Current Topics in Medicinal Chemistry - Volume 1, Issue 5, 2001

The attrition rates of new chemical entities (NCEs) in preclinical and clinical development are staggeringly high. NCEs are abandoned due to insufficient efficacy, safety issues, and economic reasons. Uncovering drug defects that produce these failures as early as possible in drug discovery would be highly effective in lowing the cost and time of developing therapeutically useful drugs. Unfortunately, there is no single factor that can account for these NCE failures in preclinical and clinical development since factors, such as solubility, pKa, absorption, metabolism, formulation, pharmacokinetics, toxicity and efficacy, to name a few, are all interrelated. In addition, there are many problems in scaling-up drug candidates from the laboratory bench scale to the pilot plant scale. To address the problem of attrition rates of NCEs in preclinical and clinical development and drug scale-up issues, pharmaceutical companies need to reorganize their preclinical departments from a traditional linear approach to a parallel approach. In this review, a strategy is put forth to integrate certain aspects of drug metabolism / pharmacokinetics, toxicology functions and process chemistry into drug discovery. Compound optimization in early and late phase drug discovery occurs by relating factors such as physicochemical properties, in vitro absorption, in vitro metabolism, in vivo pharmacokinetics and drug scale-up issues to efficacy optimization. This pre-preclinical paradigm will improve the success rate of drug candidates entering development.

This review gives an overview of the current approaches to evaluate drug absorption potential in the different phases of drug discovery and development. Methods discussed include in silico models, artificial membranes as absorption models, in vitro models such as the Ussing chamber and Caco-2 monolayers, in situ rat intestinal perfusion and in vivo absorption studies. In silico models such as iDEATM can help optimizing chemical synthesis since the fraction absorbed (Fa) can be predicted based on structural characteristics only. A more accurate prediction of Fa can be obtained by feeding the iDEATM model with Caco-2 permeability data and solubility data at various pH's. Permeability experiments with artificial membranes such as the filter-IAM technology are high-throughput and offer the possibility to group compounds according to a low and a high permeability. Highly permeable compounds, however, need to be further evaluated in Caco-2 cells, since artificial membranes lack active transport systems and efflux mechanisms such as P-glycoprotein (PgP). Caco-2 and other ”intestinal-like“ cell lines (MDCK, TC-7, HT29-MTX, 2 / 4 / A1) permit to perform mechanistic studies and identify drug-drug interactions at the level of PgP. The everted sac and Ussing chamber techniques are more advanced models in the sense that they can provide additional information with respect to intestinal metabolism. In situ rat intestinal perfusion is a reliable technique to investigate drug absorption potential in combination with intestinal metabolism, however, it is time consuming, and therefore not suited for screening purposes. Finally, in vivo absorption in animals can be estimated from bioavailability studies (ratio of the plasma AUC after oral and i.v. administration). The role of the liver in affecting bioavailability can be evaluated by portal vein sampling experiments in dogs.

The advent of more efficient methods to synthesize and screen new chemical compounds is increasing the number of chemical leads identified in the drug discovery phase. Compounds with good biological activity may fail to become drugs due to insufficient oral absorption. Selection of drug development candidates with adequate absorption characteristics should increase the probability of success in the development phase. To assess the absorption potential of new chemical entities numerous in vitro and in vivo model systems have been used. Many laboratories rely on cell culture models of intestinal permeability such as, Caco-2, HT-29 and MDCK. To attempt to increase the throughput of permeability measurements, several physicochemical methods such as, immobilized artificial membrane (IAM) columns and parallel artificial membrane permeation assay (PAMPA) have been used. More recently, much attention has been given to the development of computational methods to predict drug absorption. However, it is clear that no single method will sufficient for studying drug absorption, but most likely a combination of systems will be needed. Higher throughput, less reliable methods could be used to discover loser compounds, whereas lower throughput, more accurate methods could be used to optimize the absorption properties of lead compounds. Finally, accurate methods are needed to understand absorption mechanisms (efflux -limited absorption, carrier-mediated, intestinal metabolism) that may limit intestinal drug absorption. This information could be extremely valuable to medicinal chemists in the selection of favorable chemo-types. This review describes different techniques used for evaluating drug absorption and indicates their advantages and disadvantages.

To reduce the high attrition rates of NCEs in preclinical and clinical development uncovering pharmacokinetics, toxicokinetics, drug metabolism, and drug-drug interactions early in drug discovery would be highly valuable. There have been many in vitro screens developed for these areas that have higher sample throughput, which is consistent with the iterative cycle of a typical drug discovery research project. We have presented the present status and given detailed descriptions of biotransformation, metabolic stability assays, identification of drug metabolizing P450 enzymes, prediction of pharmacokinetic parameters from in vitro metabolism data, structure elucidation of metabolites, CYP450 inhibition assays and CYP450 induction assays from a drug discovery perspective. Strategies for the proper sequencing of primary and secondary assays employed for drug metabolism and CYP450 inhibition & induction is discussed.

Nuclear magnetic resonance techniques have become critically important in the design of new pharmaceuticals, the characterization of drug-receptor interactions and metabolite identification. Advances in solvent suppression, coherent and incoherent magnetization transfer pathway selection, isotope editing and filtering, and diffusion filtering have made it possible to examine the interactions between small molecules and proteins or nucleic acids in great detail. Multiple schemes for high-throughput lead compound identification, metabolite screening and drug disposition have been proposed and reduced to practice. In particular, the coupling of NMR with other analytical methods, especially HPLC, combine the structural and dynamic detail available from NMR methods with the resolution and sensitivity of other analytical techniques.

Over the last years, there has been an exponentially growing need and interest to bring pharmacokinetic expertise into discovery. In order to allow a multidisciplinary selection and a higher attrition rate, both the in vivo and in vitro pharmacokinetic parameters of an ever increasing number of tentative new chemical entities are evaluated in an earlier phase of Drug Discovery. A higher attrition rate at the beginning of the pipeline should result in a lower attrition rate at a later stage in development. In this process, the bioanalytical laboratory has become increasingly important. Analytical strategies needed to be adapted to cope with novel experimental designs such as cassette dosing, cassette analysis or 96-well techniques. At the same time, HT-synthesis programs surfaced a broader variety of chemical classes to be investigated, disfavoring further generalization of analytical approaches. Progress in lab automation, improved chromatographic techniques and the proliferation of LC-MS / MS enabled the analyst to deal with these challenges much faster and with a higher level of confidence. Quality standards regarding method development and method validation, setting the boundaries for more than a decade, needed to be titrated to reach an optimal balance between speed and quality. This review will give an illustrative overview of the bioanalytical techniques and strategies used to support Drug Discovery, together with some pitfalls related to the overzealous use of new techniques.

We have investigated various sample chromatographic extraction and sample preparation methods for liquid chromatography mass spectrometry analysis in order to increase the throughput of various in vivo and in vitro assays in support of drug discovery. The results indicated that direct plasma injection, although certainly faster than conventional protein precipitation for sample preparation, had problems associated with column longevity and overall robustness. Frequently a single study could not be completed without column replacement. On-line solid phase extraction, on the other hand, compared well with off-line solid phase extraction, using our LC extraction column design, as contamination of the extraction column was minimized by back flushing using the Gilson syringe pump. Finally, on-line solid phase extraction for support of Caco-2 permeability studies worked very well for both single components and mixtures as the matrix was much simpler, presenting fewer contamination problems.