The toxicity, pharmacokinetics, anti-inflammatory and anti-tumour properties of a methotrexate polymer

Abstract

A major effort to develop anticancer drugs through both empiric screening and rational design of new compounds has been under way for over 30 years (Katzung, 2004). In recent years, research and development in the field of sitespecific drug therapy has progressed significantly. Safe and non-toxic formulations of cytotoxic drugs based on polymers with their improved sitespecific delivery and effective activation to biologically active cytotoxic compounds at the targeted tumours have become a promising approach to cancer therapy. Drug delivery systems based on polymer micelles, coated microand nanoparticles, liposomes and various pro-drug systems including watersoluble polymer–drug conjugates and immunoconjugates have been prepared and extensively studied as novel drug delivery systems designed for cancer chemotherapy. Amongst these drug delivery systems that enable specific drug delivery and release, water-soluble polymer–drug conjugates rank among the most promising, versatile and efficient systems. This dissertation reviews the preclinical testing and pharmacokinetic study of D85, a novel water-soluble macromolecular pro-drug that is a polymer with pHcontrolled methotrexate (MTX) release with potential for treatment of cancer in humans (Ulbrich&Subr, 2004). As MTX is also indicated in low doses for the treatment of chronic inflammatory conditions, the polymer was further tested in an acute inflammatory model to determine whether the polymer would be more effective than MTX in controlling inflammation. The objective of this study was to compare the potency and efficacy of D85 to MTX. D85, a MTX conjugated polymeric lead compound, was designed and synthesised as a potential anti-neoplastic and anti-inflammatory agent. It was initially tested in vitro on three different cancer cell lines where selective toxicity towards the cancer cell cultures compared to primary cell cultures and greater toxicity than MTX was observed. The initial in vitro tests showed very promising results with D85 demonstrating approximately 300 times greater cytotoxicity than MTX against a colon cancer cell line (COLO 320 DM). This high cytotoxic effect warranted further investigation in an in vivo colon cancer tumour model. An induced murine tumour model of COLO 320 DM was successfully developed in nude mice, and the anti-tumour efficacy of D85 tested in this model. The maximum tolerated dose of D85 was established by carrying out an in vivo dose ranging toxicity test in BALB/c mice. The anti-inflammatory effects of D85 were also determined using the carrageenan-induced paw oedema model in rats where carrageenan was injected into a footpad of a rat causing acute oedema, which was measured using a water displacement plethysmometer. D85 was found to exacerbate the inflammatory response. Finally, the pharmacokinetic parameters of MTX and D85 were assessed using a LC/MS/MS method specifically developed and validated to determine low concentrations of MTX in small volumes of plasma. This new method made use of online solid phase extraction and sample cleanup on 2μl injections of diluted plasma allowing an entire pharmacokinetics study to be completed on an individual rat. Fairly similar pharmacokinetics were determined from both compounds. Copyright