Using a high-throughput, whole-cell hydrogenase assay to identify potential small molecule inhibitors of [NiFe]-hydrogenase.

[NiFe]-hydrogenases are used by several human pathogens to catalyze the reversible conversion between molecular hydrogen and protons and electrons. Hydrogenases provide an increased metabolic flexibility for pathogens, such as Escherichia coli and Helicobacter pylori, by allowing the use of molecular hydrogen as an energy source to promote survival in anaerobic environments. With the rise of antimicrobial resistance and the desire for novel therapeutics, the [NiFe]-hydrogenases are alluring targets. Inhibiting the nickel insertion pathway of [NiFe]-hydrogenases is attractive as this pathway is required for the generation of functional enzymes and is orthogonal to human biochemistry. In this work, nickel availability for the production and function of E. coli [NiFe]-hydrogenase was explored through immunoblot and activity assays. Whole-cell hydrogenase activities were assayed in high throughput against a small molecule library of known bioactives. Iodoquinol was identified as a potential inhibitor of the nickel biosynthetic pathway of [NiFe]-hydrogenase through a two-step screening process, but further studies with immunoblot assays showed confounding effects dependent on the cell growth phase. This study highlights the significance of considering the growth phenotype for whole-cell based assays overall and its effects on various cellular processes influenced by metal trafficking and homeostasis.

Transforming iodoquinol into broad spectrum anti-tumor leads: Repurposing to modulate redox homeostasis.

We managed to repurpose the old drug iodoquinol to a series of novel anticancer 7-iodo-quinoline-5,8-diones. Twelve compounds were identified as inhibitors of moderate to high potency on an inhouse MCF-7 cell line, of which 2 compounds (5 and 6) were capable of reducing NAD level in MCF-7 cells in concentrations equivalent to half of their IC50s, potentially due to NAD(P)H quinone oxidoreductase (NQO1) inhibition. The same 2 compounds (5 and 6) were capable of reducing p53 expression and increasing reactive oxygen species levels, which further supports the NQO-1 inhibitory activity. Furthermore, 4 compounds (compounds 5-7 and 10) were qualified by the Development Therapeutic Program (DTP) division of the National Cancer Institute (NCI) for full panel five-dose in vitro assay to determine their GI50 on the 60 cell lines. All five compounds showed broad spectrum sub-micromolar to single digit micromolar GI50 against a wide range of cell lines. Cell cycle analysis and dual staining assays with annexin V-FITC/propidium iodide on MCF-7 cells confirmed the capability of the most active compound (compound 5) to induce cell cycle arrest at Pre-G1 and G2/M phases as well as apoptosis. Both cell cycle arrest and apoptosis were affirmed at the molecular level by the ability of compound 5 to enhance the expression levels of caspase-3 and Bax together with suppressing that of CDK1 and Bcl-2. Additionally, an anti-angiogenic effect was evident with compound 5 as supported by the decreased expression of VEGF. Interesting binding modes within NQO-1 active site had been identified and confirmed by both molecular docking and dymanic experiments.

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 µM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 µM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 µM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 µM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.

Discovery of the FDA-approved drugs bexarotene, cetilistat, diiodohydroxyquinoline, and abiraterone as potential COVID-19 treatments with a robust two-tier screening system.

Coronavirus Disease 2019 (COVID-19) caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a crude case fatality rate of about 0.5-10 % depending on locality. A few clinically approved drugs, such as remdesivir, chloroquine, hydroxychloroquine, nafamostat, camostat, and ivermectin, exhibited anti-SARS-CoV-2 activity in vitro and/or in a small number of patients. However, their clinical use may be limited by anti-SARS-CoV-2 50 % maximal effective concentrations (EC50) that exceeded their achievable peak serum concentrations (Cmax), side effects, and/or availability. To find more immediately available COVID-19 antivirals, we established a two-tier drug screening system that combines SARS-CoV-2 enzyme-linked immunosorbent assay and cell viability assay, and applied it to screen a library consisting 1528 FDA-approved drugs. Cetilistat (anti-pancreatic lipase), diiodohydroxyquinoline (anti-parasitic), abiraterone acetate (synthetic androstane steroid), and bexarotene (antineoplastic retinoid) exhibited potent in vitro anti-SARS-CoV-2 activity (EC50 1.13-2.01 µM). Bexarotene demonstrated the highest Cmax:EC50 ratio (1.69) which was higher than those of chloroquine, hydroxychloroquine, and ivermectin. These results demonstrated the efficacy of the two-tier screening system and identified potential COVID-19 treatments which can achieve effective levels if given by inhalation or systemically depending on their pharmacokinetics.

Repurposing the Antiamoebic Drug Diiodohydroxyquinoline for Treatment of Clostridioides difficile Infections.

Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for the treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug. DIHQ exhibited potent activity against 39 C. difficile isolates, inhibiting growth of 50% and 90% of these isolates at concentrations of 0.5 µg/ml and 2 µg/ml, respectively. In a time-kill assay, DIHQ was superior to vancomycin and metronidazole, reducing a high bacterial inoculum by 3 log10 within 6 h. Furthermore, DIHQ reacted synergistically with vancomycin and metronidazole against C. difficilein vitro. Moreover, at subinhibitory concentrations, DIHQ was superior to vancomycin and metronidazole in inhibiting two key virulence factors of C. difficile, toxin production and spore formation. Additionally, DIHQ did not inhibit the growth of key species that compose the host intestinal microbiota, such as Bacteroides, Bifidobacterium, and Lactobacillus spp. Collectively, our results indicate that DIHQ is a promising anticlostridial drug that warrants further investigation as a new therapeutic for CDIs.

Combined vaginal administration of nystatin, diiodohydroxyquin, and benzalkonium chloride versus oral metronidazole for the treatment of bacterial vaginosis.

OBJECTIVE: To compare combined vaginal administration of nystatin, diiodohydroxyquin, and benzalkonium chloride versus oral metronidazole for the treatment of bacterial vaginosis (BV). METHODS: A randomized controlled trial was conducted among women diagnosed with BV using the Amsel criteria (n=90) at a university hospital in Khon Kaen, Thailand, between June 27, 2017, and April 30, 2018. The oral metronidazole group (n=44) received 400 mg of metronidazole, administered three times per day. The combined vaginal tablet group (n=46) received a vaginal suppository once daily, which comprised nystatin (100 000 U), diiodohydroxyquin (100 mg), and benzalkonium chloride (7 mg). Treatment was administered for 7 days in both groups. Follow-up visits at 14 and 42 days assessed treatment outcomes and adverse effects. RESULTS: Remission of BV occurred among 41 (93%) women in the oral metronidazole group and 39 (85%) women in the combined vaginal tablet group. The adjusted relative risk was 0.92 (95% confidence interval 0.80-1.06). The rate of nausea and/or vomiting was significantly higher in the oral metronidazole group than that in the combined vaginal tablet group. CONCLUSION: Treatment efficacy of the combined vaginal tablet versus oral metronidazole was equivalent. CLINICAL TRIAL REGISTRATION: TCTR20170627001 (www.clinicaltrials.in.th).

Identification and Characterization of the Human Cytosolic Sulfotransferases Mediating the Sulfation of Clioquinol and Iodoquinol.

OBJECTIVE: The aim of the current study was to identify the human cytosolic sulfotransferases (SULTs) that are capable of sulfating clioquinol and iodoquinol, and to verify the presence of clioquinol/ iodoquinol-sulfating activity in human organ homogenates and cultured cells. METHOD: An established sulfotransferase assay was employed to analyze clioquinol/iodoquinolsulfating activity of thirteen known human SULTs, as well as cytosols of human kidney, liver, lung, and small intestine. Metabolic labeling with [35S]sulfate in the presence of different concentrations of clioquinol/iodoquinol was performed using cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells. RESULTS: A systematic analysis revealed that six of the thirteen known human SULTs, SULT1A1 SULT1A2, SULTA3, SULT1B1, SULT1C4, and SULT1E1 showed considerable clioquinol/ iodoquinol-sulfating activity. Kinetic parameters of the sulfation of clioquinol and iodoquinol by three SULTs, SULT1A1, SULT1A3, and SULT1C4, that showed the strongest clioquinol/iodoquinolsulfating activity were determined. Moreover, clioquinol/iodoquinol-sulfating activity was detected in the cytosol fractions of human liver, lung, kidney, and small intestine. Cultured HepG2 and Caco-2 cells were shown to be capable of sulfating clioquinol/iodoquinol under metabolic conditions. CONCLUSION: Collectively, these results provided a molecular basis underling the metabolism of clioquinol and iodoquinol through sulfation.

Chromatographic methods for simultaneous determination of diiodohydroxyquinoline and metronidazole in their binary mixture.

Two chromatographic methods were developed for analysis ofdiiodohydroxyquinoline (DIHQ) and metronidazole (MTN). In the first method, diiodohydroxyquinoline and metronidazole were separated on TLC silica gel 60F254 plate using chloroform: acetone: glacial acetic acid (7.5: 2.5: 0.1, by volume) as mobile phase. The obtained bands were then scanned at 254 nm. The second method is a RP-HPLC method in which diiodohydroxyquinoline and metronidazole were separated on a reversed-phase C18 column using water : methanol (60 :40, V/V, PH=3.6 )as mobile phase at a flow rate of 0.7 mL.min-1 and UV detection at 220 nm. The mentioned methods were successfully used for determination of diiodohydroxyquinoline and metronidazole in pure form and in their pharmaceutical formulation.

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism.

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase-DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate. The compound also induces the religation of the stalled enzyme-CPT (camptothecin)-DNA ternary complex. Analysis of the molecule docked over the binary complex, together with its chemical properties, suggests that the religation enhancement is due to the presence on the compound of two oxygen atoms that act as hydrogen acceptors. This property facilitates the deprotonation of the 5' DNA end, suggesting that this is the limiting step in the topoisomerase religation mechanism.

Pleural empyema secondary to rupture of amoebic liver abscess.

Amoebiasis is a worldwide parasitic infection although it is more prevalent in the subtropical and tropical countries. Extraintestinal amoebic infections currently have been reported in increased numbers of male homosexuals and immunocompromised patients. Here, we present an interesting case of a 27-year-old homosexual man with pleural empyema secondary to rupture of amoebic liver abscess. Using chest tube and percutaneous liver abscess drainage, the patient was treated with metronidazole followed by iodoquinol. His general condition improved dramatically. After one-year of follow-up, there was no evidence of relapse on plain chest radiography and abdominal CT scan.

A case-controlled study of Dientamoeba fragilis infections in children.

Dientamoeba fragilis is a pathogenic protozoan parasite that is implicated as a cause of human diarrhoea. A case-controlled study was conducted to determine the clinical signs associated with D. fragilis infection in children presenting to a Sydney Hospital. Treatment options are also discussed. Stool specimens were collected from children aged 15 years or younger and analysed for the presence of D. fragilis. In total, 41 children were included in the study along with a control group. Laboratory diagnosis was performed by microscopy of permanently stained, fixed faecal smears and by real-time PCR. Gastrointestinal symptoms were present in 40/41 (98%) of these children with dientamoebiasis, with diarrhoea (71%) and abdominal pain (29%) the most common clinical signs. Chronic gastrointestinal symptoms were present in 2% of cases. The most common anti-microbial used for treatment was metronidazole (n=41), with complete resolution of symptoms and clearance of parasite occurring in 85% of cases. A treatment failure rate occurred in 15% of those treated with metronidazole. Follow-up treatment comprised of an additional course of metronidazole or iodoquinol was needed in order to achieve complete resolution of infection and symptoms in this group. This study demonstrates the pathogenic potential of D. fragilis in children and as such it is recommended that all laboratories must routinely test for this organism and treat if detected.

Amoebic dysentery.

INTRODUCTION: Amoebic dysentery is caused by the protozoan parasite Entamoeba histolytica. It is transmitted in areas where poor sanitation allows contamination of drinking water and food with faeces. In these areas, up to 40% of people with diarrhoea may have amoebic dysentery. METHODS AND OUTCOMES: We conducted a systematic review and aimed to answer the following clinical question: What are the effects of drug treatments for amoebic dysentery in endemic areas? We searched: Medline, Embase, The Cochrane Library, and other important databases up to April 2010 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). RESULTS: We found 6 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. CONCLUSIONS: In this systematic review, we present information relating to the effectiveness and safety of the following interventions: diiodohydroxyquinoline (iodoquinol), diloxanide, emetine, metronidazole, nitazoxanide, ornidazole, paromomycin, secnidazole, and tinidazole.

Antimicrobial activity of iodoquinol 1%-hydrocortisone acetate 2% gel against ciclopirox and clotrimazole.

Commercially available topical formulations consisting of iodoquinol 1%-hydrocortisone acetate 2%, ciclopirox 0.77%, and clotrimazole 1%-betamethasone dipropionate 0.5% were assessed for their antimicrobial activity against cultures of Micrococcus luteus, Propionibacterium acnes, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Corynebacterium aquaticum, Trichophyton mentagrophytes, Malassezia furfur, Microsporum canis, Candida albicans, Trichophyton rubrum, or Epidermophyton floccosum. At 1 and 5 minutes following inoculation into suspensions of each product, aliquots were removed, serially diluted, and plated onto appropriate agar to determine the log reduction in colony-forming units (CFUs) for each organism. Iodoquinol 1% produced the broadest and greatest antimicrobial activity as measured by a 3-log reduction of CFU, active against all microbes tested following incubation times of 1 or 5 minutes, except M luteus. By contrast, ciclopirox 0.77% and clotrimazole 1% showed activity against P aeruginosa and T rubrum, with ciclopirox also killing M luteus, P acnes, M canis, C albicans, and E floccosum at 5 minutes. Iodoquinol 1%-hydrocortisone acetate 2% also was the only product that showed effective antibacterial reduction of MRSA at 1 minute.

Eradication of amebiasis in a large institution for adults with mental retardation in Taiwan.

OBJECTIVE: Investigation of a program to eradicate amebiasis using consecutive intensive mass screenings followed by medication in a large institute for adults with mental retardation in Taiwan. DESIGN: Prospective cohort study, with 3 years of follow-up. SETTING: A large, 450-bed institution for adults with mental retardation located in southern Taiwan. PARTICIPANTS: All 443 adults with mental retardation in the institution, who have various motor and/or mental handicaps, were included in this study. INTERVENTIONS: A total of 7 consecutive intensive mass screenings for amebiasis for all residents (performed in March, August and November 2001, March and August 2002, January 2003, and May 2004). Infected patients were treated using the standard protocol of the Center for Disease Control of Taiwan. RESULTS: Enzyme immunoassay testing was used for the amebiasis screening, with the rapid detection of the specific antigen for Entamoeba histolytica in human fecal specimens confirmed by microscopic examination. The serial prevalence and cumulative incidence were then calculated. The prevalence of amebic infection declined in serial screenings, but new infections and reinfections were detected in 5 of 6 follow-up screenings. The prevalence was 10.8% at the beginning of the program and then gradually reduced, falling to 6.3%, 3.6%, 2.7%, 3.4%, and 2.2%. Finally, no more positive cases were identified in the last screening (May 2004). The cumulative incidence rate stabilized at around 40% by the fifth screening. Of the 179 infected patients, 120 had primary infections, with 59 cases of multiple amebic infections. CONCLUSIONS: Active surveillance with intensive mass screening is an effective method of identifying asymptomatic and latent cases of amebiasis in areas where it is endemic, such as an institution for adults with mental retardation.

Differentiating amoebic ulcero-haemorrhagic recto-colitis from idiopathic inflammatory bowel disease: still a diagnostic dilemma.

The colon responds monomorphically to a variety of insults thus making it difficult to differentiate invasive amoebic colitis and inflammatory bowel disease (IBD). The authors present a case with chronic dysentery, haematochezia, anaemia and hypoproteinaemia. The endoscopic findings were suggestive of IBD. The stool examination was negative for trophozoites or cysts of parasites. The recto-colonic biopsy specimens showed mucosal inflammation with exudates containing amoebic trophozoites. The patient was successfully treated with metronidazole and iodoquinol. He recovered within two weeks and repeat colonoscopy four weeks after the treatment showed a normal rectum and colon. Clinicians should have a high level of suspicion for amoebic colitis in cases of colitis especially in regions where amoebiasis is still present. Efforts should be made to find the amoebic trophozoites in multiple stool and colonic biopsy specimens.

Differentiating amoebic ulcero-haemorrhagic recto-colitis from idiopathic inflammatory bowel disease: still a diagnostic dilemma / Diferenciar la Rectocolitis Úlcero-hemorrágica Amebiana de la Enfermedad Inflamatoria Idiopática del Intestino: un Dilema Diagnóstico sin Resolver

The colon responds monomorphically to a variety of insults thus making it difficult to differentiate invasive amoebic colitis and inflammatory bowel disease (IBD). The authors present a case with chronic dysentery, haematochezia, anaemia and hypoproteinaemia. The endoscopic findings were suggestive of IBD. The stool examination was negative for trophozoites or cysts of parasites. The recto-colonic biopsy specimens showed mucosal inflammation with exudates containing amoebic trophozoites. The patient was successfully treated with metronidazole and iodoquinol. He recovered within two weeks and repeat colonoscopy four weeks after the treatment showed a normal rectum and colon. Clinicians should have a high level of suspicion for amoebic colitis in cases of colitis especially in regions where amoebiasis is still present. Efforts should be made to find the amoebic trophozoites in multiple stool and colonic biopsy specimens

El colon responde de manera monomórfica a una variedad de insultos, lo cual hace difícil distinguir entre la colitis amebiana invasiva y la enfermedad intestinal inflamatoria (EII). Los autores presentan un caso con disentería crónica, hematoquexia, anemia e hipoproteinemia. Los resultados endoscópicos apuntaban a una EII. El análisis de las heces fecales arrojó resultados negativos en cuanto a presencia de trofozoitos o quistes de parásitos. Esto condujo a un diagnóstico erróneo y el paciente fue tratado por una EII. Sin embargo, los especímenes de la biopsia rectocolónica mostraron una inflamación mucosal con exudados en los que se hallaban presentes trofozoitos amebianos. El paciente tuvo un tratamiento exitoso con metronidazol y iodoquinol. Se recuperó en dos semanas, y se le repitió la colonoscopia cuatro semanas después de que el tratamiento mostró un recto y colon normales. Los clínicos debían mostrar un alto nivel de sospecha ante la colitis amebiana, especialmente en aquellas regiones donde la amebiasis todavía está presente. Deben hacerse esfuerzos por encontrar trofozoitos amebianos en múltiples especímenes de heces fecales y biopsia colónica.

Efficacy of saccharomyces boulardii with antibiotics in acute amoebiasis.

AIM: To compare the efficacy of antibiotics therapy alone with antibiotics and saccharomyces boulardii in treatment of acute amebiasis. METHODS: In a double blind, random clinical trial on patients with acute intestinal amoebiasis, 57 adult patients with acute amoebiasis, diagnosed with clinical manifestations (acute mucous bloody diarrhea) and amebic trophozoites engulfing RBCs found in stool were enrolled in the study. Regimen 1 included metronidazole (750 mg Tid) and iodoquinol (630 mg Tid) for 10 days. Regimen 2 contained capsules of lyophilized saccharomyces boulardii (250 mg Tid) orally in addition to regimen 1. Patients were re-examined at two and four weeks after the treatment, and stool examination was performed at the end of week 4. Student's t-test, chi(2) and McNemar's tests were used for statistical analysis. RESULTS: Three patients refused to participate. The other 54 patients were randomized to receive either regimen 1 or regimen 2 (Groups 1 and 2 respectively, each with 27 patients). The two groups were similar regarding their age, sex and clinical manifestations. In Group 1, diarrhea lasted 48.0+/-18.5 hours and in Group 2, 12.0+/-3.7 hours (P<0.0001). In Group 1, the durations of fever and abdominal pain were 24.0+/-8.8 and 24.0+/-7.3 hours and in Group 2 they were 12.0+/-5.3 and 12.0+/-3.2 hours, respectively (P<0.001). Duration of headache was similar in both groups. At week 4, amebic cysts were detected in 5 cases (18.5 %) of Group 1 but in none of the Group 2 (P<0.02). CONCLUSION: Adding saccharomyces boulardii to antibiotics in the treatment of acute amebiasis seems to decrease the duration of clinical symptoms and cyst passage.

LC of pharmaceutically important halogenated 8-hydroxyquinolines after precolumn derivatization with Pd (II).

An accurate, sensitive, and selective reversed phase high performance liquid chromatographic (HPLC) method was developed for the analysis of two halogenated 8-hydroxyquinoline derivatives; clioquinol (CQN) and iodoquinol (IQN). The proposed method depends on the complexation ability of the studied compounds with Pd(II) ions. Reversed phase chromatography was conducted using a 300 x 3.9 mm i.d. stainless steel column packed with 10 microm Bondclone phenyl at ambient temperature. A solution containing 0.005% w/v of Pd(II)-chloride in a mixture of acetonitrile-methanol-water (3:3:4 v/v/v) of pH 3.7 as a mobile phase pumped at a flow rate of 0.75 ml min(-1). UV-detection was performed at 282 and 285 nm for CQN and IQN, respectively. The method showed excellent linearity in the range 0.05-1.8 and 0.1-3.0 microg ml(-1) with limit of detection (S/N=2) 4.8 ng ml(-1) (1.57 x 10(-8) M) and 6.4 ng ml(-1) (1.61 x 10(-8) M) for CQN and IQN, respectively. The suggested method was successfully applied for the analysis of the studied drugs in bulk with average% recoveries of 99.68+/-0.44 for CQN and 99.65+/-0.53 for IQN. The proposed method was successfully applied for the analysis of the studied drugs in single or combined dosage forms with average% recoveries of 99.41+/-0.51-100.02+/-0.63. The proposed method could be used successfully for the determination of the studied compounds in the presence of their degradation product as they could be eluted with different retention times. The presence of metronidazole (MNZ) or tolnaftate (TFT) with the studied drugs does not affect their accurate determination. The results obtained were favorably compared with those obtained by the reference method. The results were satisfactorily, accurate, and precise.