Screening for non-genotoxic carcinogens

Medicilon's toxicology department has professional teams with rich experience in toxicology studies. We offer high-quality data and rapid turnaround period to support drug discovery and development. Our toxicological studies are conducted in various animal species. The toxicological evaluation from dose design, in-life studies to histology and pathology testing along with toxicokinetics studies are all compliant with GLP or NON-GLP standards. Our study platform is certified as one of the Shanghai Public Service Research Platforms.

The invention relates to a method for screening for the effects of non-genotoxic carcinogens in an animal model. The invention also relates to animal models that are suitable for use in such a method, and cell lines derived from these animals for in vitro screening purposes. More specifically, the invention relates to a transgenic rodent animal which has been humanised for the nuclear transcription factors CAR, PXR and PPARα, and in which the endogenous equivalent genes have been rendered inoperable.

A further example is provided by the family of peroxisome proliferator activated receptors (PPARs), to which various drugs were in the past developed as hypolipidic agents. The development of these drugs was stopped, as they were identified in mouse and frequently rat models to be non-genotoxic (also called epigenetic) carcinogens. It was initially thought that these differences were due to differences in expression level. However, it turns out that, for unknown reasons the human receptor upon ligand binding does not activate the cell proliferation machinery in the same way as the mouse receptor does. In vitro screens often use human cells in an attempt to overcome the problems with cross-species variation. However, in vitro systems can only ever incorporate a small part of the drug metabolism landscape, and do not present a holistic view. Accordingly, the real in vivo effects may be disguised. For example, consider the frequent situation that arises when drugs look hazardous in vitro because a toxic by-product is generated but not in vivo because the drug activates a secondary enzyme that metabolises away the toxic by-product. It is also true that almost any compound will interact with a particular target at some level - the question of importance for drug safety is whether this interaction is physiologically relevant at the concentrations to which tissues will be exposed. The limitations inherent in the in vitro scenario make this solution inappropriate.

It would be of great utility if it were possible to demonstrate that a hyperplastic response does not occur in humans in response to drug exposure. The inventors have concluded that one effective way to generate a faithful test for safety of non-genotoxic carcinogens is through the use of rodents that have been humanised for the transcription factors with which non-genotoxic carcinogens principally interact. At the same time, it is essential to annul the expression of the equivalent endogenous rodent transcription factor genes in order to ensure that interference from non-human metabolic pathways on the functions of introduced human proteins is significantly reduced.

The inventors have noted that in general, compounds that are non-genotoxic carcinogens and cause liver tumours in rodents are PXR, CAR and PP ARa ligands. These cause hyperplasia, by either or both stimulating cell proliferation and inhibiting apoptosis. Additionally, they cause hypertrophy, stimulating organelle (eg. smooth endoplasmic reticulum, peroxisomes) proliferation through the smooth endoplasmic reticulum, and enzyme induction. The barbiturates induce primarily the P450 enzyme CYP2B; steroids primarily induce CYP3A, and peroxisome proliferators primarily induce CYP4A. Some chemicals interact substantially with multiple receptors and induce multiple cytochromes P450.

Mice that have been individually humanised for CAR, PXR or PP ARa currently exist. Indeed, Cheung et al 2004 monitored various physiological effects including the increase in liver body weight on exposure to drug in wild type and PP ARa knockout mice, and compare this response to that seen in humanised mice. The humanised mice showed a lesser increase in liver body weight and a lack of increased replicative DNA synthesis (a marker for hyperplasia).

However, the multiply humanised models of the present invention provide a significant advantage over the deletion of individual transcription factors because of the functional redundancy between members of the same gene families. The inventors consider that there are a number of reasons why an animal that has been humanised for all three receptors simultaneously will provide a significant improvement over the application of the current singly humanised models.

In the first instance, the transcription factors that principally mediate non-genotoxic carcinogens-regulated hyperplasia are all humanised, so resolving the problems associated with the differences in ligand specificity noted above. Therefore, one advantage of a humanised model is that it negates the issues of ligand specificity. AU of

PPARα, CAR, and PXR interact with exogenous ligands that transactivate gene expression, and thereby mediate pathways that the inventors consider to be potentially deleterious in the metabolism of non-genotoxic carcinogens.

The ratio of protein levels that are generated by a particular drug are also of significant importance. For example, the action of mouse PXR stimulates expression of different proteins than the action of human PXR and at different levels. The levels of a particular drug and its metabolites depends crucially on which drug metabolising enzymes and transporters are expressed and so, again, the inventors consider that it is of utmost importance for human transcription factors to be used rather than endogenous transcription factors from the test animal.

The use of the specified human transcription factors is also important from a toxicological standpoint. For example, PXR is naturally regulated by bile acids and other physiological compounds and toxic conditions such as biliary necrosis and biliary cholestasis can result from exposure to a particular drug. It may therefore be that as a result of differences between drug metabolism between human and a test animal, a toxic effect will be noted in that animal that would not be evident in the human.

One major advantage of these triple humanised animals on a triple null background is that there is significant redundancy between these transcription factors in their response to chemicals, as one chemical agent may interact with multiple receptors. Therefore, a mouse humanised for just one of these receptors would not give the correct magnitude of response.

Furthermore, there are ways in which cross-talk may occur between different nuclear receptors that are implicated in the metabolism of non-genotoxic carcinogens. The first is cross-talk between receptors at a molecular level. The second is cross-talk at a metabolic interface, for example through generation of cross-reacting secondary metabolites, or from changes in drug disposition. Thirdly, the nuclear receptors themselves can cross-talk and modulate each others' levels of expression and functions. A particular level of drug may activate genes that transactivate other genes, so leading to further levels of complication. Therefore, only by having a complex panel of humanised receptors will it give the bona fide response that is anticipated in man. Such cross-talk might in principle be predicted at some qualitative level, but because the magnitude of the effects and the extent of any feedback mechanisms are inherently unpredictable, this undermines the value of any system that does not incorporate all the necessary elements of the system at physiologically relevant levels.

One example where cross-talk between these receptors may be of key importance in defining the eventual outcome is the fact that CAR, PXR and PP ARa transcription factors, in addition to their activation by exogenous ligands, are regulated by perturbations in fatty acid homeostasis. It will be advantageous, therefore, to have mice that are humanised for all these receptors simultaneously.

Also, the simultaneous humanisation of animals at all three gene loci dramatically reduces the number of animals required to establish clearly whether a chemical agent has the capacity to induce hyperplasia, as it negates the need for carrying out multiple experiments on individual humanised animals. The inventors have noted that the capacity of promoters to induce enzyme expression is different in different tissues. This adds significant weight to the contention that human transcription factors should be used rather than the endogenous transcription factors from the host animal. Accordingly, the regulatory sequences of the transcription factors and the genes that they regulate should mirror the natural physiological condition as closely as possible.

Animals according to the invention may be any non-human species, for example a rodent, for instance a rat, hamster or a guinea pig, or another species such as a monkey, pig, rabbit, or a canine or feline, or an ungulate species such as ovine, caprine, equine, bovine, or a non-mammalian animal species. More preferably, the transgenic non-human animal or mammal and tissues or cells are derived from a rodent, more preferably, a mouse. Although the use of transgenic animals poses questions of an ethical nature, the benefit to man from studies of the types described herein is considered vastly to outweigh any suffering that might be imposed in the creation and testing of transgenic animals. As will be evident to those of skill in the art, drug therapies require animal testing before clinical trials can commence in humans and under current regulations and with currently available model systems, animal testing cannot be dispensed with. Any new drug must be tested on at least two different species of live mammal, one of which must be a large non-rodent. Experts consider that new classes of drugs now in development that act in very specific ways in the body may lead to more animals being used in future years, and to the use of more primates. For example, as science seeks to tackle the neurological diseases afflicting a 'greying population', it is considered that we will need a steady supply of monkeys on which to test the safety and effectiveness of the next-generation pills. Accordingly, the benefit to man from transgenic models such as those described herein is not in any limited to mice, or to rodents generally, but encompasses other mammals including primates. The specific way in which these novel drugs will work means that primates may be the only animals suitable for experimentation because their brain architecture is very similar to our own.

The invention aims to reduce the extent of attrition in drug discovery. Whenever a drug fails at a late stage in testing, all of the animal experiments will in a sense have been wasted. Stopping drugs failing therefore saves test animals' lives. Therefore, although the present invention relates to transgenic animals, the use of such animals should reduce the number of animals that must be used in drug testing programmes.

The regulatory sequences governing expression of the transcription factor(s) may preferably be either of human origin, or may originate from the target animal species e.g. the mouse. Regulation of the expression of introduced human proteins should be retained such that patterns of expression in the human are reproduced.