Lead compounds permeability testing

Many types of modeling techniques are available in the discovery phase of drug development, from structure activity relationships (SAR) to physiology based pharmacokinetics (PBPK) and pharmacokinetics-/pharmacodynamics (PK/PD) to help choosing some of the lead compounds. Some tests that are carried out by discovery include techniques related to structure determination, metabolism, and permeability NMR, MS/MS, elemental analysis, PAMPA, CACO-2, and in vitro metabolic stability. Although they are important as a part of physicochemical molecular characterization under the biopharmaceutics umbrella, they will not be discussed here. The reader can find relevant information in numerous monographs [9,10]. 

Seventy-five percent of drug candidates do not reach the clinical trial phase mainly due to poor pharmacokinetics in animal studies (1). Since so many compounds fail in late stage testing, the current trend is to study the pharmacokinetics of lead compounds as early as possible. One of the most important elements of pharmacokinetics is lipophilicity, or a compound s affinity for fat. Usually, the more water soluble a compound is, the lower its lipophilicity. Low water solubility (high-lipophilicity) compounds have a limited oral bioavailability but are usually easily metabolized. On the other hand, low-lipophilicity compounds have poor membrane permeability since membranes are partly composed of fat. 

Each of these assays has drawbacks associated with them. The major obstacle for developing rapid screens for inhibitors of cytochrome c release from mitochondria is that enriched mitochondria have a finite time in which they can be used. We are in the process of testing mitochondria preparations to determine the stability of the mitochondria with respect to use in the cytochrome c release assay. Mitochondria stored on ice for 4 h (the longest time we have tested at the time of this writing) can be used in the cytochrome c release assay. Therefore, it is possible to run multiple assay cycles with one preparation of mitochondria. The short format cytochrome c release assay is not affected by cell permeability, makes no assumptions about the mechanism of action, does not use cultured cells, is targeted at the process to be inhibited (i.e., cytochrome c release from mitochondria), and is a colormetric assay easily monitored by spectrophotometric plate readers. This assay would, therefore, increase the chances of detecting lead compounds to be subsequently modified to increase potency, cell permeability, and pharmacological efficacy. 

Partly due to the limited throughput of Caco-2 permeability measurements, the structure-activity evaluation of compounds tested in the hit-to-lead phase is done with minimal permeability information, at best. Given the importance of membrane permeability in drug absorption, early consideration of the permeability characteristics of hit compounds would enhance the drug-like quality, and ultimately the probability of success, of selected lead candidates. To incorporate permeability information into the hit-to-lead phase of the drug discovery process it is necessary that permeability measurements be made quickly and with small amounts of material. Thus, efforts have been made to automate and miniaturize the Caco-2 permeability assay. 

Standard markers should be included in all experiments. Usually marker compounds for different permeability classes are used like metoprolol for high permeability and radioactive mannitol for low permeability. Quality assurance criteria define accepted upper permeability values for mannitol (in the case of mannitol many laboratoratories use 1.0 x E-06 cm/sec). Permeability values higher than upper limit should lead to rejection of the test. 

Similar approaches to enhance solubility have been studied in the PAMPA system. For instance, Kansy et al. (2001) explored the use of glycocholic acid to solubilize compounds. As PAMPA is a completely artificial system, it is expected that, compared to cell-based models, higher concentrations of cosolvents can be used. Sugano and coworkers reported that DMSO, ethanol and PEG 400 could be used up to 30% without causing disruptions of the lipid layer (Sugano et al., 2001). An effect of these cosolvents on the physicochemical properties of the test compounds (e.g. impact on pKa) may however lead to an unpredictable effect on drug permeability (Sugano et al., 2001). Currently, DMSO is commonly used as a cosolvent in the PAMPA system at a concentration of 1 to 2%. 

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