**1.1 Current situation in the development of pharmaceuticals**

A typical testing scheme for a small-molecule therapeutics (outlined in Fig. 1) begins with a large number of compounds and high-throughput assays (Kramer et al., 2007). As the number of viable lead molecules is reduced, incrementally more predictive but lower throughput assays identify those leads with the most drug-like properties and optimal in vitro and in vivo efficacy. Confirmed hit compounds identified in high-throughput screens are evaluated for potency, selectivity, ADME (absorption, distribution, metabolism and excretion), physical and chemical properties, and activity in relevant animal models (Fig. 1). This testing paradigm typically delivers drug-like compounds that have promising pharmacokinetic parameters and efficacy in preclinical models within a 1–2-year cycle time. Compounds that successfully meet preclinical efficacy, ADME, pharmacokinetics and safety criteria are nominated as candidates for formal development. Historically, the move from discovery to development consisted of a discreet hand-off from the 'discovery' organization to the 'development' organization, and little preclinical safety assessment was performed on lead molecules beyond a few basic in vitro toxicity assays. As toxicity is a primary cause for compound attrition and long development (Kola & Landis, 2004), companies in the past 5– 10 years have increasingly integrated safety assessment principles into earlier phases of the drug discovery process.

Also as shown in Fig.1, the costs of R & D for a drug in 2001 were of the order of US \$802 million (DiMasi et al., 2003); current estimates are closer to about US \$900 million; Considerably more of these costs are incurred later in the pipeline, and most of the attrition occurs during full clinical development (Phases II and III). In the other literature, it has been estimated that the average cost associated with the discovery and preclinical evaluation of a single drug candidate were US \$620 million (Rawlins, 2004).

Kola and Landis researched the reason why compounds undergo attrition and how this has changed over time (Kola. & Landis, 2004). In 1991, adverse pharmacokinetic and bioavailability results were the most significant cause of attrition and accounted for ~40% of all attrition. However, in 2000 the major causes of attrition in the clinical trials were lack of efficacy (accounting for approximately 30% of failures) and safety (toxicology in preclinical

Application of a New Genotoxicity Test System with

in demonstrating the absence of genotoxic activity.

as used in the genotoxicity test.

biological variation.

**vitro genotoxicity testing** 

**1.3 Recent achievement in the in vitro genotoxicity testing** 

Human Hepatocyte Cell Lines to Improve the Risk Assessment in the Drug Development 363

battery generally includes (i) an in vitro test for gene mutation in bacteria; (ii) an in vitro test in mammalian cells with cytogenetic evaluation of chromosomal damage and/or a test that detects gene mutations; (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. For compounds giving negative results in all 3 of the assays, the completion of this test battery is generally considered to provide a sufficient level of safety

The most widely used in vitro genotoxicity test is the Ames test (Ames et al., 1975). The relatively simplicity and low cost of the test make it a valuable screening tool for mutagens. However, DNA is naked in the prokaryote and the form of DNA is different from that in eukaryote. Thus, the test using mammalian cell lines has been developed Chromosomal alterations are quite common in malignant neoplasm, as such the detection of chromosomal abnormalities by test chemicals is considered an excellent test for the assessment of carcinogenic potential. In mammalian cell lines, most of the test systems used the same lines

An important discovery in the understanding of chemical carcinogenesis came from the investigation of the Millers who established that many carcinogens are not intrinsically carcinogenic, but require metabolic activation to be carcinogenic (Miller and Miller, 1947). They demonstrated that azo dyes covalently bind to proteins in liver, leading to the conclusion that carcinogens may bind to proteins that are critical for cell growth control (Miller and Miller, 1947). An additional investigation with other genotoxic carcinogens which requires metabolic activation confirmed that metabolism of the parent compound was necessary to produce a metabolite (activation) that was able to interact with DNA. In standard in vitro genotoxicity testing, an activation system is included with the purpose of generating electrophilic metabolites that can react with macromolecules including nucleic acids. To address the potential role of metabolism, the induced rat liver S9 has been adopted for in vitro genotoxicity tests as an exogenous activation system for detecting promutagens (Ames et al., 1973, Paolini, 1997). Its initial choice was logical; levels of several cytochrome P450 (CYP) enzymes are elevated after induction, in particular the CYP1A subfamily of enzymes (CYP1A1 and 1A2), which are efficient catalysts of the bioactivation of polycyclic aromatic hydrocarbons and azaarenes, aromatic amines and aflatoxins. These types of compounds were some of the first known and best understood mutagens and the Aroclor 1254-induced rat S9 fraction effectively allowed for their identification as mutagens. Its choice was also logical in that it provided a reliable, robust and readily available bioactivation system at a time when human-derived systems were rare or unavailable. Also, a rodent system can be more easily standardized than an exogenous human derived system that normally would rely on human tissue samples, which are subject to significant

**1.4 Problems in the use of rat liver S9 fraction as a metabolic activation system in** 

As mentioned in the above sections, the initial choice of rat liver S9 fraction as a metabolic activation system in the in vitro genotoxicity testing was logical. However, it can be questioned if the standard Aroclor-induced rat liver S-9 fraction represents an appropriate surrogate for the metabolic capabilities of humans for the following reasons (Ku et al., 2007; Obach and Dobo, 2008). First, it is now known that the rat and human CYP enzymes can

development and safety in clinical development accounting for a further approximately 30%). As a result, many companies developing small-molecule therapeutics have adopted a strategy that includes the earlier incorporation of preclinical safety assessment before advancement into regulated preclinical studies.

Fig. 1. A typical testing scheme in the development for a small-molecule therapeutics. PK/PD, pharmacokinetic/pharmacodynamic; POC, proof-of-concept; POP, proof-ofprinciple. ADME; absorption, distribution, metabolism and excretion, GLP; good laboratory practice.

#### **1.2 Safety assessment of pharmaceutical candidates before administration to humans according to the regulatory guidance**

Currently, drug companies tend to perform a fairly standard package of nonclinical studies before commencing First-In-Man (FIM) clinical trial investigations with pharmaceuticals. The non-clinical safety study recommendations for the marketing approval of a pharmaceutical usually include single and repeated dose toxicity studies, reproduction toxicity studies, genotoxicity studies, and local tolerance studies. For drugs that have special cause for concern or are intended for a long duration of use, an assessment of carcinogenic potential must be included. Other non-clinical studies include pharmacology studies for safety assessment (safety pharmacology) and pharmacokinetic (ADME) studies.

For the conventional, chemically-synthesized small molecules, such a package of studies is in agreement with international regulatory guidance as given by the International Conference on Harmonization (ICH M3-R2) (Table 1). The genotoxic potential has to be assessed comprehensively before administration to humans regardless for both chemicallysynthesized small molecules and biotechnology-derived pharmaceuticals.

According to the current international guidelines on genotoxicity testing of pharmaceutical candidates (ICH S2A, S2B and M3), a standard battery of tests has to be performed. This battery generally includes (i) an in vitro test for gene mutation in bacteria; (ii) an in vitro test in mammalian cells with cytogenetic evaluation of chromosomal damage and/or a test that detects gene mutations; (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. For compounds giving negative results in all 3 of the assays, the completion of this test battery is generally considered to provide a sufficient level of safety in demonstrating the absence of genotoxic activity.
