at different time points of JEV infection to determine the stage at which NTZ affected JEV replication. Mice were infected with a lethal dose of JEV and intragastrically administered with NTZ from 1 day post-infection. The protective effect of NTZ on the JEV-infected mice was evaluated. Findings: NTZ significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 ± 0.04 μg/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 ± 0.31 μg/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated by daily intragastric administration of 100 mg/kg/day of NTZ were protected from a lethal challenge dose of JEV. Conclusions: Both in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of Japanese encephalitis. [2] Nitazoxanide (NTZ), a drug currently being tested in human clinical trials for efficacy against chronic cryptosporidiosis, was assessed in cell culture and in two animal models. The inhibitory activity of NTZ was compared with that of paromomycin (PRM), a drug that is partially effective against Cryptosporidium parvum. A concentration of 10 microg of NTZ/ml (32 microM) consistently reduced parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity, in contrast to an 80% reduction produced by PRM at 2,000 microg/ml (3.2 mM). In contrast to its efficacy in vitro, NTZ at either 100 or 200 mg/kg of body weight/day for 10 days was ineffective at reducing the parasite burden in C. parvum-infected, anti-gamma-interferon-conditioned SCID mice. Combined treatment with NTZ and PRM was no more effective than treatment with PRM alone. Finally, NTZ was partially effective at reducing the parasite burden in a gnotobiotic piglet diarrhea model when given orally for 11 days at 250 mg/kg/day but not at 125 mg/kg/day. However, the higher dose of NTZ induced a drug-related diarrhea in piglets that might have influenced its therapeutic efficacy. As we have previously reported, PRM was effective at markedly reducing the parasite burden in piglets at a dosage of 500 mg/kg/day. Our results indicate that of all of the models tested, the piglet diarrhea model most closely mimics the partial response to NTZ treatment reported to occur in patients with chronic cryptosporidiosis.[3] Nitazoxanide is a new thiazolide antiparasitic agent that shows excellent in vitro activity against a wide variety of protozoa and helminths. It is given by the oral route with good bioavailability and is well tolerated, with primarily mild gastrointestinal side effects. At present, there are no documented drug-drug interactions. Nitazoxanide has been licensed for the treatment of Giardia intestinalis-induced diarrhea in patients >or=1 year of age and Cryptosporidum-induced diarrhea in children aged 1-11 years. At present, it is pending licensure for treatment of infection due to Cryptosporidium species in adults and for use in treating immunocompromised hosts. It represents an important addition to the antiparasitic arsenal.[4]

Solubility Data

Solubility (In Vitro)

DMSO : 61~100 mg/mL ( 198.51~325.44 mM )

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3.25 mg/mL (10.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 3.25 mg/mL (10.58 mM)  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.2544 mL	16.2718 mL	32.5436 mL
	5 mM	0.6509 mL	3.2544 mL	6.5087 mL
	10 mM	0.3254 mL	1.6272 mL	3.2544 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.