Many organic molecules, biological reagents, and some drugs form sub-micrometer sized colloidal aggregates in biochemical buffers at micromolar concentrations. Upon formation of these particles, non- specific, picomolar enzyme inhibition is observed. Although much is known about the in vitro behavior of colloidal aggregates, the biological ramifications of colloidal aggregates remain essentially unexplored. This project addresses key biological and mechanistic aspects of colloidal aggregates. Most ambitiously, this project investigates the impact of colloidal aggregates on drug distribution. The research will test the theory proposed by Janssen et al. that hydrophobic drugs aggregate in the gut, and are absorbed into the lymph by specialized particle-absorbing cells in the intestinal tract. Janssen et al. proposed that such a mechanism accounts for the higher than expected efficacy of non-nucleoside reverse transcriptase inhibitors and other hydrophobic drugs [Ref. 6]. To test Janssen's hypothesis, the absorption and physiologic distribution of several hydrophobic, known aggregating drugs will be examined in Sprague Dawley rats. If hydrophobic drugs are found to exist in the lymph as aggregates, it will profoundly affect current understanding of pharmacology, drug distribution and bioavailability. This project investigates the potential cellular toxicity of colloidal aggregates. Prior to advancing to """"""""lead"""""""" status, candidate drugs and tools are tested for toxicity. Here, molecules are intentionally tested at concentrations much higher than their EC50 values, and at these concentrations many compounds aggregate. Because aggregates have been shown to be hemolytic, they may be responsible for false-positive toxicity results. Well-characterized aggregators will be tested in cell toxicity assays above and below their critical aggregation concentration (CAC). If aggregates are found to be cytotoxic, while their soluble monomeric counterparts are innocuous, it could prevent lead compounds from being unnecessarily abandoned based on false-positive cellular toxicity results that arise from aggregation brought about by the assay conditions. The thermodynamics of aggregate formation and the kinetics of aggregate-based enzyme inhibition are explored in this project. If the hydrophobic effect drives aggregate formation, a large change in heat capacity ( Cp) would be observed. If a small Cp is measured, it suggests a radically different mechanism of aggregate formation. The kinetics of the incubation effect of observed with aggregate-based inhibition will be probed with classical enzymology in kinetic assays using 2-lactamase and several well-characterized aggregators. The knowledge acquired from these experiments may illuminate methods for controlling aggregate formation and subsequent enzyme inhibition. These insights could be exploited to prevent negative drug side effects caused by aggregates, as well as to optimize conditions for drug absorption and bioavailability.
This research project investigates whether certain drugs associate into particles called colloidal aggregates inside the body, and if so, how those particles affect the distribution of drug within the body, and if drug toxicity is due to formation of these particles. The project will also investigate the mechanism of particle formation. The results of this research may profoundly impact current practices and concepts related to drug discovery.
