Lipophilicity screening

High-throughput formats use HPLC-based methods via correlation of log D with retention time [79,80], The experimental setup of a lipophilicity screening assay involves the equilibration of the drug compound in water (or buffer) and octanol. The phases are diluted in water/methanol and analyzed by LC-MS. The values for log P are determined by correlation to log k [81]. A calibration curve is established from known log P of each standard versus the log k value calculated from the retention time (tr). Hexachlorobenzene (log P = 6.11) can be used as a standard for log P calibration (log P values >5). 

HPLC techniques have also been used in the determination of log P values. Lambert et al. (1990), for example, have described the development of a preformulation lipophilicity screen utilizing a C18 derivatized HPLC column. They appeared to prefer this column to the traditional reverse-phase HPLC columns, which may yield a poor correlation between log P and the capacity factor (k ). A potential problem with the use of HPLC retention data is that it is not a direct method and thus requires calibration. Futhermore, there may be problems with performing experiments above pH 8. 

If areas identified as likely to receive significant atmospheric contaminant concentrations include areas supporting edible biota, the biouptake of contaminants must be considered as a possible environmental fate pathway. Direct biouptake from the atmosphere is a potential fate mechanism for lipophilic contaminants. Biouptake from soil or water following transfer of contaminants to these media must also be considered as part of the screening assessments of these media. 

A thorough review, even with a very superflcial mention of the vast literature on ADME, QSPR and QSAR applications involving the use of lipophilicity, would be a daunting task and it is far beyond the scope of this chapter. However, it is no surprise that three international conferences, in recent years, have been specifically dedicated to this topic, and that the development of newer and faster screening methods, in some cases seeking to produce alternative lipophilicity parameters to the classical or nonclassical log Pod determinations are still an active area of interest [6]. 

The first non-peptide oxytocin antagonists, based on a spiropiperidine template, were described by Merck in 1992 [68-70]. The binding affinity data for key compounds from this series are summarised in Table 7.2. The initial screening hit, L-342,643, (23), had modest (4/iM) affinity for rat uterine oxytocin receptors and very little vasopressin selectivity [71]. A structure activity relationship (SAR) study was carried out around this template, focussing on the toluenesulphonamide group. This work led to the identification of bulky lipophilic substitution as key to improved oxytocin potency, while the introduction of a carboxylic acid group led to improved... 

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

Two main factors have guided the need for optimization of the early screening techniques on one hand the use of simple, quick and high-capacity cell monolayer methods, e.g., Caco-2 cell and MDCK and on the other hand the increased synthesis of more lipophilic, insoluble compounds from combinatorial libraries. This has created a vast number of different variants of cell-based assays and has resulted in variability among the data obtained. A need for optimization of as many as possible of the different parameters in order to increase the predictivity and throughput of the model has been suggested in the literature [98-100]. 

The use of high-throughput screening (HTS) results in hits and leads that are more lipophilic than optimized drugs [24]... 

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