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From chemist to pharmacologist

Although most people are aware that there are thousands of different pharmaceuticals on the market, people are less familiar with what is required to develop them. Multidisciplinary teams within the ARC Centre of Excellence for Free Radical Chemistry and Biotechnology are working together to produce and test compounds that may one day become pharmaceuticals.

It is for good reason that the candidate compounds produced in the laboratory are tested comprehensively. The majority will never make it to pharmacy shelves. Those compounds that are marketed as pharmaceuticals have often undergone more than ten years of tests, including several rounds of both clinical and pre-clinical trials.

The Centre is engaged in pre-clinical trials to test a number of its potential pharmaceuticals.  Professor James Angus, Associate Professor Christine Wright and Associate Professor James Ziogas’ laboratories in the Centre at the University of Melbourne’s Department of Pharmacology are at the forefront of the Centre’s pre-clinical testing. Currently the group members Mark Ross-Smith and Nitya Jani are testing novel SARTAN compounds (selective AT1 receptor antagonists) being produced in the Schiesser laboratory.

Sartans act to reduce hypertension by reducing (antagonising) the binding ability of the peptide, angiotensin II, to its receptor. The interaction between angiotensin II and the receptor can also result in the release of free radical species that in turn can create further hypertensive consequences. Unlike the sartans currently on the market, the new class of sartan being developed by Dr Michelle Taylor and Nicole Tan from the Schiesser group is designed to act as an antioxidant as well as an AT1 receptor antagonist.

Once synthesised, the various sartan compounds must undergo pre-clinical tests to assess their efficacy prior to entering clinical trials. The Wright/Ziogas group utilise high-throughput antioxidant and calcium imaging screens as a means by which to rapidly assess the antioxidant and antagonistic capabilities of the compounds. Although not acceptable for clinical trials, the high-throughput in vitro methods are ideal for pre-clinical trialling.

The first screen, the calcium imaging screen, utilises cell culture lines, a fluorescent dye and an analyser. The chinese hamster ovary (CHO) cell line contains endogenous AT1 receptors, and when these cells are stimulated with angiotensin II these AT1 receptors are activated. This causes cell membrane calcium channels to open, resulting in an influx of calcium. The shift in calcium concentration is detected by the fluorescent dye, Fluo 4. Fluo 4 also has the advantageous properties of an absorption spectrum compatible with excitation at 488nm by argon-ion laser sources (which are present in confocal microscopes and microplate screening machines) and a very large increase in fluorescence intensity in response to Ca2+ binding (typically >100 fold) with no spectral shift. The data can be observed and analysed in a microplate screening machine or on a confocal microscope, and the level of AT1 receptor activation inferred.

Microplate screening as performed in the Ziogas laboratory allows rapid analysis of large numbers of cells and compounds. Compounds which inhibit the normal increase in fluorescence seen in cells stimulated by angiotensin II represent potential antagonists and are subjected to further pre-clinical testing - antioxidant testing and animal model testing.

Recently, the team have developed a number of lead compounds, which have performed well in preliminary pre-clinical tests. As the compounds move into clinical trials in the not too distant future it is exciting to contemplate the difference these drugs will make in patients’ lives.