Novel microbially triggered colon specific delivery system of 5-Fluorouracil: Statistical optimization, in vitro, in vivo, cytotoxic and stability assessment

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Abstract

The present study aimed to statistically optimize a colon specific formulation of 5-Fluorouracil for the treatment of colon cancer. A 32 full factorial design was used for optimization. The independent variables employed were amount of pectin and amount of starch paste, each at three levels. The evaluated responses were hardness, percent cumulative drug release (% CDR) at 5th h and t90% (time required for 90% of drug release). Drug release studies were carried out using change over media [pH 1.2, 7.4 and 6.5 in presence of 4% (w/v) rat caecal contents]. The optimized formulation was subjected to in vivo roentgenographic studies in New Zealand white rabbits to analyze the in vivo behaviour of the developed tablets. This formulation was also evaluated for cytotoxic potential using HT-29 human colon cancer cell lines. Pharmacokinetic studies in New Zealand white rabbits were conducted to determine the extent of systemic exposure provided by the developed formulation in comparison to an immediate release tablet. The optimized formulation consisting of pectin (66.67%, w/w) and starch paste (15%, w/w) released negligible amount of drug at pH 1.2 and pH 7.4 whereas significant (p < 0.05) drug release was observed at pH 6.5 in presence of 4% (w/v) rat caecal contents. Roentgenographic studies corroborated the in vitro observations, thus providing the “proof of concept”. Pharmacokinetic studies revealed significant reduction in systemic exposure and cytotoxicity studies demonstrated enhanced cellular uptake of drug by the developed formulation. Shelf life of the formulation was found to be 2.83 years. The results of the study established pectin-based coated matrix tablet to be a promising system for the colon specific delivery of 5-FU so as to treat colon carcinoma.

Introduction

Colon provides a myriad of therapeutic opportunities for the treatment of various diseases like inflammatory bowel disease, colon carcinoma, and amoebiasis (Philip et al., 2008). Colorectal cancer manifests as cancerous growths in the colon, rectum and appendix. It is reported to be the third most common type of cancer accounting for 6,55,000 deaths globally per year and the second leading cause of cancer-associated deaths in the western world (Zhao and Li, 2010). 5-Fluorouracil (5-FU) is the drug of choice for the treatment of colon cancer (Calabresi and Chabner, 1996). Currently, only intravenous preparations of 5-FU are available in market for clinical use (5-FU-CBC, Dabur, India; Oncourcil, Sunpharma, India; Flurac, Cadila, India). Intravenous administration is associated with pain and formulations have to be sterile. Moreover, owing to skillfulness required for administration, it is time consuming for doctors and patients and self-administration is not possible. Psychological distress, hypertrophy or atrophy of the subcutaneous fat at the site of injection, occasional allergies are some of the additional factors responsible for non-conformity by the patient to the therapy using this route. Furthermore, intravenous administration of the drug has been reported to cause severe gastrointestinal, dermatological, hematological, cardiac and neural side effects (Diasio and Harris, 1989). Most of these side effects are due to exposure of the drug to the unwanted sites. Severe systemic toxic effects along with a short plasma half life of 10–20 min predominantly make this drug to be delivered by a local delivery system capable of providing a continuous sustained release (Wei et al., 2008). Delivery of drugs to the receptors at a particular site has the potential to reduce side effects and to increase pharmacological response (Sinha et al., 2004). Recently, it has also been demonstrated in mice that targeting improves mesenchymal stem cells treatment of inflammatory bowel disease (Ko et al., 2010). Amongst the different routes of targeting a drug, the oral route remains the choice of administration (Krishnaiah et al., 2002). Conventional oral dosage forms are ineffective in delivering drugs to the colon due to absorption or degradation of the active ingredient in the upper gastrointestinal tract (Sinha et al., 2004).

It was, thus, hypothesized that targeted delivery of 5-FU through oral route would not only reduce systemic exposure of drug but would also help to circumvent limitations associated with i.v. route. Moreover, reduced systemic exposure would help to decrease above mentioned side effects. It was also anticipated that minimal systemic exposure of drug achieved due to site-specific delivery of 5-FU to colon would lead to reduction in dose as well as the duration of therapy in comparison to conventional oral administration. Longer exposure to lower concentration of 5-FU has been reported to favor DNA-directed effects which is thought to contribute to its anti-tumour effect (Wei et al., 2008). All these factors are expected to significantly enhance safety and effectiveness of the therapy which in turn would contribute to increased patient compliance. Literature review reveals that mainly tablets and multiparticulate systems have been investigated for colonic delivery of 5-FU (Sinha et al., 2004, Rahman et al., 2008, Patel et al., 2008). Tablets owing to the merits of being cost effective and requiring minimal processing steps during manufacture have been more preferred.

Pectin is a non-starch, linear polysaccharide extracted from the plant cell walls. It largely remains intact in the physiological environment of the stomach as well as small intestine, but is degraded by the bacterial inhabitants of the human colon which contain pectinolytic enzymes (Sinha and Kumaria, 2001). Various systems using pectin as a carrier have been investigated in order to develop oral formulations intended to release their active substance in the colon (Wakerly et al., 1996, Adkin et al., 1997, Monin and Pundarikakshudu, 2007, Ahrabi et al., 2000).

However, most of the studied approaches either employ compression coating having demerits of multiple granulation, improper centration, capping and multitude of steps required for processing or employ polymers having undesirable effects. Polymer like guar gum causes suppression of appetite and obstruction of esophagus due to premature swelling (Rowe et al., 2006) while chitosan causes loss of weight (Egras et al., in press). Such untoward effects are likely to further aggravate the debilitated condition of a patient suffering from colon cancer thereby decreasing the patient compliance to the therapy. Pectin, being one of the components of dietary fiber, was selected as the carrier for matrix formation since an inverse relationship has been reported between risk of colorectal cancer and intake of dietary fiber (Dahm et al., 2010). Hence, the present research work was aimed to formulate a colon specific drug delivery system of 5-FU using pectin-based matrix, dually coated by spray coating technique, as the carrier to overcome limitations of the reported investigations. An attempt was also made to estimate shelf life of developed formulation.

Section snippets

Materials

5-FU (98–99% pure) was obtained as a gift sample from Shalaks Pharmaceuticals (P) Ltd., New Delhi, India. Eudragit S 100 was gifted by Degussa India (P) Ltd., Mumbai, India. Other materials namely lactose, starch, magnesium stearate, talc and barium sulphate were purchased from s.d. fine-chem. Ltd., Mumbai, India. New Zealand white rabbits used for the animal studies were procured from the animal house of Rajiv Academy for pharmacy, Mathura, India.

Experimental design

A 32 full factorial design was used for the

Selection of dose for colon specific delivery

As per the calculation by Chabner and Alabresi, the dose of 5-FU for colon specific drug delivery on the basis of colon surface area was found to be 75 mg. Considering the reports stating 5-FU to be highly toxic against the intestinal mucosa, only 50 mg of 5-FU was used by the authors (Sinha et al., 2004). So, 50 mg has been used as the dose of the drug for colon specific delivery in the present study as well.

Preparation of pectin-based colon-targeted 5-FU tablets

On the basis of 32 experimental design, nine formulations were prepared which were

Conclusion

Colon targeted, pectin-based matrix tablets of 5-FU dually coated with Eudragit S 100 were successfully optimized using 32 full factorial design. The optimized tablets contained 66.67% (w/w) of pectin as the matrix former and 15% (w/w) of 10% (w/v) starch paste as the binder. Six percent (w/v) solution of Eudragit S 100 was found to be capable of preventing the drug release in the gastric environment. The optimized tablets released only 8.01 ± 2.34% of their drug content in the small intestine

Acknowledgement

The authors are thankful to Pt. Deen Dayal Upadhaya Pashu Chikitsa Vigyan, Mathura, Uttar Pradesh, India for their assistance in performing the in vivo roentgenography study.

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