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Global approach to modern drug discovery and development

The choice of appropriate screening procedures could reduce drug attrition later in the drug development process. Dr S S Murugan, Scientific Director, RCC Laboratories India, and Dr T Kumaravel, Consultant, Pre-clinical Consultations UK, review some of these procedures

Dr S S Murugan, Scientific Director, RCC Laboratories, India

Dr T Kumaravel, Consultant, Preclinical Consultations, UK

Modern drug discovery and development efforts generally comes from basic research and then move gradually on to specific sequential activities, which if successful ends in a new drug for the treatment of a human disease. The overall pathway is structured by well delineated milestones, which include selection of the drug target, identification of a lead compound, its modification to a compound suitable for toxicity testing in animals, and selection as drug candidate for clinical testing. Even before the onset of human studies, a drug candidate suitable for clinical testing is expected to satisfy specific and demanding safety criteria. It must bind selectively to the receptor site on the target and elicit the desired functional response. It must have sufficient bioavailability and distribution within the body to reach the site of action, and it must elicit the desired responses in vivo, in animal models. Most importantly, a drug candidate suitable for testing in humans must pass formal toxicity evaluations, to demonstrate that humans participating in the phase I clinical studies are exposed to minimal risks only.

Figure 1 gives a schematic of the drug development process. Not all 'lead' compounds generated by the drug discovery teams are tested in full regulatory packages. This is because the regulatory testing is very time consuming and an expensive affair. Usually a series of tests are initially conducted, to help select few candidate molecules with the desired pharmacological potentials and safety profile for further regulatory testing. Drugs not fulfilling the necessary requirements in these initial assays are less likely to be carried over for testing in more expensive, time consuming regulatory tests. In this manuscript, general principles of these initial screening tests are discussed (see figures 2 and 3). However, it should be noted that the initial screening studies are selected on a case-by-case basis.

Primary screening

1. Molecular structure—The wide availability of chemical libraries and automatic screening technologies has made it relatively easy to identify initial lead candidates for new drug targets. Drug discovery chemists have developed certain rules that lead molecules must fulfill to become potential candidate drug molecules.

• molecular weight of less than 500
• no more than five hydrogen bond donors
• no more than 10 hydrogen bond acceptors

2. Octanol/water partition coefficient—The lipid solubility of drugs is expressed as octanol/water partition coefficients of the uncharged molecules, or log P. Higher the log P values, higher is the lipid solubility and more likely to be accumulated in the body.

3. Structural activity relationship (SAR)—SAR provides information about the potential toxicity of a chemical based on chemical structures, when no experimental data is available. It can deliver predictions about a broad variety of toxicological properties of compounds such as carcinogenicity, irritancy, lachrymation, neurotoxicity, thyroid toxicity, teratogenicity, respiratory and skin sensitization, and mutagenicity.

4. Cytotoxicity—An essential part of the drug discovery/approval process is determining the toxic effects of compounds that are potential drugs. Following a toxic insult, cells may respond with changes in size and/or morphology depending on the cell type and the compound. Loss of cell membrane integrity is another common phenotypic feature of cytotoxicity. Some toxins can interfere with the cell's functionality by affecting the physiology of organelles such as lysosomes and endosomes, or by causing an increase in the number of lysosomes, as in the case of phospholipidosis. Multiparameter cytotoxicity kits are commercially available and can be used.

5. Parallel artificial membrane permeability assay (PAMPA)—The ability of a molecule to be orally absorbed is one of the most important aspects in deciding whether the molecule is a potential lead candidate. The PAMPA, as a passive-permeability screen, is an excellent alternative to cellular models for the earliest absorption, distribution, metabolism, and excretion (ADME) primary screening of research compounds. This method is used to measure the effective permeability, P(e), as a function of pH from 4 to 10. This provides rapid, low cost and automation friendly method to measure a compound's passive permeability.

6. Derived solubility—The aqueous solubility of a drug is one of the key physical properties that affect both its ADME profile and 'screenability' in high throughput systems.

7. Genetic toxicology—Cut down versions of the ames and mouse lymphoma assays are initially conducted, in order to select candidates for regulatory testing. Alternatively, SOS/umu assay is performed to look for potential genotoxic effects. The advantage of the SOS/umu assay is that 50-60 compounds can be screened in a single day.

Secondary screening

At the end of the 'primary screening' approximately fifty percent of the lead molecules are rejected and the remaining are carried forward to the secondary screening level. The secondary screening is predominantly relates to pharmacokinetic measurements. The tests in the secondary screening includes the following:

1. Absorption using Caco-2 model—The human colon adenocarcinoma cell line Caco-2, grown on semi-permeable filter supports. Caco-2 cells spontaneously differentiate into enterocyte-like cells and in spite of their colonic origin, a number of active transport mechanisms normally found in the absorptive enterocytes of the small intestine are present in this cell line. The use of the Caco-2 cell model permits the investigation of simultaneous absorption routes at the same time (eg. passive diffusion, active efflux, metabolism), and much insight into the different steps of absorption has been gained by the use of the Caco-2 cell model.

2. Aqueous/Plasma stability—The stability of lead molecules in plasma is an important parameter, which strongly can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases), they can undergo intramolecular re-arrangement or bind irreversibly (covalently) to proteins. Thus the investigation of plasma stability should be performed early in drug discovery. Measurement of plasma stability is performed in plasma of different species at physiological pH level. 3. Protein binding—A thorough understanding of plasma and tissue (brain, liver, etc.) protein binding is crucial for evaluating the distribution of drug candidates. Human or animal plasma or tissue homogenate is incubated with the test agent. The bound and unbound test agents are separated using ultrafiltration or equilibrium dialysis, and the amount of test agent in both fractions is estimated using LC/MS or HPLC. Human, mouse, rat, dog, and monkey plasma are routinely used.

4. Metabolic profiling—When a drug enters an organism it is subject to metabolism, resulting in the production of metabolites from the parent drug. However, metabolites can be toxic or pharmacologically active, just like drug candidates. Consequently, it is required to identify metabolites and evaluate their safety. Before any lead molecule can be approved for clinical research, the molecules's metabolites must be identified and subjected to preclinical toxicity testing to ensure safety. Because producing the metabolites needed for preclinical toxicity testing is costly and time consuming, metabolite identification, synthesis, and toxicity testing are generally reserved for the final stages of preclinical testing. However, it is advantageous to profile the metabolites of the lead molecule at this stage rather than have unexpected surprises late in drug development. At the end of the secondary screening stage further 50 percent of molecules are rejected. The remaining molecules are then subjected to more detailed yet rapid turnover studies. These include drug interactions and cardiotoxicity studies.

Tertiary screening

Tertiary screening essentially consists of screening assays to test for potential drug interactions and cardiotoxicity. Several in vitro techniques are used on a case by case basis. Generally, the following tests are used.

1. P-glycoprotein (Pgp)—This is the product of the multidrug resistance gene, is an ATP-dependent efflux transporter that affects the absorption, distribution, and excretion of a number of clinically important drugs. For example, Pgp limits the intestinal absorption of digoxin, talinolol, and cyclosporin after oral dosing, limits the central nervous system penetration of human immunodeficiency virus protease inhibitors, and excretes paclitaxel into the intestine. Due to the significance this drug efflux transporter can have on in vivo disposition and pharmacokinetics, identification of compounds that are Pgp substrates can aid the optimisation and the selection of new drug candidates.

2. p450 inhibition and induction— Unmanageable drug-drug interactions have led to the withdrawal of many drugs from the market. Many of these interactions involve inhibition and, to a lesser extent, induction of drug metabolising enzymes. Consequently, the ability to predict metabolically-based drug-drug interactions early in the drug development process is essential. This is the simplest form of enzyme inhibition, where the inhibitor drug occupies the active site of the enzyme, blocking the metabolism of the 'victim' drug. Quantitative prediction of the extent of inhibition depends on the inhibitor concentration at the active site and the inhibition constant defining the interaction. Enzyme induction is another major mechanism of pharmacokinetic drug-drug interactions. Enzyme induction studies are generally performed using human hepatocytes.

3. hERG—hERG is a gene (KCNH2) that codes for a protein in cells known as the Kv11.1 potassium ion channel; this ion channel protein is best known for its contribution to the electrical activity of the heart that coordinates the heart's beating. When this channel's ability to conduct electrical current across the cell membrane is inhibited or compromised, either by application of drugs or by rare mutations in some families, it can result in a potentially fatal disorder called long QT syndrome; a number of clinically successful drugs in the market have had the tendency to inhibit hERG, and create a concomitant risk of sudden death, as an unwanted side effect, which has made hERG inhibition a central issue in both drug regulation and drug development. Although there exist other potential targets for cardiac adverse effects, the vast majority of drugs associated with acquired QT prolongation are known to interact with the hERG potassium channel. One of the main reasons for this phenomenon is the larger inner vestibule of the hERG channel, thus providing more space for many different drug classes to bind and block this potassium channel. Due to the awareness of the potential danger of such QT drugs the regulatory authorities issued recommendations for the establishment of cardiac safety during preclinical drug development. Approximately 90 percent of clinical candidates fail during the development stage with the estimated costs due to poor absorption, distribution, metabolism, elimination or toxicity (ADME/Tox) properties believed to be between $ 50 million and $ 70 million. Innovative solutions and early introduction of ADME/Tox technologies now offer the promise of reducing attrition rates during clinical development. Such advances are poised to help multinational pharmaceutical and biotechnology companies benefit from the 'fail early, fail cheaply' syndrome.

Dr S S Murugan, Scientific Director, RCC Laboratories India, can be contacted at siva.murugan@rccltd.in