A phase II, randomized, double-blind, placebo-controlled, dose-ranging study to evaluate the efficacy and safety of VT-1161 oral tablets in the treatment of patients with distal and lateral subungual onychomycosis of the toenail.

BACKGROUND: Onychomycosis is a fungal disease that affects the fingernails and toenails and is predominantly caused by dermatophytes. VT-1161 is a novel inhibitor of fungal CYP51 through the inhibition of lanosterol demethylase, and has demonstrated potent activity against Trichophyton rubrum and Trichophyton mentagrophytes. OBJECTIVES: To evaluate the safety and efficacy of four dosing regimens of orally administered VT-1161 compared with placebo in patients with moderate-to-severe distal and lateral subungual onychomycosis of the toenail. METHODS: This was a phase II, randomized, double-blind, placebo-controlled, multicentre study (ClinicalTrials.gov identifier NCT02267356). Patients aged 18-70 years (n = 259) who had 25-75% mycotic involvement were randomized to five treatment groups. They received 300 mg VT-1161 as a 2-week daily dose, followed by a once-weekly dose for either 10 or 22 weeks, or 600 mg VT-1161 as a 2-week daily dose, followed by a once-weekly dose for either 10 or 22 weeks. All treatments were followed by a nontreatment period of 36 weeks. A matching placebo arm was included. RESULTS: In the intent-to-treat population, at week 48 the complete cure rates were 0% in the placebo group and ranged from 32% to 42% in the VT-1161 treatment groups (P < 0·001 vs. placebo). VT-1161 was well tolerated, with no evidence of an adverse effect on liver function or QT intervals. CONCLUSIONS: VT-1161 treatment led to high nail clearance rates and a favourable safety profile. VT-1161 exhibits characteristics that appear promising for the treatment of this chronic and difficult-to-treat condition and warrants further evaluation in larger studies.

The novel fungal CYP51 inhibitor VT-1598 displays classic dose-dependent antifungal activity in murine models of invasive aspergillosis.

Aspergillus spp. infections remain a global concern, with ∼30% attributable mortality of invasive aspergillosis (IA). VT-1598 is a novel fungal CYP51 inhibitor designed for exquisite selectivity versus human CYP enzymes to achieve a maximal therapeutic index and therefore maximal antifungal efficacy. Previously, its broad-spectrum in vitro antifungal activity was reported. We report here the pharmacokinetics (PK) and pharmacodynamics (PD) of VT-1598 in neutropenic mouse models of IA. The plasma area-under-the-curve (AUC) of VT-1598 increased nearly linearly between 5 and 40 mg/kg after 5 days of QD administration (155 and 1033 µg*h/ml, respectively), with a further increase with 40 mg/kg BID dosing (1354 µg*h/ml). When A. fumigatus isolates with in vitro susceptibilities of 0.25 and 1.0 µg/ml were used in a disseminated IA model, VT-1598 treatment produced no decrease in kidney fungal burden at QD 10 mg/kg, intermediate decreases at QD 20 mg/kg and maximum or near maximum decreases at 40 mg/kg QD and BID. The PK/PD relationships of AUCfree/MIC for 1-log killing for the two strains were 5.1 and 1.6 h, respectively, similar to values reported for approved CYP51 inhibitors. In a survival study where animals were observed for 12 days after the last treatment, survival was 100% at the doses tested (20 and 40 mg/kg QD), and fungal burden remained suppressed even though drug wash-out was complete. Similar dose-dependent reductions in lung fungal burden were observed in a pulmonary model of IA. These data strongly support further exploration of VT-1598 for the treatment of this lethal mold infection.

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.

Fungal CYP51 Inhibitors VT-1161 and VT-1129 Exhibit Strong In Vitro Activity against Candida glabrata and C. krusei Isolates Clinically Resistant to Azole and Echinocandin Antifungal Compounds.

The in vitro activities of fungal CYP51 inhibitors VT-1161 and VT-1129 were determined for Candida glabrata (n = 34) and C. krusei (n = 50). C. glabrata isolates were screened for FKS gene mutations. All isolates were resistant clinically and/or in vitro to at least one standard antifungal compound. VT-1161 and VT-1129 MICs for all isolates were at least 5-fold below achievable human plasma levels for VT-1161. VT-1161 and VT-1129 are promising for the treatment of resistant C. glabrata and C. krusei infections.

Efficacy of the clinical agent VT-1161 against fluconazole-sensitive and -resistant Candida albicans in a murine model of vaginal candidiasis.

Vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC) remain major health problems for women. VT-1161, a novel fungal CYP51 inhibitor which has potent antifungal activity against fluconazole-sensitive Candida albicans, retained its in vitro potency (MIC50 of ≤0.015 and MIC90 of 0.12 µg/ml) against 10 clinical isolates from VVC or RVVC patients resistant to fluconazole (MIC50 of 8 and MIC90 of 64 µg/ml). VT-1161 pharmacokinetics in mice displayed a high volume of distribution (1.4 liters/kg), high oral absorption (73%), and a long half-life (>48 h) and showed rapid penetration into vaginal tissue. In a murine model of vaginal candidiasis using fluconazole-sensitive yeast, oral doses as low as 4 mg/kg VT-1161 significantly reduced the fungal burden 1 and 4 days posttreatment (P < 0.0001). Similar VT-1161 efficacy was measured when an isolate highly resistant to fluconazole (MIC of 64 µg/ml) but fully sensitive in vitro to VT-1161 was used. When an isolate partially sensitive to VT-1161 (MIC of 0.12 µg/ml) and moderately resistant to fluconazole (MIC of 8 µg/ml) was used, VT-1161 remained efficacious, whereas fluconazole was efficacious on day 1 but did not sustain efficacy 4 days posttreatment. Both agents were inactive in treating an infection with an isolate that demonstrated weaker potency (MICs of 2 and 64 µg/ml for VT-1161 and fluconazole, respectively). Finally, the plasma concentrations of free VT-1161 were predictive of efficacy when in excess of the in vitro MIC values. These data support the clinical development of VT-1161 as a potentially more efficacious treatment for VVC and RVVC.

VT-1161 dosed once daily or once weekly exhibits potent efficacy in treatment of dermatophytosis in a guinea pig model.

Current therapies used to treat dermatophytoses such as onychomycosis are effective but display room for improvement in efficacy, safety, and convenience of dosing. We report here that the investigational agent VT-1161 displays potent in vitro antifungal activity against dermatophytes, with MIC values in the range of ≤0.016 to 0.5 µg/ml. In pharmacokinetic studies supporting testing in a guinea pig model of dermatophytosis, VT-1161 plasma concentrations following single oral doses were dose proportional and persisted at or above the MIC values for at least 48 h, indicating potential in vivo efficacy with once-daily and possibly once-weekly dosing. Subsequently, in a guinea pig dermatophytosis model utilizing Trichophyton mentagrophytes and at oral doses of 5, 10, or 25 mg/kg of body weight once daily or 70 mg/kg once weekly, VT-1161 was statistically superior to untreated controls in fungal burden reduction (P < 0.001) and improvement in clinical scores (P < 0.001). The efficacy profile of VT-1161 was equivalent to those for doses and regimens of itraconazole and terbinafine except that VT-1161 was superior to itraconazole when each drug was dosed once weekly (P < 0.05). VT-1161 was distributed into skin and hair, with plasma and tissue concentrations in all treatment and regimen groups ranging from 0.8 to 40 µg/ml (or µg/g), at or above the MIC against the isolate used in the model (0.5 µg/ml). These data strongly support the clinical development of VT-1161 for the oral treatment of onychomycosis using either once-daily or once-weekly dosing regimens.

The clinical candidate VT-1161 is a highly potent inhibitor of Candida albicans CYP51 but fails to bind the human enzyme.

The binding and cytochrome P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 (ERG11) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, characteristic of heme iron coordination. The binding affinity of VT-1161 for Candida albicans CYP51 was high (dissociation constant [Kd], ≤ 39 nM) and similar to that of the pharmaceutical azole antifungals (Kd, ≤ 50 nM). In stark contrast, VT-1161 at concentrations up to 86 µM did not perturb the spectrum of recombinant human CYP51, whereas all the pharmaceutical azoles bound to human CYP51. In reconstitution assays, VT-1161 inhibited Candida albicans CYP51 activity in a tight-binding fashion with a potency similar to that of the pharmaceutical azoles but failed to inhibit the human enzyme at the highest concentration tested (50 µM). In addition, VT-1161 (MIC = 0.002 µg ml(-1)) had a more pronounced fungal sterol disruption profile (increased levels of methylated sterols and decreased levels of ergosterol) than the known CYP51 inhibitor voriconazole (MIC = 0.004 µg ml(-1)). Furthermore, VT-1161 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. In summary, VT-1161 potently inhibited Candida albicans CYP51 and culture growth but did not inhibit human CYP51, demonstrating a >2,000-fold selectivity. This degree of potency and selectivity strongly supports the potential utility of VT-1161 in the treatment of Candida infections.

Target determination of neurotransmitter phenotype in sympathetic neurons.

While the majority of sympathetic neurons are noradrenergic, a minority population are cholinergic. At least one population of cholinergic sympathetic neurons arises during development by a target-dependent conversion from an initial noradrenergic phenotype. Evidence for retrograde specification has been obtained from transplantation studies in which sympathetic neurons that normally express a noradrenergic phenotype throughout life were induced to innervate sweat glands, a target normally innervated by cholinergic sympathetic neurons. This was accomplished by transplanting footpad skin containing sweat gland primordia from early postnatal donor rats to the hairy skin region of host rats. The sympathetic neurons innervating the novel target decreased their expression of noradrenergic traits and developed choline acetyltransferase (ChAT) activity. In addition, many sweat gland-associated fibers acquired acetylcholinesterase (AChE) staining and VIP immunoreactivity. These studies indicate that sympathetic neurons in vivo alter their neurotransmitter phenotype in response to novel environmental signals and that sweat glands play a critical role in the cholinergic and peptidergic differentiation of the sympathetic neurons that innervate them. The sweat gland-derived cholinergic differentiation factor is distinct from leukemia inhibitory factor and ciliary neurotrophic factor, two well-characterized cytokines that alter the neurotransmitter properties of cultured sympathetic neurons in a similar fashion. Recent studies indicate that anterograde signalling is also important for the establishment of functional synapses in this system. We have found that the production of cholinergic differentiation activity by sweat glands requires sympathetic innervation, and the acquisition and maintenance of secretory competence by sweat glands depends upon functional cholinergic innervation.

Acquisition of cholinergic and peptidergic properties by sympathetic innervation of rat sweat glands requires interaction with normal target.

The sweat glands, a target of cholinergic sympathetic neurons, were replaced with parotid gland, a target of noradrenergic sympathetic neurons, in neonatal rats. This transplantation paradigm allowed sympathetic neurons that would normally innervate the sweat glands and develop a cholinergic phenotype to innervate the parotid gland instead. The innervation of the transplanted parotid gland did not develop a cholinergic phenotype, as assessed by choline acetyltransferase activity and acetylcholinesterase immunoreactivity, but continued to express intense catecholamine fluorescence. In addition, immunoreactivity for vasoactive intestinal peptide, normally expressed by the sympathetic innervation of the sweat glands but not the parotid, was observed in only a small percentage of the parotid-associated fibers. These results suggest that cellular interactions between neurons and their targets play an important role in the differentiation of mature neurotransmitter and neuropeptide phenotypes in vivo.

Stereostructure activity relationships of catecholamines on human platelet function.

The concentration-dependent effects of clonidine, isomers of epinephrine, norepinephrine (NE), isoproterenol, cobefrin and alpha-methyldopamine, and related desoxy analogs (epinine, dopamine, N-isopropyldopamine) were examined on human platelets. The rank order of aggregatory potency (pD2 values) was R(-)-epinephrine (6.3) greater than R(-)-NE (5.9) greater than (+/-)-erythro-cobefrin (5.3) greater than S(+)-epinephrine (4.7) = S(+)-NE (4.7) = clonidine (4.7) = dopamine (4.6) greater than epinine (4.4) greater than S(+)-alpha-methyldopamine (4.3) = R(-)-alpha-methyldopamine (4.3) greater than (+/-)-threo-cobefrin (3.7). The isoproterenol isomers and N-isopropyl-dopamine were inactive as agonists. In 9 of 16 platelet-rich plasma preparations, R(-)-epinephrine, R(-)-NE, and (+/-)erythro-cobefrin were agonists and the remaining analogs blocked R(-)-NE-induced aggregation with a rank order of inhibitory potencies (pKB values) of clonidine (6.2) greater than S(+)-alpha-methyldopamine (5.0) greater than dopamine (4.6) = R(-)-alpha-methyldopamine (4.4) greater than or equal to S(+)-NE (4.3) greater than N-isopropyldopamine (4.1) greater than S(+)-isoproterenol (3.7) = R(-)-isoproterenol (3.5). Each compound was also able to reverse prostaglandin E1 (PGE1) (0.1 microM)-induced blockade of the maximal aggregation response to ADP. At high concentrations, R(-)-isoproterenol was more potent than either the S(+)-isomer or desoxy analog, N-isopropyldopamine, in the reversal of PGE1 inhibition of ADP aggregation. Phentolamine blocked these alpha 2-adrenoceptor-mediated actions against PGE1 on ADP aggregation. The rank order of potency for the reversal of PGE1-mediated inhibition of ADP aggregation by these catecholamines was similar to that observed for platelet aggregation. Our results indicate that (i) the stereochemical requirements for the interaction of catecholamines with platelet alpha 2-adrenoceptors are in agreement with the Easson-Stedman hypothesis and other alpha-adrenoceptor tissues; (ii) catecholamines lacking a benzylic hydroxyl group in the R-configuration and/or possessing an N-isopropyl group were alpha 2-adrenoceptor antagonists; (iii) clonidine gave quantitatively different responses compared with catecholamines for interaction with alpha 2-adrenoceptors; and (iv) inhibition of platelet adenylate cyclase is correlated to the inhibition of epinephrine-induced aggregation response for this series of compounds.

Postnatal development of autonomic and sensory innervation of thoracic hairy skin in the rat. A histochemical, immunocytochemical, and radioenzymatic study.

Histochemical, immunocytochemical, and radioenzymatic techniques were used to examine the neurotransmitter-related properties of the innervation of thoracic hairy skin in rats during adulthood and postnatal development. In the adult, catecholamine-containing fibers were associated with blood vessels and piloerector muscles, and ran in nerve bundles throughout the dermis. The distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers was identical. Neuronal fibers displaying neuropeptide Y (NPY) immunoreactivity were seen in association with blood vessels. Double-labeling studies suggested that most, if not all, NPY-IR fibers were also TH-IR and likewise most, if not all, vessel-associated TH-IR fibers were also NPY-IR. Calcitonin gene-related peptide (CGRP)-IR fibers were observed near and penetrating into the epidermis, in close association with hair follicles and blood vessels, and in nerve bundles. A similar distribution of substance P (SP)-IR fibers was evident. In adult animals treated as neonates with the sympathetic neurotoxin 6-hydroxydopamine, a virtual absence of TH-IR and NPY-IR fibers was observed, whereas the distribution of CGRP-IR and SP-IR fibers appeared unaltered. During postnatal development, a generalized increase in the number, fluorescence intensity, and varicose morphology of neuronal fibers displaying catecholamine fluorescence, NPY-IR, CGRP-IR, and SP-IR was observed. By postnatal day 21, the distribution of the above fibers had reached essentially adult levels, although the density of epidermal-associated CGRP-IR and SP-IR fibers was significantly greater than in the adult. The following were not evident in thoracic hairy skin at any timepoint examined: choline acetyltransferase activity, acetylcholinesterase histochemical staining or immunoreactivity, fibers displaying immunoreactivity to vasoactive intestinal peptide, cholecystokinin, or leucine-enkephalin. The present study demonstrates that the thoracic hairy skin in developing and adult rats receives an abundant sympathetic catecholaminergic and sensory innervation, but not a cholinergic innervation.

Cholinergic phenotype developed by noradrenergic sympathetic neurons after innervation of a novel cholinergic target in vivo.

Mammalian sympathetic neurons in vivo may express either a noradrenergic or cholinergic phenotype. In view of the opposing effect of noradrenaline and acetylcholine on most autonomic target organs, the target-appropriate expression of neurotransmitter is critical. We have examined the maturation of the sympathetic innervation of rat sweat glands to define the developmental mechanisms regulating neurotransmitter choice in vivo. Eccrine sweat glands and their sympathetic innervation develop together postnatally in the rat. Early postnatal innervation expresses only noradrenergic properties, but as the glands and their innervation mature, noradrenergic properties decrease dramatically and cholinergic features appear in the same population of neurons. To investigate the role of the sweat gland in this change we have used a transplantation paradigm which allows sweat glands to be innervated by sympathetic neurons that would normally innervate noradrenergic target organs and remain noradrenergic throughout life. We observe that the sympathetic neurons that innervate the novel cholinergic target alter their neurotransmitter properties and develop a cholinergic phenotype. These results indicate that target organs are able to induce appropriate neurotransmitter traits in the neurons that innervate them.