In vivo efficacy of paclitaxel-loaded injectable in situ-forming gel against subcutaneous tumor growth

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Abstract

Injectable in situ-forming gels have received considerable attention as localized drug delivery systems. Here, we examined a poly(ethylene glycol)–b-polycaprolactone (MPEG–PCL) diblock copolymer gel as an injectable drug depot for paclitaxel (Ptx). The copolymer solution remained liquid at room temperature and rapidly gelled in vivo at body temperature. In vitro experiments showed that Ptx was released from MPEG–PCL copolymer gels over the course of more than 14 days. Experiments employing intratumoral injection of saline (control), gel-only, Taxol, or Ptx-loaded gel into mice bearing B16F10 tumor xenografts showed that Ptx-loaded gel inhibited the growth of B16F10 tumors more effectively than did saline or gel alone. Further, intratumoral injection of Ptx-loaded gel was more efficacious in inhibiting the growth of B16F10 tumor over 10 days than was injection of Taxol. A histological analysis demonstrated an increase in necrotic tissue in tumors treated with Ptx-loaded gel. In conclusion, our data show that intratumoral injection of Ptx-loaded MPEG–PCL diblock copolymer yielded an in situ-forming gel that exhibited controlled Ptx release profile, and that was effective in treating localized solid tumors.

Introduction

During the course of cancer treatment, almost all patients with cancer receive some form of surgery to remove as much of the tumor as possible (Benotti and Steele, 1992). However, the risk of recurrence stemming from residual cancer cells remains, and may be averted through administration of local radiotherapy or systemic chemotherapy. In systemic chemotherapy, the antitumor activity of anticancer drugs may be enhanced by changing the manner of drug administration, with particular focus on direct intratumoral injection (Goldberg et al., 2002, Ta et al., 2008, Rossi et al., 2003). Such targeted delivery would be expected to provide a high local concentration of the anticancer drug, reducing systemic drug levels and thereby decreasing the incidence of side-effects commonly observed with systemic therapy (Chvapil, 2005).

Paclitaxel (Ptx) is one of the most effective naturally occurring antineoplastic drugs discovered in recent decades (Spencer and Faulds, 1994). The drug interacts with tubulin dimers in the G2 phase of the mitotic cell cycle to promote microtubule polymerization; the resulting formation of highly stable microtubules prevents cell division and accounts for the cytotoxic properties of Ptx (Singh et al., 2008). Ptx has excellent therapeutic efficacy against a variety of solid tumors, including breast cancer, ovarian carcinoma, lung cancer, head-and-neck carcinoma, and acute leukemia (de Bree et al., 2006, Singla et al., 2002, Fjällskog et al., 1993). However, Ptx is a hydrophobic molecule that is poorly soluble in water. Currently, the only commercial available formulation in clinical use is Taxol, which consists of a solution of Ptx prepared using a mixture of the polyethoxylated castor oil Cremophor EL, and ethanol (Terwogt et al., 1997, Chun et al., 2009, Ding et al., 2005). Taxol must be repeatedly administered and thus causes serious side-effects, particularly hypersensitivity reactions, some of which are life-threatening. Efforts to eliminate these problems have focused on developing new drug delivery systems for Ptx that achieve site-specific delivery, prolong action periods, and improve patient compliance (Cheng et al., 2007).

During the last decade, injectable in situ-forming gels have attracted considerable attention as polymeric drug carriers (Vintiloiu and Leroux, 2008, Park et al., 2008). Among the advantages of such gels are their ability to conform to any shape at a specific site, and the fact that they can be introduced using minimally invasive non-surgical procedures. Several block copolymers consisting of polyethylene glycol (PEG) and biodegradable polyesters, such as poly(l-lactic acid) (PLLA), poly(glycolic acid) (PGA), or their copolyesters (PLGA), and the pluronic series, have been prepared and examined as candidate injectable in situ-forming gels (Sasatsu et al., 2008, Ong et al., 2009, Bajpai et al., 2008).

Recently, we reported on a novel biodegradable diblock copolymer, MPEG–PCL, formed from PEG and poly(caprolactone) (Ahn et al., 2009, Hyun et al., 2007, Kim et al., 2006a, Kim et al., 2006b). MPEG–PCL copolymer solutions exhibited sol-to-gel transitions at body temperature. In addition, we found that the subcutaneous injection of a MPEG–PCL copolymer solution resulted in in situ gel formation without inside-needle gelation. It is suitable for long-term drug delivery system because of its low degradation rate. Over the course of a series of investigations, our laboratory has formulated an injectable in situ-forming MPEG–PCL gel for various biomedical applications.

The overall aim of the current study was to develop a simple and generally applicable intratumoral injection strategy for systemic chemotherapy using Ptx. Accordingly, we sought to evaluate an injectable in situ-forming gel prepared with MPEG–PCL diblock copolymer in the context of the following specific questions: (1) does the MPEG–PCL diblock copolymer act as a suitable drug depot for Ptx in vitro and in vivo? (2) Does intratumoral injection of MPEG–PCL result in in vivo gel formation? (3) Do Ptx-loaded MPEG–PCL gels exert a growth inhibitory effect against tumors? Resolving these issues will have a significant impact on the application of intratumoral injections for systemic chemotherapy with Ptx, holding the promise of achieving prolonged action periods and thus facilitating patient compliance and comfort.

Section snippets

Synthesis of MPEG–PCL diblock copolymers

MPEG–PCL diblock copolymer (750–2400) was prepared using a block copolymerization method reported previously (Kim et al., 2006a, Kim et al., 2006b).

Viscosity measurements

The MPEG–PCL diblock copolymer was dissolved in 5 ml vials at 80 °C with deionized water to yield 15, 20, and 25 wt% concentrations, and was stored at 4 °C. After 15 h, viscosity was measured using a circulating bath with a programmable controller (TC-502P, Brookfield Engineering Laboratories, Middleboro, MA) and a Brookfield DV-III ultraviscometer

Preparation of an injectable in situ-forming gel

Our previous work suggested that aqueous solutions of MPEG–PCL diblock copolymers could exhibit sol-to-gel phase transitions as a function of temperature (Kim et al., 2006a, Kim et al., 2006b). Block copolymer solutions of MPEG (MW = 750) and PCL (MW  1400–3000) were liquid at room temperature and exhibited a sol-to-gel phase transition as the temperature was increased. On the basis of previous results, we chose a diblock copolymer solution containing 750 g/mol MPEG and 2400 g/mol PCL.

An aqueous

Discussion

Primary clinical treatment of tumors is usually achieved by surgery or radiation (Parvez, 2008, Cho, 2008). However, local recurrence of tumors generally occurs near the site of the previous surgical excision. Intratumoral injection of anticancer agents has been proposed by several investigators as one treatment option for preventing this phenomenon (Pawar et al., 2004, Ranganath and Wang, 2008, Springate et al., 2008, Lee et al., 2009). An important feature of a successful engineering solution

Conclusion

We have explored the potential clinical utility of a Ptx-containing drug depot using an in situ gel-forming MPEG–PCL diblock copolymer/Ptx solution. Ptx in vitro release profiles from Ptx-loaded gels demonstrated controlled delivery for more than 1 month. The present findings show that an MPEG–PCL diblock copolymer gel can act as injectable drug depot that maintains structural integrity under physiological conditions. Animals that received intratumoral injections of Ptx-loaded gels displayed

Acknowledgements

This study was supported by a grant from KMOHW (grant no. A050082) and Priority Research Centers Program (2009-0093826) and Pioneer Research Center Program (2009-0082804) through NRF funded by the Ministry of Education, Science and Technology.

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