Pharmaceutical Nanotechnology
Docetaxel-loaded-lipid-based-nanosuspensions (DTX-LNS): Preparation, pharmacokinetics, tissue distribution and antitumor activity

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Abstract

The purpose of the study was to design lipid-based-nanosuspensions (LNS) for Docetaxel (DTX) without Tween 80 for clinical intravenous administration (i.v.). DTX-LNS were prepared by high pressure homogenization method, and then lyophilization was carried out to improve the stability. The physical–chemical properties in terms of particle size, size distribution, zeta potential and morphology were evaluated, respectively. The in vitro cytotoxic activity was assessed by MTT against SKOV-3 and malignant melanoma B16 cells. The in vivo pharmacokinetics, tissue distribution as well as antitumor efficacy were investigated in B16 melanoma-bearing Kunming mice. The particle size and zeta potential of DTX-LNS were (200.0 ± 3.42) nm and (−11.15 ± 0.99) mV, respectively. Compared with Duopafei®, it was shown that DTX-LNS exhibited higher antitumor efficacy by reducing tumor volume (P < 0.05) and increasing survival rate in B16 melanoma-bearing mice and strongly reduced the anticancer drug toxicity. The results of biodistribution studies clearly indicated the superiority of DTX-LNS to Duopafei® in increasing the accumulation of DTX within tumor and the organs rich in macrophages (liver, lungs and spleen), while, the drug concentration in heart and kidney decreased. Together these results suggested that DTX-LNS could effectively inhibit tumor growth, reduce toxicity during the therapeutic procedure and hold the potential to be an appropriate choice for the clinical administration of DTX.

Introduction

During the past decades, significant number of drug candidates were identified in drug discovery programs, but most of them (40–70%) are quite often poorly soluble. This challenges drug delivery institutions in industry or academia to develop innovative approaches to reach a high bioavailability after oral administration or make intravenously injectable forms available. Among these innovative formulations, lipid-based nanocarriers are an important class of carriers.

DTX, which belongs to the taxoid family, is widely used in the treatment of ovarian cancer (Kaye et al., 1997), breast cancer (Campora et al., 2008), non-small cell lung cancer (Fossella et al., 1995) and other tumors (Clarke and Rivory, 1999). However, the clinical application of DTX is limited by the poor aqueous solubility, low bioavailability and high toxicity. Presently used Taxotere® and Duopafei® in clinical contain high concentration of nonionic surfactant Tween 80. Adverse reactions due to either the drug itself or the solvent system have been reported in patients (e.g., hypersensitivity, fluid retention, neurotoxicity, musculoskeletal toxicity and neutropenia) (Chu et al., 2000). In order to eliminate the Tween 80-based vehicle and increase the drug solubility, alternative dosage forms have been developed, such as microparticulate lipoidal vesicles (liposomes) (Naik et al., 2010, Zhai et al., 2010), cyclodextrins (Grosse et al., 1998), polymeric nanoparticles (Hwang et al., 2008), micelles (Li et al., 2008), solid lipid nanoparticles (SLN) (Xu et al., 2009) and nanostructured lipid carriers (NLC) (Li et al., 2009). Among these forms, liposomes, NLC and SLN belong to lipid-based nanocarriers which have such favorable characteristics as: (a) improved drug dispersibility; (b) enhanced drug solubilization; (c) enhanced drug transmembrane transport capability and (d) increased therapeutic efficacy and reduced toxicity. In the present study, as an innovative lipid-based nanocarrier, lipid-based-nanosuspensions (LNS) have been developed. Using single injectable phospholipids as the stabilizer, LNS hold the advantages of lipid-based nanocarriers, while avoiding their shortcomings. For example, (1) LNS have no drug leakage problems, which were considered to be the common disadvantages of SLN (Müller et al., 2000, Muller et al., 2002); (2) carry adequate amounts of drug and without excessive loading of the organism with foreign material; (3) formulate compounds that are insoluble in both water and oil (Kocbek et al., 2006). Besides, (4) the drug loading of LNS is high and the administration volume is significantly reduced (Rabinow, 2004); (5) LNS is appropriate for large-scale production.

Recently, several techniques such as precipitation methods (Sjostrom et al., 1993, Trotta et al., 2001), milling methods (Ain-Ai and Gupta, 2008) and homogenization methods (Keck and Muller, 2006) were developed to produce drug nanosuspensions. Among these methods, high pressure homogenization is the simplest one and has been successfully employed in large-scale production.

Above all, a new concept of lipid-based nanocarrier: lipid-based-nanosuspensions (LNS) were proposed. The lipid-based nanocarriers used DTX as a model drug and were produced by high pressure homogenization method. LNS were total avoidance of organic solvents during the production process, could be successfully employed for large-scale production and conveniently applied in clinical.

It has been previously reported that several DTX delivery systems such as SLN and DTX-loaded PEGylated-NPs had achieved the satisfied antitumor effect in liver cancer and colon cancer (Senthilkumar et al., 2008, Xu et al., 2009). However, the antitumor efficacy evaluation of DTX loaded nanosuspensions against malignant melanoma was rarely reported. In our study, DTX loaded LNS was first developed for murine malignant melanoma treatment. High pressure homogenization was used to the LNS preparation. The morphology, particle size and zeta potential were characterized. In vitro drug release was assessed using the dialysis bag diffusion technique. In vitro cytotoxicity of Duopafei® and LNS were performed using SKOV-3 and B16 cells. Finally, in vivo antitumor efficacy and the pharmacokinetics as well as the drug tissue distribution was evaluated in Kunming mice bearing B16 cells. DTX loaded LNS could avoid the serious hypersensitivity reactions caused by Tween 80 and be stable, safe and convenient for clinical administration.

Section snippets

Materials

Injectable soya lecithin (phosphatidylcholine accounts for 95%, pH 5.0–7.0) was provided by Shanghai Taiwan Pharmaceutical Co., Ltd. (Shanghai, China). Duopafei® was provided by Qilu Pharmaceutical Co., Ltd. (Jinan, China). All reagents for HPLC analysis, including acetonitrile and methanol were of HPLC grade. All the other chemicals and reagents used were of analytical purity grade or higher, obtained commercially.

Human ovary cancer cells (SKOV-3) and mouse malignant melanoma (B16) cell line

Characterization of DTX-LNS

The DTX-LNS were homogenized applying 5, 10, 15, 20, 25 homogenization cycles at 100 MPa, respectively, and the particle diameter and polydispersity index (PI) were analyzed. The mean particle size of the suspensions decreased with the cycle number increased. There was no significant difference in the diameters of DTX-LNS between 20 cycles and 25 cycles (P > 0.05), while, PI of 20 cycles was smaller than that of 25 cycles (P < 0.05). It was indicated that both the diameter and PI were closely

Conclusion

In the present study, DTX-LNS were successfully prepared by high pressure homogenization. Compared with Duopafei®, DTX-LNS showed higher antitumor efficacy, increased survival rate in B16 cells bearing mice and strongly reduced anticancer drug toxicity. So we have applied for patents to protect our achievement (Wang et al., 2010).

Above all, LNS were total avoidance of organic solvents during the production process, could be successfully employed for large-scale production and conveniently

Acknowledgement

The authors are grateful to R & D projects in key areas of Jining City for providing financial assistance to carry out this work.

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