Research paper
A thermosensitive chitosan-based hydrogel for the local delivery of paclitaxel

https://doi.org/10.1016/S0939-6411(03)00095-XGet rights and content

Abstract

A novel injectable thermosensitive in situ gelling hydrogel has been developed. The system, which falls under the BST-Gel™ platform technology developed at Biosyntech Inc. (Laval, QC, Canada), consists of a chitosan solution (C) neutralized with β-glycerophosphate (GP) that is liquid at room temperature but gels when heated to body temperature. We propose to use this thermosensitive hydrogel for the sustained release of paclitaxel at tumor resection sites in order to prevent local tumor recurrence. The in vitro release profiles demonstrated controlled delivery over 1 month. The initial drug loading substantially affected the release. Local delivery of paclitaxel from the formulation injected intratumorally was investigated using EMT-6 tumors implanted subcutaneously on Balb/c mice. These experiments showed that one intratumoral injection of the thermosensitive hydrogel containing paclitaxel was as efficacious as four intravenous injections of Taxol® in inhibiting the growth of EMT-6 cancer cells in mice, but in a less toxic manner. Further histological analysis revealed that while the proportion of necrotic areas was similar for the C/GP/paclitaxel and the Taxol®-treated tumors, a disparity between tumor-associated inflammatory cell populations may suggest differing anti-tumor mechanisms.

Introduction

Excluding cancers of the skin, breast cancer is the most frequently diagnosed cancer among women. An estimated 211,300 new cases of invasive breast cancer are expected to occur among women in the United States during 2003, and breast cancer will be the second leading cause of cancer death in American women behind lung cancer (American Cancer Society Inc., Surveillance Research, 2003). Almost all women with breast cancer will have some type of surgery in the course of their treatment. The purpose of many of these surgeries is to remove as much of the cancerous tissue as possible, as in a lumpectomy. However, the risk of recurrence stemming from residual malignant cells still exists, and may be averted through administration of local radiotherapy or systemic chemotherapy.

Paclitaxel is one of the best antineoplastic drugs found in nature in the past decades. It interacts with tubulin dimers in the G2 mitotic phase of cell division to promote microtubule polymerisation that results in the formation of highly stable microtubules, thus preventing cell division [1]. Paclitaxel success to date is largely due to its unique mechanism of action against tumors and its ability to work in combination with other anticancer therapeutic agents. It has excellent therapeutic efficacy for a wide spectrum of cancers, especially for ovarian and breast cancers.

However, paclitaxel is a hydrophobic molecule that is poorly soluble in water. Currently, Cremophor® EL, a non-ionic polyethoxylated castor oil solubilizer, is used to enable its clinical administration. Although it has a history of use with other drugs, the amount of Cremophor® EL necessary to deliver the required doses of paclitaxel is significantly higher than that administered with any other marketed drug. This causes serious side effects, particularly hypersensitivity reactions, some of which are life-threatening [2], [3], [4]. The incidence of these reactions can be substantially reduced by the use of prophylactic antiallergic premedications, but this is undesired as it increases the treatment burden.

In order to eliminate the toxicity of Cremophor® EL, improve efficacy and eliminate premedication, current paclitaxel research is focused on developing new drug delivery systems, which circumvent the Cremophor® EL difficulty associated with its use. A variety of approaches have been investigated including emulsification [5], [6], [7], microspheres [8], [9], liposomes [10], [11], [12], nanoparticles [13], [14] and polymeric micelles [15], [16].

However, each drug delivery approach has unique inherent difficulties. Stable emulsions are hard to achieve and drug loading is often limited (i.e. high volumes needed to achieve therapeutic concentration) [5]. Likewise, satisfactory entrapment efficiency can sometimes be problematic with nanoparticles, microspheres and liposomes. Harper et al. [8] noted in their paper that high doses of the Paclimer Delivery System (microspheres) were not investigated, in large part because of anticipated difficulties in administering large volumes into tumor nodules. As for micelles, their disassembly upon injection, due to dilution, can result in burst release and toxic effects.

Another therapeutic approach to combating solid tumors and preventing metastasis and tumor re-growth, involves surgical removal of the tumor followed by implantation of a biodegradable device loaded with an antineoplastic agent in the resulting cavity. This method would provide high local drug concentration, effectively destroying surviving malignant cells and would also prevent the systemic side effects of chemotherapy normally associated with its intravenous administration. Since local recurrence of tumors generally occurs near the original excision site, this treatment modality is more desirable than a systemic one. Implantation of drug-loaded devices (including drug-polymer composites) into tumors or tumor resection sites has been investigated by several workers [17], [18], [19], [20], [21], [22], [23], [24].

A material, which has large potential for use as an injectable in situ gelling drug delivery device, can be obtained from a chitosan solution (C) neutralized with a polyol counterionic dibase salt such as β-glycerophosphate (GP). This thermosensitive C/GP solution, which falls under the BST-Gel™ platform technology developed at Biosyntech Inc. (Laval, QC, Canada), is liquid at room temperature and solidifies into a hydrogel as temperature is increased to body temperature (Fig. 1) [25], [26], [27]. The objective of this work was to evaluate the C/GP thermosensitive formulation as the basis for local chemotherapy. The in vitro release profiles of paclitaxel from within the gel were first investigated, and the anti-tumoral activity of paclitaxel released from the gel was then assessed in vivo using the EMT-6 murine mammary carcinoma model.

Section snippets

Materials

Medical grade chitosan (Ultrasan™, Mw 228,700, P.I. 1.61) having a deacetylation degree of 95% was obtained from Biosyntech Inc. (Laval, QC, Canada). GP and sodium dodecyl sulfate (SDS) were from Sigma (St. Louis, MO). Paclitaxel was purchased from Bioxel Pharma (Ste-Foy, QC, Canada). Taxol® (Bristol-Myers Squibb) was purchased in a retail pharmacy. All other chemicals were reagent grade. All products were used as received. Deionized distilled water from a Milli-Q water system of Millipore

In vitro release studies

In order to understand the ability of the C/GP gel to effectively deliver paclitaxel in a sustained fashion, in vitro release studies were performed in PBS/SDS 0.3% at 37 °C under sink conditions. Fig. 2 shows the resulting release profiles of paclitaxel from the C/GP hydrogel. It clearly demonstrates that the initial drug loading substantially affects the release rate, the latter being reduced at higher drug loading. The initial burst effect was lower (7.0 vs 16.6%) and the release rate was

Discussion

Local control of tumors is a primary clinical objective and is usually achieved by surgery or radiation. However, local recurrence of tumors generally occurs near the previous surgical excision site of the primary tumor. In order to prevent this phenomenon, locoregional adjuvant chemotherapy has been proposed by several investigators [18], [21], [23], [30]. In the present work, we evaluated a new thermosensitive formulation as the basis for local chemotherapy. The chitosan-based solution we

Conclusions

In summary, we have provided proof of principle and preclinical efficacy data for a site-directed, injectable, and controlled-release formulation of paclitaxel as an effective treatment for localized solid tumors. In vitro release profiles demonstrated controlled delivery over 1 month. Local delivery of paclitaxel from the formulation injected intratumorally in EMT-6 tumors implanted subcutaneously on Balb/c mice showed that one intratumoral injection of the thermosensitive hydrogel containing

Acknowledgements

This study was supported in part by the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chair Program. J.-C. Leroux acknowledges a scholarship from the Fonds de la Recherche en Santé du Québec.

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