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

362 Toxicity and Drug Testing

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

Hit to lead

Lead optimization

Candidate seeking

**Preclinical development**

**Clinical development**

Phase III: Definitive clinical POP

High-throughput screening High content analysis, IC50 determination, Hit triage

GLP toxicology studies: General toxicity, Specific toxicity

Phase I: Safety and tolerability in healthy volunteers Phase II: Safety and tolerability in patients, early clinical POP

including genotoxicity, Safety pharmacology

Selectivity assays, *in vitro* efficacy assays, Tier I ADME/physical chemistry assays

*In vivo* efficacy assays (preclinical POC), Tier II ADME/physical chemistry assays

Second species PK, PK/PD modeling, Salt-form selection, Crystal form assessment

advancement into regulated preclinical studies.

1,000

Number of chemicals

100

Dozen

1-3

1

**according to the regulatory guidance** 

practice.

\$467 million

Cost for R & D

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

**1.2 Safety assessment of pharmaceutical candidates before administration to humans** 

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

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 chemically-

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

safety assessment (safety pharmacology) and pharmacokinetic (ADME) studies.

synthesized small molecules and biotechnology-derived pharmaceuticals.

\$335 million

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 as used in the genotoxicity test.

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 biological variation.

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

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

Application of a New Genotoxicity Test System with

**Chemical**

NADPH

providing an unrealistic metabolic profile.

binding to DNA to form DNA adducts.

CYP

Phase I drug metabolizing enzymes **Oxidation Reduction Hydrolysis**

Human Hepatocyte Cell Lines to Improve the Risk Assessment in the Drug Development 365

**Chemical**

**Cytosol**

**Microsomes**

CYP **Nucleus** NADPH

UGT UDPGA

SULT PAPS

NAT Acetyl-CoA

Phase I drug metabolizing enzymes **Oxidation Reduction Hydrolysis**

> GST GSH

Phase II drug metabolizing enzymes **Conjugation**

**Nucleus**

 A. Rat liver S9 mix B. Human hepatocyte Fig. 2. Detection of genotoxicity using rat liver S9 mix compared to the expression of genotoxicity occurring hepatocyte. A. The detection method of genotoxicity is currently used a rat liver S9 fraction in the *in vitro* genotoxicity testing. B. The expression of genotoxicity is occurred in the hepatocyte. It can be questioned if the standard Aroclorinduced rat liver S-9 fraction represents an appropriate surrogate for the metabolic capabilities of humans. However, in human hepatocyte, the genotoxicity was expressed through the comprehensive metabolic pathway including phase I and phase II drugmetabolizing enzymes. Thus human hepatocyte can be a good genotoxicity test system reflecting human metabolism. In addition, human hepatocyte has complete metabolism consisting oxidation, reduction, hydrolysis and conjugation, whereas rat liver S9 mix is set up to favor CYP-mediated metabolism and the other enzymes present in the system that could be responsible for detoxification of reactive intermediates are not supplemented with the appropriate cofactors (e.g., UGT, GST, methyl transfereases, etc), thus potentially

short-life phase-2 metabolites) will not penetrate cell membranes sufficiently. If these types of metabolites are generated extracellularly, most in vitro genotoxicity testing showed negative results since the access to nuclear DNA was difficult. Another reason is that the diffusion pathways are longer for externally generated active metabolites resulting in more opportunities for alternative chemical reactions (e.g. with components of S9 or cell membranes) than for metabolites formed in the target cell. Electrophilic metabolites of a chemical bind to serum or S9 proteins (forming protein adducts) and this reduces the rate of

Therefore it is considered that the use of genetically engineered cells is the most reliable remedy to avoid the shortcomings of the extracellular metabolic activation systems such as human S9 and recombinant human CYPs (Fig. 3). To be useful tools for the prediction of drug metabolism and toxicity in the human liver, Yoshitomi et al. established a series of HepG2 transformants expressing the cytochromes 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 with the apparent Vmax values for characteristic substrates (Table 2) in a previous work (Yoshitomi et al., 2001). Since most human drug metabolism is catalyzed by CYP1A2, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, this HepG2 transformant system would be

**Microsomes**

*In vitro* cytogenetic test **Mammalian cells** (human lymphocyte or Chinese hamster lung fibroblast)

**Cytosol**

**DNA**

Ames test **Bacteria** (Salmonella or E. coli)


Table 1. Non-clinical toxicology testing. Toxicological testing is conducted on large numbers of animals of different species in an attempt to predict adverse effects that might be triggered by the drug in humans. Genotoxicity assays are mandatory regulatory studies designed to detect potential mutagens and/or carcinogens.

differ in their substrate specificities and the reactions catalyzed (Guengerich, 1997). Second, with phenobarbital/ 5,6-benzoflavone induction, although the expression levels of CYP1A and 2B enzymes are markedly elevated, others such as CYP3A are affected only in a minor way, whereas others (e.g., CYP2C11) may decrease (Guengerich et al., 1982). Third, the system is set up to favor CYP-mediated metabolism. Some phase II enzymes, such as UDPglucuronosyltransferases (UGT), glutathione S-transferases (GST), sulfotransferase (SULT), or N-acetyl transferases, are not active in the reduced form of the nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented S9 system (S9 mix) because other cofactors and additives (e.g., uridine diphosphate glucuronic acid, glutathione, acetylcoenzyme A, etc.) would be needed (Ku et al., 2007; Obach and Dobo, 2008). This can be essential not only for reducing potential false positives (e.g., reactive electrophiles that would be rapidly quenched by conjugation in vivo before being able to cause mutation) but also for false negatives because some conjugation reactions can yield metabolites that are more reactive than their substrate (e.g., sulfation of N-hydroxy-2-acetylaminofluorene or acetylation of N-hydroxylated heterocyclic amines) (Dashwood, 2002; Ku et al., 2007). The rat liver S9 mix may represent an incomplete picture of the metabolism that can occur in vivo (Fig. 2).

To detect those genotoxic potential, some genotoxic metabolites have to be formed in the target cell by endogenous enzymes that are not represented in standard in vitro test systems. One of the major reasons is that certain types of active metabolites (including many

Acute toxicity To identify doses causing no adverse effect and doses

(Sub) Chronic toxicity To characterize the toxicological profile of a chemical

mechanisms

causing cancer.

body.

designed to detect potential mutagens and/or carcinogens.

causing major (life-threatening) toxicity.

To reveal any effect of an active chemical on mammalian

effects of a chemical on physiological functions in relation

to exposure in the therapeutic range and above.

following repeated administration.

Genotoxicity To detect chemicals that induce genetic damage by various

reproduction and development. Carcinogenicity To examine carcinogen that is an agent directly involved in

Immunotoxicology To detect immune dysfunction resulting from exposure of an organism to a chemical Local tolerance To ascertain whether chemicals are tolerated at site in the

Safety pharmacology To investigate the potential undesirable pharmacodynamic

Table 1. Non-clinical toxicology testing. Toxicological testing is conducted on large numbers

differ in their substrate specificities and the reactions catalyzed (Guengerich, 1997). Second, with phenobarbital/ 5,6-benzoflavone induction, although the expression levels of CYP1A and 2B enzymes are markedly elevated, others such as CYP3A are affected only in a minor way, whereas others (e.g., CYP2C11) may decrease (Guengerich et al., 1982). Third, the system is set up to favor CYP-mediated metabolism. Some phase II enzymes, such as UDPglucuronosyltransferases (UGT), glutathione S-transferases (GST), sulfotransferase (SULT), or N-acetyl transferases, are not active in the reduced form of the nicotinamide adenine dinucleotide phosphate (NADPH)-supplemented S9 system (S9 mix) because other cofactors and additives (e.g., uridine diphosphate glucuronic acid, glutathione, acetylcoenzyme A, etc.) would be needed (Ku et al., 2007; Obach and Dobo, 2008). This can be essential not only for reducing potential false positives (e.g., reactive electrophiles that would be rapidly quenched by conjugation in vivo before being able to cause mutation) but also for false negatives because some conjugation reactions can yield metabolites that are more reactive than their substrate (e.g., sulfation of N-hydroxy-2-acetylaminofluorene or acetylation of N-hydroxylated heterocyclic amines) (Dashwood, 2002; Ku et al., 2007). The rat liver S9 mix may represent an incomplete picture of the metabolism that can occur in

To detect those genotoxic potential, some genotoxic metabolites have to be formed in the target cell by endogenous enzymes that are not represented in standard in vitro test systems. One of the major reasons is that certain types of active metabolites (including many

of animals of different species in an attempt to predict adverse effects that might be triggered by the drug in humans. Genotoxicity assays are mandatory regulatory studies

Menu Purpose

General Toxicity

Specific Toxicity

vivo (Fig. 2).

Reproductive and developmental toxicity

A. Rat liver S9 mix B. Human hepatocyte

Fig. 2. Detection of genotoxicity using rat liver S9 mix compared to the expression of genotoxicity occurring hepatocyte. A. The detection method of genotoxicity is currently used a rat liver S9 fraction in the *in vitro* genotoxicity testing. B. The expression of genotoxicity is occurred in the hepatocyte. It can be questioned if the standard Aroclorinduced rat liver S-9 fraction represents an appropriate surrogate for the metabolic capabilities of humans. However, in human hepatocyte, the genotoxicity was expressed through the comprehensive metabolic pathway including phase I and phase II drugmetabolizing enzymes. Thus human hepatocyte can be a good genotoxicity test system reflecting human metabolism. In addition, human hepatocyte has complete metabolism consisting oxidation, reduction, hydrolysis and conjugation, whereas rat liver S9 mix is set up to favor CYP-mediated metabolism and the other enzymes present in the system that could be responsible for detoxification of reactive intermediates are not supplemented with the appropriate cofactors (e.g., UGT, GST, methyl transfereases, etc), thus potentially providing an unrealistic metabolic profile.

short-life phase-2 metabolites) will not penetrate cell membranes sufficiently. If these types of metabolites are generated extracellularly, most in vitro genotoxicity testing showed negative results since the access to nuclear DNA was difficult. Another reason is that the diffusion pathways are longer for externally generated active metabolites resulting in more opportunities for alternative chemical reactions (e.g. with components of S9 or cell membranes) than for metabolites formed in the target cell. Electrophilic metabolites of a chemical bind to serum or S9 proteins (forming protein adducts) and this reduces the rate of binding to DNA to form DNA adducts.

Therefore it is considered that the use of genetically engineered cells is the most reliable remedy to avoid the shortcomings of the extracellular metabolic activation systems such as human S9 and recombinant human CYPs (Fig. 3). To be useful tools for the prediction of drug metabolism and toxicity in the human liver, Yoshitomi et al. established a series of HepG2 transformants expressing the cytochromes 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 with the apparent Vmax values for characteristic substrates (Table 2) in a previous work (Yoshitomi et al., 2001). Since most human drug metabolism is catalyzed by CYP1A2, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4, this HepG2 transformant system would be

Application of a New Genotoxicity Test System with

Human Hepatocyte Cell Lines to Improve the Risk Assessment in the Drug Development 367

target cell, (2) requirement of high exposure concentration to compensate for short exposure times and (3) differences in metabolism compared to intact tissues. To overcome these limitations, the use of genetically engineered stable cell lines expressing CYPs has studied. The liver is the tissue containing the greatest concentrations of drug-metabolizing enzymes, such as the CYP enzyme family, among many others. In human liver, about 70% of the total CYP could be accounted for by CYP1A2, 2A6, 2B6, 2C, 2D6, 2E1 and 3A proteins (Rendic and Guengerich, 1997). In the extrahepatic organs such as lungs and kidneys, CYP1A1 is present. CYPs catalyze to form toxic reactive intermediates from many chemicals. As it is well known that there are significant quantitative and qualitative differences between laboratory animals and humans in their CYP subtypes, it is necessary to use human CYP

CYP1A2 CYP2B6

CYP2C9

**Chemical**

**Cytosol Microsomes**

CYP **Nucleus**

NAT UGT GST SULT

Phase II drug metabolizing enzymes **Conjugation**

Phase I drug metabolizing enzymes **Oxidation Reduction Hydrolysis**

*HepG2 transformants expressing human CYP isoforms*

CYP2C19

CYP2D6

Fig. 3. HepG2 transformants expressing human CYP isoforms relating drug metabolism. The

In vitro systems, particularly those derived from liver, are a commonly applied tool to gain a better understanding of the metabolism of drugs and other xenobiotics. Also in genotoxicity, a number of publications are discussed which are relevant for the use of human derived liver cell lines. One of the most promising lines is the human HepG2 cell line, originally isolated by Aden et al. in 1972 from a primary hepatoblastoma of an 11-yearold Argentine boy. This cell line retains many of the specialized functions normally lost by primary hepatocytes in culture such as secretion of the major plasma proteins. Since several publications alerted that HepG2 lacks a few drug-metabolizing enzymes such as CYP2E1 and 1A2, transfectants constitutively expressing these enzymes have been constructed. Cederbaum and coworkers developed a line, which possesses CYP2E1 activity and used it

pie chart shows the contribution of each CYP isoform to the human drug metabolism (Lewis, 2004). It has been concerned about the low CYP activities in HepG2 cells, so we established the HepG2 transformant system expressing a series of human CYP isoforms.

in a number of mechanistic studies (for review see Kessova and Cederbaum, 2003).

CYP1A1

CYP2E1

isoforms to predict the metabolism and toxicity of chemicals in humans.

CYP3A4

**Cytosol Microsomes**

**Nucleus** CYP

*HepG2 (low CYP activity)*

NAT UGT GST SULT

**Chemical**

Phase II drug metabolizing enzymes **Conjugation**

Phase I drug metabolizing enzymes **Oxidation Reduction Hydrolysis**

more suitable for the genotoxic assessment of chemicals than the induced rat liver S9 fraction in the routine screening when considering human hepatic metabolism in the future. Therefore in the present thesis, we explored the usefulness of a series of 10 transformants expressing major human CYP isoforms such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in HepG2 cells established previously to assess the genotoxicity of metabolites (Fig.3) (Hashizume et al., 2009; Hashizume et al., 2010).


Table 2. Characteristics of a series of 10 transformants expressing major human CYP isoforms such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 in HepG2 cells (Yoshitomi et al., 2001). a). Iwata et al., 1998.
