Mefenoxam, Itraconazole, and Terbinafine Combination Therapy for Management of Pythiosis in Dogs (Six Cases).

Pythium insidiosum is an oomycete that encysts in the skin or gastrointestinal tract, leading to pythiosis. Pythiosis is reported in tropical and subtropical climates, affecting dogs and rarely cats. Surgical resection is the treatment of choice; however, cases present late in the disease and lesions are often nonresectable. Medical management is typically unsuccessful, with uncommon exceptions; however, mefenoxam, an agricultural fungicide, has in vitro efficacy against P insidiosum. We describe the use of mefenoxam, itraconazole, and terbinafine (MIT) in five dogs with gastrointestinal pythiosis and one dog with cutaneous pythiosis. Two of the gastrointestinal cases had disease extending to surgical margins and received MIT: resolution of clinical signs and seronegativity occurred after 189-193 days. Another case underwent surgical resection and MIT. The dog improved but subsequently developed a rectal mass, which responded to addition of prednisone and immunotherapy. Two cases were treated with MIT alone, and response varied. Efficacy of MIT in cutaneous pythiosis could not be determined. MIT may result in improved survival and seronegativity in dogs with incompletely resected gastrointestinal pythiosis. Mefenoxam is EPA registered, and extralabel use under the Animal Medicinal Drug Use Clarification Act does not apply. Additional research is recommended before use.

Clinical efficacy of combination therapy of itraconazole and prednisolone for treating effusive feline infectious peritonitis.

A 3-month-old male Scottish Fold kitten with pleural fluid and low ratio of albumin to globulin (A/G ratio) was brought to our small animal hospital. Since RNA from the type I feline coronavirus (FCoV) were detected in drained pleural fluid, the cat was tentatively diagnosed with effusive feline infectious peritonitis (FIP). Following the administration of itraconazole and prednisolone, the A/G ratio increased, and the pleural fluid mostly disappeared. The fecal FCoV levels temporarily decreased. However, the cat showed neurological manifestations and was eventually euthanized due to status epilepticus after 38 days of treatment. In conclusion, itraconazole partly exerted a beneficial effect in a cat with FIP. However, further investigation of a possible role of itraconazole in FIP treatment is warranted.

Discovery of Novel Fungal Lanosterol 14α-Demethylase (CYP51)/Histone Deacetylase Dual Inhibitors to Treat Azole-Resistant Candidiasis.

Invasive fungal infections (particularly candidiasis) are emerging as severe infectious diseases worldwide. Because of serious antifungal drug resistance, therapeutic efficacy of the current treatment for candidiasis is limited and associated with high mortality. However, it is highly challenging to develop novel strategies and effective therapeutic agents to combat drug resistance. Herein, the first generation of lanosterol 14α-demethylase (CYP51)-histone deacetylase (HDAC) dual inhibitors was designed, which exhibited potent antifungal activity against azole-resistant clinical isolates. In particular, compounds 12h and 15j were highly active both in vitro and in vivo to treat azole-resistant candidiasis. Antifungal mechanism studies revealed that they acted by blocking ergosterol biosynthesis and HDAC catalytic activity in fungus, suppressing the function of efflux pump, yeast-to-hypha morphological transition, and biofilm formation. Therefore, CYP51-HDAC dual inhibitors represent a promising strategy to develop novel antifungal agents against azole-resistant candidiasis.

Evaluation of topical econazole nitrate formulations with potential for treating Raynaud's phenomenon.

The purpose of this work was to design and characterize a topical formulation of econazole nitrate (EN) with potential for treating Raynaud's phenomenon (RP). Four topical dosage forms (F1_topical solution, F2_HPMC or hydroxypropyl methylcellulose dispersion, F3_VersaBase® cream, and F4_Lipoderm® Activemax™ Cream) containing 3% w/w EN were prepared and characterized for drug content, pH, viscosity, spreadability, drug crystallinity, stability, and in vitro permeation using Franz cells across pig ear skin, and results were compared to the 1% marketed EN cream. All four formulations had acceptable physical and visual characteristics required for topical application, with 3% w/w EN. The order of amount of drug permeated from highest to lowest was F2 (10.27%) > F4 (2.47%) > F1 (2.28%) > F3 (1.47%) > marketed formulation (0.22%). Formulation F2 showed better penetration of the drug into the stratum corneum, epidermis, and dermis layers. The drug concentration in the stratum corneum and epidermis was approximately 10-20 times higher with F2 compared to the marketed formulation. All formulations were found to be stable for up to 6 months. All four EN formulations were found to be better than the 1% marketed cream. Formulation F2_HPMC dispersion could be further explored as a treatment option for RP.

Successful Aspects of the Coadministration of Sterol 14α-Demethylase Inhibitor VFV and Benznidazole in Experimental Mouse Models of Chagas Disease Caused by the Drug-Resistant Strain of Trypanosoma cruzi.

Up to now, no vaccines are available for Chagas disease, and the current therapy is largely unsatisfactory. Novel imidazole-based scaffolds of protozoan sterol 14α-demethylase (CYP51) inhibitors have demonstrated potent antiparasitic activity with no acute toxicity. Presently our aim was to investigate the effectiveness of the experimental 14α-demethylase inhibitor VFV in the mouse models of Trypanosoma cruzi infection using a naturally drug-resistant Colombiana strain, under monotherapy and in association with the reference drug, benznidazole (Bz). The treatment with VFV resulted in complete parasitemia suppression and 100% animal survival when administered orally (given in 10% DMSO plus 5% Arabic gum) at 25 mg/kg (bid) for 60 days. However, as parasite relapse was found using VFV alone under this treatment scheme, the coadministration of VFV with Bz was assayed giving simultaneously (for 60 days, bid) by oral route, under two different drug vehicles (10% DMSO plus 5% Gum Arabic with or without 3% Tween 80). All tested mice groups resulted in >99.9% of parasitemia decrease and 100% animal survival. qPCR analysis performed on cyclophosphamide immunosuppressed mice revealed that, although presenting lack of cure, VFV given as monotherapy was 14-fold more active than Bz, and the coadministration of Bz plus VFV (given simultaneously, using 10% DMSO plus 5% Gum Arabic as vehicle) resulted in 106-fold lower blood parasitism as compared to the monotherapy of Bz. Another interesting finding was the parasitological cure in 70% of the animals treated with Bz and VFV when the coadministration was given using the VFV suspension in 10% DMSO + Arabic gum + Tween 80 (a formulation that we have found to provide a better pharmacokinetics), even after immunosuppression using cyclophosphamide cycles, supporting the promising aspect of the drug coadministration in improving the efficacy of therapeutic arsenal against T. cruzi.

The novel fungal CYP51 inhibitor VT-1598 is efficacious alone and in combination with liposomal amphotericin B in a murine model of cryptococcal meningitis.

Objectives: Annual global deaths from cryptococcal meningitis (CM) are estimated at 180 000 and mortality is as high as 30%, even with optimal therapy. VT-1598 is a novel fungal CYP51 inhibitor with potent intrinsic antifungal activity against Cryptococcus. We report here VT-1598's in vivo antifungal activity in a murine model of CM. Methods: Single-dose plasma and brain pharmacokinetics in mice and MIC for Cryptococcus neoformans H99 were determined prior to efficacy studies. Short-course monotherapy and combination doses were explored with the endpoint of brain fungal burden. A survival study was also conducted using monotherapy treatment with fungal burden measured after a 6 day drug washout. Results: Oral doses of VT-1598 had good plasma and brain exposure and resulted in significant (P < 0.0001) and dose-dependent reductions in brain fungal burden, reaching a 6 log10 reduction. Unlike either positive drug control (fluconazole or liposomal amphotericin B), both mid and high doses of VT-1598 reduced fungal burden to below levels measured at the start of treatment. When VT-1598 was dosed in the survival study, no VT-1598-treated animal succumbed to the infection. Whereas fluconazole showed a 2.5 log10 increase in fungal burden after the 6 day washout, the VT-1598 mid- and high-dose animals showed almost no regrowth (<0.5 log10). In a separate fungal burden study using suboptimal doses of VT-1598 and liposomal amphotericin B to probe for combination effects, each combination had a positive effect relative to corresponding monotherapies. Conclusions: These pre-clinical in vivo data strongly support clinical investigation of VT-1598 as a novel therapy for this lethal infection.

A phase 2, randomized, double-blind, placebo-controlled, dose-ranging study to evaluate the efficacy and safety of orally administered VT-1161 in the treatment of recurrent vulvovaginal candidiasis.

BACKGROUND: Lanosterol demethylase is an enzyme that is essential for fungal growth and catalyzes an early step in the biosynthetic pathway of ergosterol, which is a sterol that is required for fungal cell membrane formation and integrity. Lanosterol demethylase is the molecular target of the class of drugs referred to as "azole antifungals." VT-1161 is a novel, oral, selective inhibitor of fungal lanosterol demethylase and is being developed for the treatment of recurrent vulvovaginal candidiasis. OBJECTIVE: We evaluated the efficacy and safety of 4 dosing regimens of oral VT-1161 compared with placebo in women with recurrent vulvovaginal candidiasis, which was defined as at least 3 symptomatic episodes of acute vulvovaginal candidiasis within a 12-month period. STUDY DESIGN: Two hundred fifteen women with a documented history of recurrent vulvovaginal candidiasis and who, at screening, were experiencing an episode of acute vulvovaginal candidiasis (acute vulvovaginal candidiasis; composite vulvovaginal signs and symptoms score of ≥3 and a positive potassium hydroxide test for yeast) were enrolled. After treatment of the acute infection with fluconazole, subjects were assigned randomly to 1 of 5 treatment regimens: (1) VT-1161 150 mg once daily for 7 days, then 150 mg once weekly for 11 weeks, followed by a once-weekly dose of placebo for 12 weeks; (2) VT-1161 300 mg once daily for 7 days, then 300 mg once weekly for 11 weeks, followed by a once-weekly dose of placebo for 12 weeks; (3) VT-1161 150 mg once daily for 7 days, then 150 mg once weekly for 23 weeks; (4) VT-1161 300 mg once daily for 7 days, then 300 mg once weekly for 23 weeks; or (5) a matching placebo regimen for 24 weeks. The primary efficacy outcome was the proportion of subjects with ≥1 culture-verified acute vulvovaginal candidiasis episodes through week 48. RESULTS: In the intent-to-treat population, the proportion of subjects with ≥1 acute vulvovaginal candidiasis episodes ranged from 0-7% across the 4 VT-1161 arms vs 52% in the placebo arm, with all arms achieving statistical significance vs placebo. VT-1161 was well-tolerated with a favorable safety profile, and the incidence of adverse events was lower in all VT-1161 arms compared with placebo. In addition, no patient in any VT-1161 arm discontinued the study early because of an adverse event or laboratory abnormality. There was also no evidence of an adverse effect of VT-1161 on liver function or electrocardiogram recordings. CONCLUSION: In this study, VT-1161 was shown to be efficacious and safe in the treatment of patients with recurrent vulvovaginal candidiasis. These data strongly support further clinical investigation of VT-1161 for the treatment of recurrent vulvovaginal candidiasis.

Alopecia universalis in a dog with testicular neoplasia.

OBJECTIVE: To describe a case of testicular neoplasia and alopecia universalis in a dog, and successful treatment of the latter with ciclosporin. ANIMAL: Twelve-year-old intact male wirehaired fox terrier. METHODS: Castration, skin biopsy for histopathology, lymphocyte immunophenotyping and clonality analysis of the canine T-cell receptor gamma locus (TCRγ) rearrangement. RESULTS: The dog presented with symmetrical generalized alopecia. Testicular enlargement was noted which on castration was determined to be caused by bilateral interstitial cell tumours, Sertoli cell tumours and a unilateral seminoma. During the four months after castration the alopecia became more severe and widespread. Histopathology of the skin showed moderate, multifocal, mural folliculitis, peribulbar mucinosis and lymphocytic bulbitis, and targeting of anagen hair follicles. Immunophenotyping of the infiltrate showed a population of well-differentiated, small CD3-positive T lymphocytes, some expressing CD4 and others CD8. Molecular analysis revealed a polyclonal lymphocytic infiltrate, substantiating the diagnosis of alopecia areata rather than lymphoma. Treatment with ciclosporin (4.6 mg/kg) and ketoconazole (4.6 mg/kg) resulted in complete hair regrowth. CONCLUSION AND CLINICAL IMPORTANCE: Ciclosporin treatment, in combination with ketoconazole, can be effective for treatment of alopecia universalis in the dog. Alopecia universalis may present with clinically noninflammatory, symmetrical, generalized alopecia, mimicking an endocrine alopecia, and skin biopsies are needed to confirm the diagnosis.

Nano-amorphous spray dried powder to improve oral bioavailability of itraconazole.

The objective of this study was to formulate nano-amorphous spray-dried powders of itraconazole to enhance its oral bioavailability. A combination approach of solvent-antisolvent precipitation followed by spray drying was used. DoE studies were utilized to understand the critical processing parameters: antisolvent-to-solvent ratio, drug concentration and stabilizer concentration. Particle size was the critical quality attribute. Spray drying of the nano-precipitated formulation was performed with several auxiliary excipients to obtain nano-sized amorphous powder formulations. PLM, DSC and PXRD were utilized to characterize the spray-dried powders. In vitro dissolution and in vivo bioavailability studies of the nano-amorphous powders were performed. The particle size of the nano-formulations was dependent on the drug concentration. The smallest size precipitates were obtained with low drug concentration. All high molecular weight auxiliary excipients and mannitol containing formulations were unstable and crystallized during spray drying. Formulations containing disaccharides were amorphous and non-aggregating. In vitro dissolution testing and in vivo studies showed the superior performance of nano-amorphous formulations compared to melt-quench amorphous and crystalline itraconazole formulations. This study shows superior oral bioavailability of nano-amorphous powders compared to macro-amorphous powders. The nano-amorphous formulation showed similar bioavailability to the nano-crystalline formulation but with a faster absorption profile.

Efinaconazole (Jublia) for the treatment of onychomycosis.

Efinaconazole 10% nail solution (Jublia(®)) is a new topical triazole antifungal designed for the topical treatment of distal and lateral subungual onychomycosis. It inhibits ergosterol biosynthesis enzyme sterol 14α-demethylase. Efinaconazole has lower minimum inhibitory concentrations than terbinafine, ciclopirox, itraconazole and amorolfine in Trichophyton rubrum, Trichophyton mentagrophytes and Candida albicans. The solution based formula has low surface tension and keratin binding properties that increase penetrance through the nail plate. Safety studies have shown that this formulation is not associated with atopic dermatitis or contact sensitivity. Duplicate Phase III clinical trials in adults with mild to moderate distal and lateral subungual onychomycosis indicate that efinaconazole 10% solution is an effective therapy with a pooled complete cure rate of 17% and a pooled mycological cure rate of 54%. Efinaconazole 10% nail solution is a safe and effective new topical therapy for onychomycosis, which will fill a pressing need for more effective topical therapy in this disease.

Dialkylimidazole inhibitors of Trypanosoma cruzi sterol 14α-demethylase as anti-Chagas disease agents.

New dialkylimidazole based sterol 14α-demethylase inhibitors were prepared and tested as potential anti-Trypanosoma cruzi agents. Previous studies had identified compound 2 as the most potent and selective inhibitor against parasite cultures. In addition, animal studies had demonstrated that compound 2 is highly efficacious in the acute model of the disease. However, compound 2 has a high molecular weight and high hydrophobicity, issues addressed here. Systematic modifications were carried out at four positions on the scaffold and several inhibitors were identified which are highly potent (EC50 <1 nM) against T. cruzi in culture. The halogenated derivatives 36j, 36k, and 36p, display excellent activity against T. cruzi amastigotes, with reduced molecular weight and lipophilicity, and exhibit suitable physicochemical properties for an oral drug candidate.

Drug-drug interaction between voriconazole and oral hypoglycemic agents in diabetic rats.

OBJECTIVE: The objective was to study the of drug-drug interaction between voriconazole and oral hypoglycemic agents in normal and alloxan induced diabetic rats. MATERIALS AND METHODS: The study was designed in two phases. In the first phase, influence of glibenclamide (0.45 mg/kg, p.o.) and pioglitazone (2.7 mg/kg, p.o. once daily) on blood glucose levels in normoglycemic rats was studied and then influence of voriconazole (18 mg/kg, p.o. twice daily.) pre-treatment on the hypoglycemic activity studied. Simultaneously the influence of voriconazole treatment for seven consecutive days (per se effect) on blood glucose levels was also studied in normoglycemic rats. In the second phase of the study alloxan-induced diabetic rats were used to find out the influence of voriconazole pre-treatment on glibenclamide and pioglitazone induced hypoglycemic effect in pathophysiological condition. Blood samples were collected from retro orbital plexus at regular intervals of 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0, 18.0 and 24.0 h after drug treatment. All the blood samples were analyzed for plasma glucose by glucose oxidase peroxidase method (GOD/POD). RESULTS: The therapeutic dose of voriconazole potentiates the hypoglycemic activity of glibenclamide and pioglitazone both in normoglycemic and diabetic rats respectively. CONCLUSION: The results indicate that the dose of oral hypoglycemic agents needs to be adjusted if co-administered with voriconazole.

Androgen synthesis in the gonadotropin-suppressed human testes can be markedly suppressed by ketoconazole.

CONTEXT: The concentration of intratesticular testosterone (IT-T) required for human spermatogenesis is unknown because spermatogenesis can persist despite the markedly reduced IT-T concentrations observed with LH suppression. Methods to lower IT-T further are needed to determine the relationship between IT-T and spermatogenesis. OBJECTIVE: The objective of the study was to determine the effect of inhibiting the synthesis and metabolism of testosterone (T) on IT-T in gonadotropin-suppressed human testes. DESIGN/SETTING/PATIENTS: Forty normal men participated in a blinded, placebo-controlled, randomized trial at an academic center. INTERVENTION/OUTCOME MEASURES: All men were first administered the GnRH antagonist acyline to suppress LH. Forty-eight hours after acyline administration, subjects were randomly assigned to placebo, ketoconazole (to inhibit T synthesis) at 400 or 800 mg, dutasteride (to inhibit T metabolism) 2.5 mg, or anastrazole (to inhibit T metabolism) 1 mg, daily for 7 days (n = 8/group). Intratesticular steroid concentrations were measured 48 hours after acyline administration alone and again after 7 days of combination treatment. RESULTS: After 7 days of combination treatment, the median IT-T (25th, 75th percentile) in the placebo group was 14 (8.0, 21.2) ng/mL. IT-T was reduced to 3.7 (2.5, 7.1) ng/mL in the ketoconazole 400 mg group and 1.7 (0.8, 4.0) ng/mL in the ketoconazole 800 mg group (P < .001 vs placebo for both comparisons). IT-T concentrations in the dutasteride and anastrazole groups were similar to placebo. CONCLUSION: Combining inhibition of steroidogenesis with gonadotropin suppression lowers IT-T more than gonadotropin suppression alone. This combination might be useful to determine the minimum IT-T concentration necessary for human spermatogenesis, information essential for developing male hormonal contraceptives.

Voriconazole in clinical practice.

Invasive fungal diseases are associated with significant morbidity and mortality in immunocompromized patients. Voriconazole is the first line treatment of invasive aspergillosis, and has been successfully used in other invasive fungal infections, such as candidiasis, fusariosis or scedosporidiosis. Voriconazole has non-linear pharmacokinetics and undergoes extensive hepatic metabolism by the cytochrome P450 system that depends on age, genetic factors, and interactions with other drugs. Thus, significant interpatient variability is observed after administration of the same dose. Additionally, the therapeutic window is narrow, with high risk of side effects at serum levels 3-5 times higher than the minimal threshold for efficacy. Therefore, the knowledge of pharmacological properties, metabolism, interactions, dosage indications in various populations and side effects is crucial. Therapeutic drug monitoring can help maximize the efficacy and minimize the risk of toxicity. Pharmacological, mycological and clinical aspects of the treatment with voriconazole are summarized in order to optimize its use in daily clinical practice.

Pharmacological characterization, structural studies, and in vivo activities of anti-Chagas disease lead compounds derived from tipifarnib.

Chagas disease, caused by the protozoan pathogen Trypanosoma cruzi, remains a challenging infection due to the unavailability of safe and efficacious drugs. Inhibitors of the trypanosome sterol 14α-demethylase enzyme (CYP51), including azole antifungal drugs, are promising candidates for development as anti-Chagas disease drugs. Posaconazole is under clinical investigation for Chagas disease, although the high cost of this drug may limit its widespread use. We have previously reported that the human protein farnesyltransferase (PFT) inhibitor tipifarnib has potent anti-T. cruzi activity by inhibiting the CYP51 enzyme. Furthermore, we have developed analogs that minimize the PFT-inhibitory activity and enhance the CYP51 inhibition. In this paper, we describe the efficacy of the lead tipifarnib analog compared to that of posaconazole in a murine model of T. cruzi infection. The plasma exposure profiles for each compound following a single oral dose in mice and estimated exposure parameters after repeated twice-daily dosing for 20 days are also presented. The lead tipifarnib analog had potent suppressive activity on parasitemia in mice but was unsuccessful at curing mice, whereas posaconazole as well as benznidazole cured 3 of 5 and 4 of 6 mice, respectively. The efficacy results are consistent with posaconazole having substantially higher predicted exposure than that of the tipifarnib analog after repeat twice-daily administration. Further changes to the tipifarnib analogs to reduce plasma clearance are therefore likely to be important. A crystal structure of a trypanosomal CYP51 bound to a tipifarnib analog is reported here and provides new insights to guide structure-based drug design for further optimized compounds.

Epoxyeicosatrienoic acids contribute with altered nitric oxide and endothelin-1 pathways to conduit artery endothelial dysfunction in essential hypertension.

BACKGROUND: We sought to clarify, using functional and biological approaches, the role of epoxyeicosatrienoic acids, nitric oxide (NO)/reactive oxygen species balance, and endothelin-1 in conduit artery endothelial dysfunction during essential hypertension. METHODS AND RESULTS: Radial artery diameter and mean wall shear stress were determined in 28 untreated patients with essential hypertension and 30 normotensive control subjects during endothelium-dependent flow-mediated dilatation induced by hand skin heating. The role of epoxyeicosatrienoic acids and NO was assessed with the brachial infusion of inhibitors of cytochrome P450 epoxygenases (fluconazole) and NO synthase (N(G)-monomethyl-l-arginine [L-NMMA]). Compared with controls, hypertensive patients exhibited a decreased flow-mediated dilatation in response to postischemic hyperemia as well as to heating, as shown by the lesser slope of their diameter-shear stress relationship. In controls, heating-induced flow-mediated dilatation was reduced by fluconazole, L-NMMA, and, to a larger extent, by L-NMMA+fluconazole. In patients, flow-mediated dilatation was not affected by fluconazole and was reduced by L-NMMA and L-NMMA+fluconazole to a lesser extent than in controls. Furthermore, local plasma epoxyeicosatrienoic acids increased during heating in controls (an effect diminished by fluconazole) but not in patients. Plasma nitrite, an indicator of NO availability, increased during heating in controls (an effect abolished by L-NMMA) and, to a lesser extent, in patients, whereas, inversely, reactive oxygen species increased more in patients (an effect diminished by L-NMMA). Plasma endothelin-1 decreased during heating in controls but not in patients. CONCLUSIONS: These results show that an impaired role of epoxyeicosatrienoic acids contributes, together with an alteration in NO/reactive oxygen species balance and endothelin-1 pathway, to conduit artery endothelial dysfunction in essential hypertension. CLINICAL TRIAL REGISTRATION: https://www.eudract.ema.europa.eu. Unique identifier: RCB2007-A001-10-53.