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The pharmacology of nucleoside analogues in herpesvirus infection

University of British Columbia

The pharmacological aspect of anti-herpesvirus (HHV) research has entered a new era concomitant with the introduction of the nucleoside analogue-class of antiviral agent. Recent accession to the specificity of these agents has provided not only substantial improvements in the treatment and prophylaxis of many different HHV infections, but to enhancements in the methods used to diagnose and further study these virus-induced illnesses as well. The disparate applications of such compounds are perhaps best illustrated by the thymidine kinase (tk-dependent anti-HHV nucleoside analogues -- agents which, by virtue of their selective uptake and metabolism, have been used both chemotherapeutically and as molecular indicators of the virus itself. It is clear that a fundamental principle in determining an antiviral's pharmacotherapeutic relevance and of gauging its potential as a diagnostic indicator of disease lies in the ability to accurately detect and quantitate these agents in complex biological matrices. In the present study, radioiodinated 1-β-D-arabino-furanosyl- E-5-(2-iodovinyl)uracil ([*I]-IVaraU) was used to develop an autoradiographic technique (PA/IP) that rapidly and accurately demonstrated thetk- (and thymidylate kinase)-dependent metabolism of this antiviral agent. [*I]-IVaraU-PA/ IP was also used to detect antiviral-resistant HHV variants in somatic cell cultures, to examine the growth of HSV in primary cultures of mammalian CNS glial cells and to demonstrate the uptake and metabolism of 9-(4-hydroxy-3-hydroxy-methylbut- 1-yl)guanine (PCV) in a human neural (SW) cell line. We further demonstrate limited in vivo utility of [*I]-IVaraU for detecting HHV in tissue autoradiographs of CNS sections prepared from a rabbit encephalitis model. The use of this agent as a radiodiagnostic indicator of HSE in intact animals using external gamma camera imaging, however, was found to be largely unreliable. Finally, techniques in ESI mass spectrometry (both independent from and in combination with RP-IP HPLC - including the development of requisite volatile buffer systems) were used to determine that the PCV-TP synthesized by HSV(tk+) type 1-infected-PCV (10 μM and 1 µM)-treated SW cells after a 6 H drug treatment to be 11500 pmole•(106)-1 and 2440 pmole•(1O6)-1 SW cells, respectively, and that the intracellular stability of PCV-TP in this culture system was roughly 16.5 H. These determinations were made by monitoring the nucleotide→ortho/pyrophosphate transitions. We conclude that this technique can be developed further, into a method for monitoring the therapeutic efficacy of this and possibly other antiviral compounds.

Thesis/Dissertation

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2009-07-20

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http://hdl.handle.net/2429/10957

Pharmacology

University of British Columbia

UBCV

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