Pharmacokinetic comparison of intravenous carbendazim and remote loaded carbendazim liposomes in nude mice

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Abstract

Carbendazim is a novel anticancer agent. The aim of this study was to prepare and characterize a remote loaded liposome preparation of carbendazim, and compare its pharmacokinetic profile to that of unencapsulated carbendazim. Carbendazim was encapsulated in liposomes composed of sphingomyelin–cholesterol (3:1, w/w) by remote loading in response to a transmembrane pH gradient (pH 0.5 in/pH 4.0 out), which resulted in encapsulation of more than 95% of the available drug in preformed vesicles. High drug/lipid ratios were prepared which correspond to a molar ratio of up to 0.8. Physical isolation of the free drug and dialysis were used to determine the in vitro release of carbendazim from liposomes. The release was independent of the initial drug/lipid ratio and choice of internal buffer composition. Liposomal carbendazim (200 mg kg−1) was intravenously administered to athymic nude mice and the serum levels of free carbendazim were determined by HPLC analysis after a methanol-induced protein precipitation. Administration of liposomal carbendazim to mice resulted in significant alterations in the pharmacokinetics. Serum levels of free carbendazim were approximately 10-fold greater than those achieved for the same dose of unencapsulated drug. Liposomal carbendazim showed both high Cmax, AUC and low clearance rate. Liposomal carbendazim and unencapsulated carbendazim displayed a similar terminal half-life (43–48 min). The relatively large volume of distribution of carbendazim suggests that the compound may partially enter cells or be bound to some extravascular structures. The results indicate that the liposomal formulation of carbendazim significantly increases its blood concentrations.

Introduction

Carbendazim, classified as a representative benzimidazolic compound, was initially found to be a bioactive metabolite of the fungicide benomyl, and is widely used in crop protection as a fungicide to protect crops from decay caused by various fungal pathogens [1]. It is also used as a pesticide and herbicide for the protection of flowers and flower bulbs. Recently it has been found that subchronic administration of carbendazim induced testicular alterations, spermatogenic inactivity and embryotoxicity [2], [3], [4]. The molecular basis of reproductive toxicity appears to be related to the inhibition of microtubule assembly [5], [6], [7]. While the exact mechanism is unclear, recent findings further revealed that carbendazim induced apoptosis in a variety of cells. The apoptosis phenomena of the compound led us to our most recent findings that carbendazim inhibits both in vitro tumor cell growth and in vivo human tumor xenograft models [8]. Consequently, this agent is presently undergoing Phase 1 clinical trials in adults with advanced malignancies. However, the drug has limited aqueous solubility and low bioavailability due to the first-pass effect of liver metabolism. It has been reported that more than 95% of carbendazim was eliminated during the early phase of i.v. administration [9]. Even though carbendazim continues to be important to achieve an effective chemotherapy for the treatment of a variety of tumor models, its formulation has not been completely satisfactory. The purpose of this study was to develop a liposome formulation that has the following characteristics: (a) a high level potency of carbendazim; (b) a high encapsulation efficiency; and (c) small particle diameter (less than 0.2 μm) that can be filter-sterilized. This report describes an approach to encapsulate carbendazim into unilamellar vesicles based upon a remote loading procedure. The influence of liposome encapsulation on the pharmacokinetic properties was evaluated in a murine model in order to provide inference as to the anticipated pharmacokinetics of the formulation in humans.

Section snippets

Materials

Carbendazim (F.W. 191.18) and the internal standard 2-benzimidazolyl-acetonitrile (Fig. 1) were purchased from Aldrich (Milwaukee, WI, USA). Acetonitrile, methanol and KH2PO4 were purchased from EM Science (Gibbstown, NJ, USA). N,N-dimethylformamide solution was bought from Allied Signal (Muskegon, MI, USA).

Stock solution of carbendazim (1 mg ml−1) was prepared in N,N-dimethylformamide. Stock solution of internal standard (2 mg ml−1) was prepared in acetonitrile (HPLC grade). The stock

Liposome characterization

The formulation appears opaque yellow liquid. The vesicle size determined by light scattering was 148±57 nm. The level of carbendazim encapsulation was related to the interior/external pH gradient. For example, at a fixed drug/lipid molar ratio of 0.8, more than 96% of carbendazim is encapsulated using 300 mM H2SO4 (pH 0.5), whereas only 84% encapsulation was achieved at 50 mM H2SO4 (internal pH 1.33). The external pH was raised to 4.0 to achieve an initial transmembrane pH gradient about 3.5

Discussion

The developed HPLC method proves to be useful and reliable for the determination of serum concentrations of carbendazim. The sample clean-up procedure, involving a direct deproteinization with methanol, is simple and rapid, thus avoiding degradation of the drug. This method, validated for carbendazim concentrations in serum ranging from 20 to 20,000 ng ml−1, has a good reproducibility and accuracy and low limits of quantitation and detection compared to the most published methods detecting the

Conclusions

A unilamellar liposome formulation (sphingomyelin–cholesterol, 3:1, w/w) of carbendazim could be prepared by remote loading at drug/lipid molar ratios of 0.2 and 0.8. An initial transmembrane pH gradient (pH 0.5 in/pH 4.0 out) across the vesicles increased the encapsulation efficiency. A reversed-phase HPLC method was developed and validated to detect serum carbendazim concentration as low as 20 ng ml−1. Liposomal carbendazim significantly improved the pharmacokinetic profile of the drug

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