A novel method for predicting the efficacy of topical drugs on onychomycosis: A comparison of efinaconazole and luliconazole.

To be effective against onychomycosis, topically applied drugs have to reach the infection site at an effective concentration to exert antifungal activity against the parasitic form of dermatophytes. We established a novel in vitro method for predicting drug efficacy at the infection site and verified the method by comparing the efficacy of two azole class topical anti-onychomycosis drugs. To predict drug efficacy in the nail plate, a human nail permeability test was conducted and the activities of the free-drugs in the upper, middle, and lowest layers of the nail plate were determined by measuring the growth inhibitory zone. Efinaconazole permeated the nail more efficiently than luliconazole, and the amount of efinaconazole in the middle and lowest layers was higher compared with that of luliconazole. Efinaconazole demonstrated antifungal activities at the concentrations in all of the nail layers, whereas luliconazole was only active at the concentrations in the upper and middle layers. The results could be explained by differences in their affinity for keratin and nail permeability. The established method enables the evaluation of nail permeability and anti-arthrospore activity of free-drugs in the nail plate to predict drug efficacy. This method will be useful for new topical drug development.

Acetaminophen induces accumulation of functional rat CYP3A via polyubiquitination dysfunction.

Acetaminophen (APAP) is extensively used as an analgesic and antipyretic drug. APAP is partly metabolized to N-acetyl-p-benzoquinone imine, a reactive metabolite, by cytochrome P450 (CYP) 1A2, 2E1 and 3A4. Some reports have indicated that CYP3A protein production and its metabolic activity are induced by APAP in rats in vivo. The CYP3A subfamily is believed to be transcriptionally regulated by chemical compounds. However, the mechanism underlying these responses is not completely understood. To clarify these mechanisms, we assessed the effects of APAP on CYP3A1/23 protein levels according to mRNA synthesis and protein degradation in rat hepatocyte spheroids, a model of liver tissue, in vivo. APAP induced CYP3A1/23 protein levels and metabolic activity. However, no change in CYP3A1/23 mRNA levels was observed. Moreover, APAP prolonged the half-life of CYP3A1/23 protein. CYP3A is known to be degraded via the ubiquitin-proteasome system. APAP significantly was found to decrease levels of polyubiquitinated CYP3A1/23 and glycoprotein 78, an E3 ligase of CYP3A1/23. These findings demonstrate that APAP induces accumulation of functional CYP3A protein via inhibition of protein degradation. Our findings may lead to the determination of novel drug-drug interactions with APAP.

Detection of metabolic activation leading to drug-induced phospholipidosis in rat hepatocyte spheroids.

Drug-induced phospholipidosis (PLD) is one of the adverse reactions to treatment with cationic amphiphilic drugs. Recently, simple and reliable evaluation methods for PLD have been reported. However, the predictive power of these methods for in vivo PLD induction is insufficient in some cases. To accurately predict PLD, we focused on drug metabolism and used three-dimensional cultures of hepatocytes known as spheroids. Here we used the fluorescent phospholipid dye N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) to detect PLD induction. After 48 hr exposure to 20 µM amiodarone and amitriptyline, PLD inducers, NBD-PE fluorescence in the spheroids was significantly higher than that in the control. In contrast, 1 mM acetaminophen, as a negative control, did not increase fluorescence. Furthermore, the combination of NBD-PE fluorescence and LysoTracker Red fluorescence and the accumulation of intrinsic phospholipids reflected PLD induction in spheroids. To evaluate metabolic activation, we assessed PLD induction by loratadine. NBD-PE fluorescence intensity was significantly increased by 50 µM loratadine treatment. However, the fluorescence was markedly decreased by co-treatment with 500 µM 1-aminobenzotriazole, a broad cytochrome P450 inhibitor. The formation of desloratadine, a metabolite of loratadine, was observed in spheroids after treatment with loratadine alone. These results showed that metabolic activation is the key factor in PLD induction by treatment with loratadine. We demonstrated that rat primary hepatocyte spheroid culture is a useful model for evaluating drug-induced PLD induction mediated by metabolic activation of the drug using the fluorescence probe technique.

Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates.

Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10mM, 24h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.

Fractionated stereotactic radiotherapy using gamma unit after hyperbaric oxygenation on recurrent high-grade gliomas.

BACKGROUND: To reduce this complication and to enhance the radiation effect to hypoxic cells of high-grade gliomas, the authors performed noninvasive fractionated stereotactic radiotherapy (FSRT) using a Gamma unit combined with hyperbaric oxygen (HBO) therapy for the treatment of recurrent disease. PATIENTS AND METHODS: Twenty-five consecutive patients who had previously received radiotherapy with chemotherapy for recurrent high-grade gliomas, including 14 patients with anaplastic astrocytoma (AA) and 11 with glioblastoma multiforme (GBM), underwent Gamma FSRT immediately after HBO therapy (2.5 atmospheres absolute for 60 min). The Gamma FSRT was repeatedly performed using a relocatable head cast. Median tumor volume was 8.7 cc (range, 1.7-159.3 cc), and the median total radiation dose was 22 Gy (range, 18-27 Gy) to the tumor margin in 8 fractions. RESULTS: Actuarial median survival time after FSRT was 19 months for patients with AA and 11 months for patients with GBM, which was significantly different (P = 0.012, log-rank test). Two patients underwent subsequent second FSRT for regional or remote recurrence. Seven patients (28%) underwent subsequent craniotomies and resections at a mean of 8.4 months after FSRT treatment, and 4 of them had radiation effects without viable cells and remained alive for 50-78 months. CONCLUSION: Gamma FSRT after HBO therapy appears to confer a survival benefit for patients with recurrent high-grade gliomas and warrants further investigation.

Stereotactic radiation therapy with chemotherapy in the management of recurrent medulloblastomas.

Medulloblastomas are highly lethal tumors when they recur. Very few patients survive with conventional treatment. This report documents the preliminary study results of a treatment for recurrent medulloblastomas consisting of stereotactic radiation therapy (SRT) with chemotherapy. Four patients had local recurrence without apparent metastases and 8 patients had metastases with or without local recurrence. Twelve patients with 18 lesions underwent SRT as a single session (n=8) or in a hypofractionated manner (n=10) using a gamma knife or modified linear accelerator. All patients then received systemic chemotherapy. Five patients were treated with one to two sequential courses of high-dose chemotherapy with peripheral blood stem cell transplantation. The reduction in tumor size after SRT was often remarkable. Fourteen of 18 lesions treated disappeared 1-6 months after SRT. Two of 4 patients who had local recurrences without apparent metastasis at the time of SRT are alive without evidence of disease 70 and 72 months after SRT, respectively. In contrast, all 8 patients with metastasis had new lesions either in the spinal canal or on the surface of the brain outside the target area of SRT. Median progression-free survival and overall survival from the time of SRT were 9 and 19 months, respectively. The Kaplan-Meier estimates of PFS and overall survival at 3 years were 17 and 25%, respectively. One patient had brainstem edema after SRT causing bulbar palsy and quadriparesis. One patient died of toxicity of chemotherapy. Our experience suggests that local recurrence can be controlled by SRT with chemotherapy but survival of patients with metastases can not be improved effectively by SRT in conjunction with aggressive chemotherapy.

Neuroendoscopic transventricular surgery for suprasellar cystic mass lesions such as cystic craniopharyngioma and Rathke cleft cyst.

Cystic mass lesions in the suprasellar cistern are often associated with neurological deficits, cognitive disorders, and endocrinological impairments. Many surgical approaches are available to treat these mass lesions, but are technically difficult and cannot remove the lesion completely without risking damage to neurological and endocrinological functions due to the proximity to the surrounding structures. Neuroendoscopic transventricular surgery was performed using a ventricular fiberscope for three patients with craniopharyngiomas and two patients with Rathke cleft cysts, with gamma knife radiosurgery for craniopharyngiomas. The endoscopic transventricular approach is safe and minimally invasive for congenital benign suprasellar cystic lesions, especially arachnoid cysts.