Bacterial mutagenicity screening in the pharmaceutical industry

Abstract

Genetic toxicity testing is used as an early surrogate for carcinogenicity testing. Genetic toxicity testing is also required by regulatory agencies to be conducted prior to initiation of first in human clinical trials and subsequent marketing for most small molecule pharmaceutical compounds. To reduce the chances of advancing mutagenic pharmaceutical candidates through the drug discovery and development processes, companies have focused on developing testing strategies to maximize hazard identification while minimizing resource expenditure due to late stage attrition. With a large number of testing options, consensus has not been reached on the best mutagenicity platform to use or on the best time to use a specific test to aid in the selection of drug candidates for development. Most companies use a process in which compounds are initially screened for mutagenicity early in drug development using tests that require only a few milligrams of compound and then follow those studies up with a more robust mutagenicity test prior to selecting a compound for full development.

This review summarizes the current applications of bacterial mutagenicity assays utilized by pharmaceutical companies in early and late discovery programs. The initial impetus for this review was derived from a workshop on bacterial mutagenicity screening in the pharmaceutical industry presented at the 40th Annual Environmental Mutagen Society Meeting held in St. Louis, MO in October, 2009. However, included in this review are succinct summaries of use and interpretation of genetic toxicity assays, several mutagenicity assays that were not presented at the meeting, and updates to testing strategies resulting in current state-of the art description of best practices. In addition, here we discuss the advantages and liabilities of many broadly used mutagenicity screening platforms and strategies used by pharmaceutical companies. The sensitivity and specificity of these early mutagenicity screening assays using proprietary compounds and their concordance (predictivity) with the regulatory bacterial mutation test are discussed.

Introduction

In the pharmaceutical industry, genetic toxicity testing is used as an early alternate for carcinogenicity testing. Regulatory agencies require genetic toxicity testing is conducted prior to initiation of first in human (FIH) clinical trials and subsequent marketing for most small molecule compounds. A compound's potential to induce genotoxic damage is usually evaluated using a 3- or 4-test battery with individual tests detecting specific endpoints indicative of DNA damage. A typical standard test battery consists of a gene mutation assay conducted in bacteria, an in vitro mammalian chromosome damage test, and an in vivo test for structural and/or numerical chromosome damage, all conducted in compliance with Good Laboratory Practices (GLPs) [1]. Bacterial mutagenicity tests are widely used in the pharmaceutical industry during drug discovery as part of a compound selection strategy. The Salmonella-reverse-mutation assay or Ames test is the gold standard for mutagenicity testing and has been shown to be the most predictive in vitro assay for rodent and human carcinogenicity [1], [2].

The Ames assay was developed by Bruce Ames and colleagues in the mid 1970s [2] and has been subsequently revised over the years to improve sensitivity to many types of mutagens [3], [4], [5]. This assay serves as an important initial assay to determine a compound's mutagenic potential. Multiple strains of Salmonella typhimurium and Escherichia coli have been created that carry specific distinct mutations in either the histidine or tryptophan synthetic pathway, respectively, that result in the requirement for an exogenous supply of those amino acids for growth (auxotrophy). In a typical regulatory-compliant assay [5] these bacterial strains are grown in agar-filled 100 mm Petri dishes along with the compound under investigation. Compounds that are mutagenic lead to the reversion of the mutation back to the wild type such that exogenous amino acids are no longer required for growth. These revertant colonies are enumerated on agar Petri dishes after a growth period of 48–72 h [2]. The test compound is considered mutagenic, or “positive” in the assay, if the fold-increase in revertant colonies in test compound-treated dishes exceeds typically 2 to 3-fold that of vehicle-treated controls or, less commonly, if the increase is statistically significant. The bacteria used in the test are engineered to be highly sensitive to a variety of mutagens through reduction of DNA repair capability and enhanced cell wall permeability to test articles. This assay also has the ability to determine the molecular nature of mutations by employing tester strains carrying base-pair substitutions or frameshift mutations. A large proportion of compounds require metabolic activation to the ultimate mutagenic form. In these cases, a variety of exogenous metabolic activation sources are built into the standard test design (for example, rat or hamster Aroclor-induced liver S9). Typically, only phase I metabolism is simulated in the standard test; however, phase II conjugation co-factors can also be included in the test design.

The bacterial reverse mutation test has been shown to be about 65% concordant with rodent carcinogenicity or in vivo genetic toxicity [6]. Thus, in a typical predictive paradigm, without further investigation into the mode of action, a chemical is generally considered potentially carcinogenic when a positive result is observed. These features make the Ames test the gold standard in vitro mutation assay used globally to assess a chemical's mutagenic potential [7], [8], [9].

It should be noted that the original assay described by Ames and colleagues does not describe all of the current tester strain recommendations as found in OECD 471 “Bacterial Reverse Mutation Assay”, (notably the use of Escherichia coli) the pharmaceutical industry, in general, refers to that test as “the Ames assay.” As will be described, a number of the screening variations presented in this paper are referred to as “mini-Ames” or “micro-Ames” tests. Therefore, for simplicity throughout this review we will frequently refer to the standard, OECD compliant test as an “Ames assay” or “Ames test” and the test for regulatory submissions as a “GLP Ames assay”. Likewise, “standard plates” refer to the traditional, 100 mm Petri plates or dishes commonly used in the Ames assay.

The Ames test is one of the most widely used tests for early mutagenicity detection and for potential carcinogenicity prediction, due to its simple format, short assay times, relatively small compound requirement and ability to evaluate parent and phase I metabolites simultaneously in a single test system. Despite the relatively high degree of sensitivity for identification of genotoxic carcinogens achieved with the in vitro genotoxicity battery of tests, it has been desirable and essential that assay improvements and new tests with higher throughput, lower compound requirements, which provide more mechanistic data than traditional assays be developed.

However, the significance of “higher throughput” modifications/alternative tests should not be confused with “high-throughput” testing that is often employed in early drug-discovery where thousands of compounds can be assayed at one time. At best, modifications to improve the efficiency of genotoxicity evaluation can be described as “medium throughput” with some tests achieving capabilities of up to about 100 compounds per assay. Therefore it is that there are tests which offer the researcher a reliable understanding of genetic toxicity liability on many more compounds than can be evaluated by the “standard” genetic toxicology tests.

Characterization of the mutagenic potential of new chemical entities in the pharmaceutical industry is mandated by regulatory guidelines [1]. However, most companies test for mutagenic potential to help prioritize chemical series or candidate selection prior to mandated testing. This testing is carried out in different ways depending on the specific situation; for example, in early discovery, lead optimization, or on occasions where an assay may be chosen largely based on compound availability. Because these screening assays are most often used for internal decision making processes, they are not conducted to GLP standards and usually are not included with regulatory submissions. While protocol driven, there is complete freedom to vary the assay as necessary to investigate mutagenic potential, including use of non-standard bacterial strains or non-traditional methods. With our increased concern over the health risks of mutagenic exposure, Ames-positive molecules are generally not developed into commercial drugs. This may depend, however, on the relative risk-benefit analysis of a drug being developed for a life-threatening illness such as cancer. Additionally, by the time a compound is tested in a GLP Ames assay for regulatory submission, there has typically been a significant investment in the compound in terms of the conduct of other regulatory toxicity tests, manufacture of clinical supplies, etc. So it is crucial that compounds reaching regulatory submission stage show no mutagenic potential in the Ames test. Therefore, mutagenicity screening assays carried out prior to the regulatory studies must be highly predictive of GLP Ames results.

Because of the varied terminology within the pharmaceutical industry for the sub-stages of drug discovery and development the steps discussed in this review will be divided into three stages:

• Lead Identification: this stage is the earliest, where chemical series are identified as potential “hits” against the biological target of interest (“Hit identification” or “Hit-to-Lead”). This is also the stage where chemical series or scaffolds are investigated for maintaining potency while chemistry is performed to make the scaffold more pharmacologically desirable. In this stage, a discovery project team may have many potential series, many congeners to synthesize, but limited material available for testing.

• Lead Optimization: at this stage, one or more chemical series have been identified based on potency against the target and desirable properties e.g. Pharmacokinetics that makes them amenable to drug development. At the lead optimization stage, compounds are further refined to improve these drug-like properties like bioavailability, metabolic stability, clearance, solubility in drug vehicles etc. It is between lead identification and lead optimization that early mutation testing often occurs.

• Candidate Selection: at this stage, typically 1–3 unique compounds have been identified and optimized for final selection to move into early regulatory drug development and pre-clinical GLP testing. Very often the mutagenic potential of the final candidates have been identified by this stage, but sometimes more robust non-GLP testing may be required. Often at this stage non-GLP in vivo toxicology testing is performed to select the best candidate to advance.

The objective of this review is to examine the different mutagenicity assay strategies used by pharmaceutical companies, including a discussion of the overall strengths and weakness of each assay. Since validation studies are usually carried out prior to full-scale use of each assay, specific validation data will not be included here except where needed to indicate the scope and breadth of such validations.

The assays presented are those used in the pharmaceutical industry: miniscreen Ames (6 well-format), Micro-Ames (24- and 96-well format), Mini-Ames (mix of strains, standard Petri dishes), Bioluminescent (BioLum 24-well) Ames assay, Ames II, 5-Fluorouracil (FU) assay, the Xenometric Ames microplate format (MPF) assay, modified fluctuation assay, Vitotox, Xenometrics Pro-Tox, Micro Ames SOS chromotest and Salmonella SOS/umu test.