Preclinical Testing of a Novel Niclosamide Stearate Prodrug Therapeutic (NSPT) Shows Efficacy Against Osteosarcoma.

Therapeutic advances for osteosarcoma have stagnated over the past several decades, leading to an unmet clinical need for patients. The purpose of this study was to develop a novel therapy for osteosarcoma by reformulating and validating niclosamide, an established anthelminthic agent, as a niclosamide stearate prodrug therapeutic (NSPT). We sought to improve the low and inefficient clinical bioavailability of oral dosing, especially for the relatively hydrophobic classes of anticancer drugs. Nanoparticles were fabricated by rapid solvent shifting and verified using dynamic light scattering and UV-vis spectrophotometry. NSPT efficacy was then studied in vitro for cell viability, cell proliferation, and intracellular signaling by Western blot analysis; ex vivo pulmonary metastatic assay model; and in vivo pharmacokinetic and lung mouse metastatic model of osteosarcoma. NSPT formulation stabilizes niclosamide stearate against hydrolysis and delays enzymolysis; increases circulation in vivo with t 1/2 approximately 5 hours; reduces cell viability and cell proliferation in human and canine osteosarcoma cells in vitro at 0.2-2 µmol/L IC50; inhibits recognized growth pathways and induces apoptosis at 20 µmol/L; eliminates metastatic lesions in the ex vivo lung metastatic model; and when injected intravenously at 50 mg/kg weekly, it prevents metastatic spread in the lungs in a mouse model of osteosarcoma over 30 days. In conclusion, niclosamide was optimized for preclinical drug delivery as a unique prodrug nanoparticle injected intravenously at 50 mg/kg (1.9 mmol/L). This increased bioavailability of niclosamide in the blood stream prevented metastatic disease in the mouse. This chemotherapeutic strategy is now ready for canine trials, and if successful, will be targeted for human trials in patients with osteosarcoma.

Preclinical toxicity and pharmacokinetics of a new orally bioavailable flubendazole formulation and the impact for clinical trials and risk/benefit to patients.

BACKGROUND: Flubendazole, originally developed to treat infections with intestinal nematodes, has been shown to be efficacious in animal models of filarial infections. For treatment of filarial nematodes, systemic exposure is needed. For this purpose, an orally bioavailable amorphous solid dispersion (ASD) formulation of flubendazole was developed. As this formulation results in improved systemic absorption, the pharmacokinetic and toxicological profile of the flubendazole ASD formulation have been assessed to ensure human safety before clinical trials could be initiated. METHODS & FINDINGS: Safety pharmacology, toxicity and genotoxicity studies have been conducted with the flubendazole ASD formulation. In animals, flubendazole has good oral bioavailability from an ASD formulation ranging from 15% in dogs, 27% in rats to more than 100% in jirds. In in vivo toxicity studies with the ASD formulation, high systemic exposure to flubendazole and its main metabolites was reached. Flubendazole, up to high peak plasma concentrations, does not induce Cmax related effects in CNS or cardiovascular system. In repeated dose toxicity studies in rats and dogs, flubendazole-induced changes were observed in haematological, lymphoid and gastrointestinal systems and in testes. In dogs, the liver was an additional target organ. Upon treatment cessation, at least partial recovery was observed for these changes in dogs. In rats, the No Observed Adverse Effect Level (NOAEL) was 5 mg (as base)/kg body weight/day (mg eq./kg/day) in males and 2.5 mg eq./kg/day in females. In dogs, the NOAEL was lower than 20 mg eq./kg/day. Regarding genotoxicity, flubendazole was negative in the Ames test, but positive in the in vivo micronucleus test. CONCLUSIONS: Based on these results, in combination with previously described genotoxicity and reproductive toxicity data and the outcome of the preclinical efficacy studies, it was concluded that no flubendazole treatment regimen can be selected that would provide efficacy in humans at safe exposure.

Induction of Flavin-Containing Monooxygenase in Mice by Oral Administration of Phellinus baumii (Agaricomycetes) Extract.

Phellinus baumii is a yellow mushroom long used in alternative medicine in Korea and other central Asian countries. To identify genes affected by a single or 7-day oral administration of a water extract of Ph. Baumii, mouse liver tissue was analyzed using microarrays. The results showed that 8 and 23 genes were upregulated and 3 and 11 genes downregulated more than 3-fold by single and multiple oral administrations of 100 mg/kg PBE, respectively. Among the upregulated genes, the expression of 3 flavin-containing monooxygenase (Fmo) family genes, Fmo2-4, was upregulated in a concentration-dependent manner. The microarray analysis also showed that single and multiple administrations of PBE increased Fmo3 expression in the mouse liver by 5.1- and 17.6-fold, respectively. To validate the Fmo expression microarray data, polymerase chain reaction was used to confirm the induction of Fmo subclass genes. Mice were orally administered Ph. Baumii extract (PBE), Ph. Baumii water, or Ph. Baumii ß-glucan fraction (PBG) for 7 days, and induction of the expression of the Fmo subclasses in the liver, lung, and kidney was investigated. Fmo2, Fmo3, and Fmo4 expression was induced by both PBE and PBG in the lung, liver, and kidney, respectively. However, no induction of Fmo1 and Fmo5 was detected. To investigate the metabolic acceleration of xenobiotic by PBE, carbendazim was orally administered to mice and its clearance from the blood analyzed. High-performance liquid chromatography analysis showed accelerated clearance of serum carbendazim by oral administration of PBE for 7 days, as evidenced by the reduced peak plasma concentration, time to reach the peak plasma concentration, and area under the curve values. Moreover, PBE increased the carbendazim clearance rate at the higher concentration. These data indicate that oral administration of PBE resulted in modulation of gene expression: PBE was responsible for the induction of Fmo2, Fmo3, and Fmo4 expression. PBE also accelerated the metabolic clearance of carbendazim in vivo and so could be applied to the detoxification of xenobiotics such as drugs, pesticides, and nicotine.

Efeito in vitro do extrato etanólico das folhas do melão-de-São-Caetano (Momordica charantia L. ) sobre ovos e larvas de nematóides gastrintestinais de caprinos / In vitro effect of the ethanolic extract of "melão de São Caetano" (Momordica charantia L. ) leaves on the eggs and larvae of gastrointestinal nematodes from goats

O experimento in vitro foi realizado para avaliar a ação do extrato etanólico das folhas do melão-de-São-Caetano (Momordica charantia L.) sobre o desenvolvimento de ovos e motilidade de larvas de nematóides gastrintestinais de caprinos. As larvas foram obtidas de coproculturas e a recuperação de ovos foi feita pela técnica dos quatro tamises, a partir de fezes de caprinos naturalmente infectados da mesorregião do Sertão Paraibano. O extrato foi utilizado nas diluições de 50; 25; 12,5; 6,25 e 3,12 por cento para ambos os testes e como controle positivo e para controle negativo, utilizou-se água destilada estéril. As placas foram examinadas ao microscópio óptico para contagem dos ovos em desenvolvimento e larvas móveis e imóveis, após 24, 48 e 72 horas de incubação. As concentrações do extrato etanólico de M. charantia e os tratamentos controle negativo e positivo diferiram quanto ao número de ovos inviáveis. No teste de motilidade larval as concentrações acima de 12 por cento apresentaram médias significativas quanto ao número de larvas inviáveis. Nas condições ensaiadas a M. charantia apresentou atividade ovicida e larvicida.

The experiment in vitro was performed to evaluate the action of the ethanolic extract of "melão de São Caetano" (Momordica charantia L.) leaves on the development of eggs and motility of larvae of gastrointestinal nematodes from goats. The nematode larvae were obtained from coproculture and the recovery of eggs was done in sieves, from feces of naturally infected goats from the Mesoregion of Paraíba State. The extract was used at the dilutions of 50, 25, 12.5, 6.25 and 3.12 percent for both tests and as positive control; for negative control, sterile distilled water was used. The plates were examined under optical microscope to count the eggs in development and mobile larvae after 24, 48 and 72 h of incubation. The concentrations of M. charantia ethanolic extract and the negative and positive controls differed as to the number of eggs that were not viable. In the larval motility test, concentrations higher than 12 percent had significant means as to the number of larvae that were not viable. Under the tested conditions, M. charantia showed larvicidal and ovicidal activity.

Toxicity of binary mixtures of cadmium-copper and carbendazim-copper to the nematode Caenorhabditis elegans.

For ecological risk assessment, the additive model may be used to empirically predict toxic mixture effects. Detailed toxicity tests were performed to determine whether effects of mixtures of copper-cadmium and copper-carbendazim on Caenorhabditis elegans were similar to the effects of the individual compounds. Effects on the course of reproduction, the length of the juvenile period, the length of the reproductive period, and body length were analyzed. Dose-response data were compared to the additive model and tested for four deviation patterns from additivity: No deviation, synergistic/antagonistic deviation, dose ratio-dependent deviation, dose level-dependent deviation. During the exposure, the cadmium-copper effect on reproduction changed from a synergistic, to a dose ratio-dependent deviation from additivity. More cadmium in the mixture decreased the toxicity and more copper increased the toxicity. The effect of copper-carbendazim on reproduction was synergistic at low dose levels and antagonistic at high dose levels and independent of time. Mixture effects on the juvenile and reproductive period were similar to single component effects. It was concluded that the observed time-dependence of toxic interactions was small and that interactions on the timing of reproduction were not found. The additive model underestimated mixture effects on reproduction and body length.