Cytochrome P450 humanised mice
© Henry Stewart Publications 2004
Received: 26 March 2004
Accepted: 26 March 2004
Published: 1 May 2004
Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach.
Cytochromes P450 (P450s) and other foreign compound- or xenobiotic-metabolising enzymes are the primary interface between the chemical environment and the body. Xenobiotics include drugs, environmental contaminants and food additives. They can also encompass natural plant chemicals such as flavonoids, steroids and phytoalexins. Since there are countless different compounds that can enter an organism, there must be a means for their elimination. This is particularly important for hydrophobic chemicals, which can dissolve in lipids and accumulate in the body, or those that can react with cellular macromolecules and cause toxicity and cancer. In order to cope with the onslaught of chemical insults, mammals have evolved a large number of enzymes that appear to function in the metabolism of xenobiotics. Although some metabolism of endogenous chemicals has been found, the physiological relevance of these reactions is unknown. As a general paradigm, xenobiotic-metabolising enzymes convert lipid-soluble foreign chemicals into derivatives that can easily be eliminated from the body.
List of xenobiotic metabolising enzyme
Cytochrome P450s (P450 or CYP)
Flavin-containing monooxygenases (FMO)
Epoxide hydrolases (mEH, sEH)
Phase 2 'transferases'
Glutathione S-transferases (GST)
NAD(P)H-quinone oxidoreductase (NQO)
Paradoxically, the xenobiotic-metabolising enzymes are also responsible for converting inert chemicals to electrophilic derivatives that can cause cellular damage and transformation. For example, the P450s can produce electrophilic epoxides that react with cellular nucleophiles, including DNA. Phase 2 enzymes can also participate in activation reactions, in particular through the production of unstable esters derived from N-oxides produced by P450s . Thus, some chemicals are inactivated while others are activated to electrophilic derivatives by xenobiotic metabolism.
The ability of xenobiotic-metabolising enzymes to handle many different types of chemicals is due to the fact that a single enzyme can metabolise a large number of different substrates. The substrate-binding site from a single P450 can accommodate different chemical structures. Oxidation can occur at different sites on the same molecule. There is also a high degree of overlapping substrate specificities among these enzymes, whereby a single compound can be metabolised by several enzymes. Thus, a limited number of P450s are able to metabolise scores of different chemicals, and this could account for the tremendous capacity for mammals with a limited number of enzymes to metabolise many different xenobiotics.
The xenobiotic-metabolising enzymes are important determinants for both the favourable (disease or symptom amelioration induced by drug action) and unfavourable (toxicity and carcinogenicity) responses to foreign chemicals. Patients can differ in both their responses to drugs and their susceptibility to chemically-induced toxicity due to the large degree of inter-individual differences in levels of expression. Since these enzymes activate carcinogens, there could be a difference in susceptibility to cancer which depends on levels of xenobiotic-metabolising enzymes. The latter has been examined by case control epidemiology studies that have attempted to determine the association between cancer and the presence of mutant alleles for xenobiotic-metabolising enzymes [9, 10].
There are marked species differences in the expression and catalytic activities of xenobiotic-metabolising enzymes. As a result, rodents and humans can metabolise a single chemical entity quite differently. In fact, even rats and mice, which have been evolutionarily separated by at least 17 million years, have unique sets of xenobiotic-metabolising enzymes. In particular, the xenobiotic-metabolising P450s show a high degree of species differences in the number of genes, regulation of genes and substrate specificities of individual enzymes . In humans, there are a limited number of P450s that are known to metabolise clinically used drugs, and these enzymes exhibit substrate specificities and regulatory patterns that can markedly differ from P450s in mice and rats. In fact, cloning and sequencing of P450s and, more recently, total genome sequencing have revealed 102 putatively functional genes and 88 pseudogenes in the mouse and 57 putatively functional genes and 58 pseudogenes in the human . Thus, humans have fewer functional P450s genes than mice, an interesting fact that may help to develop a hypothesis on the driving forces of the evolution of xenobiotic-metabolising enzymes . The human P450s also exist in a number of allelic forms, and in some cases where there are mutant gene-inactivating mutations, these lead to P450 polymorphisms (http://www.imm.ki.se/CYPalleles). The major P450s involved in drug metabolism are CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Those P450s that are mainly involved in the metabolism of toxicants and carcinogens are CYP1A1, CYP1A2, CYP1B1, CYP2A6 and CYP2E1. The major polymorphic P450s are CYP2A6, CYP2C9, CYP2D6 and CYP2C19. Other P450s exhibit marked inter-individual differences in levels of expression which are not due to polymorphisms, notably CYP3A4 .
Since drugs are metabolised and inactivated by P450s, these enzymes dictate the rate of drug elimination. In many cases, a patient must take more than one medication, and this can result in drug interactions if both agents are metabolised by the same P450. This phenomenon, referred to as 'drug interactions', can lead to toxicity due to inhibition of metabolism and the resultant high and sustained serum levels of one of the drugs. Thus, it is critical to determine which P450 is responsible for metabolising a particular drug. In fact, it is now routine practice for pharmaceutical companies to establish which P450s metabolise a drug before it is subjected to clinical trials.
Over the past ten years, it has become standard practice to determine how a drug candidate is metabolised and which P450 form is responsible for its metabolism. This aids in the prediction of drug safety. In particular, by knowing how a drug is metabolised, it is possible to determine whether there will be drug interactions and whether there may be problems with inter-individual differences in metabolism and clearance caused by P450 polymorphisms. The best scenario for a drug company is to develop a drug that is metabolised by several forms of P450. To determine how a drug candidate is metabolised, human liver slices, human liver hepatocytes and human liver microsomes can be used in conjunction with inhibitors of specific P450 forms and antibodies to specific P450s  Recombinant human P450s, expressed in systems such as baculovirus, have also gained widespread use [15, 16]. In practice, both human liver-derived cells and extracts and recombinant P450s are used to determine how a compound is metabolised. High-throughput robotic screening procedures have been developed using recombinant P450s in order to predict how drug candidates will be metabolised in humans. To determine which P450 has the highest rate of metabolism for a particular compound, standard Michaelis-Menton kinetics are used to estimate the relative rate of clearance or Km/Vmax. In addition, it should also be noted that computer modelling has been of some value in predicting metabolism with certain human P450s, such as CYP2C9 and CYP2D6 .
Two strategies can be used to produce a humanised mouse. Historically, placing the cDNA behind a tissue-specific promoter has been used to make transgenic mice by standard pronuclei injection of recombinant DNA. For example, the rat albumin or mouse transthyretin promoter can be used to make transgenic mice that would allow for tissue-specific expression in liver. As an example, the human foetal CYP3A7 was expressed in liver using a metallothionein-1 promoter . A second strategy for making humanised mice is to use genomic clones containing the complete gene upstream sequences that include all cis-acting regulatory elements. For example, a bacterial artificial chromosome (BAC) clone can be successfully used to generate a transgenic mouse. This would allow for tissue-specific and inducible regulation of the transgene and thus is the preferred method for making a humanised mouse that would be the most biologically predictive. The gene of choice can be found on the Celera or public human gene sequence databases, and a suitable BAC clone containing the full gene and its regulatory elements identified and used as a transgene. The BAC clone containing the full gene should not contain other gene open reading frames. Several transgenic founders (independent offspring derived from the egg injections) should be characterised to find a line that exhibits expression of the transgene in a tissue-specific manner that reflects the expression found in humans. In the experience of the author, BAC transgenes are stable and expressed over many generations.
CYP2D6 metabolises a large number of drugs that are used in the treatment of psychiatric disorders, including the antidepressants amitriptyline, fluoxetine and imipramine, and the neuroleptic agents haloperidol and risperidone. In addition, CYP2D6 carries out the demethylation of codeine to the more potent analgesic morphine. These data suggested that endogenous neurochemical substrates may exist for CYP2D6. Molecular modelling has been used to analyse CYP2D6 substrates. This revealed that 5-methoxytryptamine (5-MT) is a potential substrate for this P450. Indeed, 5-MT was found to be demethylated to 5-hydroxytryptamine (5-HT), also called serotonin, by recombinant CYP2D6 and transgenic CYP2D6 . Conversion of 5-MT to 5-HT occurs at a higher rate in the CYP2D6-humanised mice as compared with the wild-type mice. These studies revealed a new role for CYP2D6 in the serotonin-melatonin cycle.
CYP3A4 is the most important of the human P450s for the metabolism of drugs. It is the most abundantly expressed P450 in human liver microsomes and is known to metabolise more than 60 per cent of all therapeutic drugs used in the treatment of many disorders, including hypercholesterolaemia (statin drugs), bacterial infections (erythromycin) and autoimmunity (cyclosporine). Thus, the potential for drug interactions is great and is of concern for the development of new drugs . In humans, four CYP3A P450s are expressed: CYP3A4, CYP3A5, CYP3A7 and CYP3A43. Interestingly, mice have eight CYP3A genes . The most abundantly expressed CYP3A gene in humans is CYP3A4. This P450 is especially of interest because it is not only expressed in liver, but also in the gut, where it can metabolise a large number of orally administered drugs.
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