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Assessment of the antiprotozoal activity of some tubulin inhibitors following cyclodextrin complexation

Menon, Kathleen Ilona (2002) Assessment of the antiprotozoal activity of some tubulin inhibitors following cyclodextrin complexation. PhD thesis, Murdoch University.

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      Abstract

      The purpose of the present study was to evaluate the potential usefulness of tubulin inhibitors when complexed with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) against a range of protozoan parasites. This approach involved investigations into the complexation of these drugs with HP-beta-CD, and subsequent investigations of these drugs and their complexes in regard to cytotoxicity, pharmacokinetics, in vitro efficacy against Giardia, Cryptosporidium and rodent malaria (Plasmodium chabaudi), and their in vivo efficacy against Giardia and malaria.

      Albendazole (ABZ) is a benzimidazole carbamate with a broad anti-parasite spectrum, while the dinitroanilines trifluralin (TF) and oryzalin (OZ) have recently been found to exhibit activity against certain parasites. All three compounds are microtubule antagonists in either nematodes or weeds and have poor aqueous solubility, with the solubility of ABZ and OZ dependent on pH. Cyclodextrins (CD) have a hydrophobic cavity that allows them to form inclusion complexes with hydrophobic drugs, resulting in increased drug aqueous solubility, and often, improved drug dissolution and bioavailability. Thus the complexation of these drugs with HP-beta-CD was investigated. All three compounds exhibited type AL phase solubility diagrams with HP-beta-CD complexation, with additional increases in ABZ and OZ solubility achieved through the manipulation of temperature and pH. OZ displayed a stronger interaction with HP-beta-CD when ionised over its neutral form. However, insufficient concentrations of the TF/HP-beta-CD complex were achieved for drug efficacy studies. The cytotoxicity of the drugs and their complexes was assessed using the assay kit Cytotox 96 with human carcinoma cells. This is a colourimetric assay that measures lactate dehydrogenase release as a consequence of compromised cellular and membrane integrity. Both ABZ and OZ are cytotoxic to rapidly proliferating and differentiating cells but are not cytotoxic to cells in the stationary phase. Complexation did not affect drug cytotoxicity.

      In pharmacokinetic studies, complexation improved ABZ (and metabolites) bioavailability, but had no significant affect on OZ bioavailability. In vitro drug assessment studies found ABZ to be highly effective against Giardia, and effectiveagainst Cryptosporidium and malaria. OZ on the other hand exhibited no activity against Giardia, but was effective against Cryptosporidium and malaria. Complexation did not improve the antiprotozoal efficacy of either ABZ or OZ. In particular, excess HP-beta-CD decreased the antigiardial effects of ABZ, possibly due to competitive complex formation. In addition, complexation did not improve the antiprotozoal effects of ABZ in vivo.

      However, the cytotoxic effect of the ABZ/HP-beta-CD complex was more evident in the treatment of malaria in vivo, resulting in increased anaemia and suppression in weight gain, due to the improved bioavailability of ABZ and metabolites. HP-beta-CD alone was found to be cytotoxic at greater than 2.5%, and inhibited Giardia both in vitro and in vivo at greater than 1% and 2% respectively. This was attributed to membrane disruption caused by the dissolution and removal of membrane components.

      In comparison, malaria grew better in the presence of HP-beta-CD in vitro, with no detrimental effect observed at up to 8% HP-beta-CD. This was attributed to either the increased solubilization of a necessary media component, or the complexation and removal of an inhibitory compound from the cultivation medium. Therefore HP-beta-CD complexation did not improve the antiprotozoal activity of the tubulin antagonists ABZ and OZ. However, the results of the pharmacokinetic studies suggest that anthelmintic activity of ABZ, particularly against systemic infections, may be improved with oral administration of the ABZ/HP-beta-CD complex. In addition, the antiparasitic activity of HP-beta-CD alone may be promising, especially against intestinal infections. Finally, the improved in vitro cultivation of P. chabaudi in the presence of HP-beta-CD presents a promising approach to its potential long term cultivation.

      Publication Type: Thesis (PhD)
      Murdoch Affiliation: School of Veterinary Biology and Biomedical Science
      Supervisor: Reynoldson, Jim
      URI: http://researchrepository.murdoch.edu.au/id/eprint/201
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