CYP 1A series
CYP1A1
The gene that codes for CYP1A1 is on chromosome 15. This isoform binds and oxidizes planar aromatic, essentially flat, lipophilic molecules. The most common representatives of these compounds are multiples of benzene, such as naphthalene (two benzenes), and what are usually termed polycyclic aromatic hydrocarbons (PAHs) that are many benzene molecules in chains. There is evidence that CYP1A1 also processes many other variants of planar aromatics, such as the polybrominated diphenylethers (PBDEs), known for their carcinogenicity and endocrine disruptor effects. Interestingly, this isoform is hepatically ‘non-constitutive’, i.e. it is not normally expressed or found in the liver. This is probably because the accumulation of large amounts of planar aromatics in the liver should not normally occur. CYP1A1 is inducible in all tissues, and this occurs in the lung in response to aromatics encountered in industrial and traffic pollution. Tobacco smokers exhibit high lung levels of this CYP due to the PAHs and other aromatics present in the smoke. Interestingly, non-smokers exposed to environmental tobacco smoke also show increased levels of CYP1A1. Metabolic products of CYP1A1, which are often epoxides, vary in their stability and the most reactive, such as those from benzpyrene derivatives, are carcinogenic. There is also evidence of higher levels of CYP1A1 in breast cancer sufferers. Experimentally, this CYP is often studied through its ability to O-deethylate the test substrate 7 – ethoxyresorufin. In general, CYPs have partly evolved to clear potential threats to the organism, so the metabolism and clearance of toxic agents such as the PDBEs by CYP1A1 would be necessary and beneficial. However, the isoform is polymorphic and its absence may predispose to toxicity with such agents. In addition, CYP1A1-mediated production of reactive metabolites is likely to be more of a threat than a protection, as it is often overexpressed in the vicinity of carcinogenesis. Hence, whether CYP1A1-mediated clearance of xenobiotics is beneficial or deleterious may change from day to day according to the individual. The proton pump inhibitor omeprazole induces this CYP in man and in vitro studies suggest it also inhibits it, thus possibly protecting cells from CYP1A1-formed reactive species.
CYP1A2
This CYP is also found on chromosome 15, only 25 kilobases away from CYP1A1 in man and it is linked with oestrogen metabolism. Increased levels of this enzyme are also associated with colon cancer. CYTP1A2 oxidizes planar aromatic molecules that contain aromatic amines, which its relative CYP1A1 does not. CYP1A2 orientates aromatic amines, some of which are quite large, in such a way as to promote the oxidation of the amine group. Consequently, this enzyme is able to metabolize a variety of drugs that resemble aromatic amines: these include caffeine, β-naphthylamine and theophylline. The enzyme is also capable of oxidizing several tricyclic antidepressants (TCAs). It tends to be inhibited by molecules that are planar and possess a small volume to surface area ratio. It can be inhibited by the methylxanthine derivative furafylline, as well as ciprofloxacin, enoxacine, cimetidine, mexiletine and fluvoxamine. The CYP1A1 inducer omeprazole promotes a similar response in CYP1A2. Other inducers of this CYP include TCDD and (probably fairly heavy) consumption of broccoli .
CYP1B1
To provide a perspective on the vast stretches of time over which CYPs have evolved, it is thought that CYP1B1 became distinct from the CYP1A sub-family over 300 million years ago. The isoform CYP1B1 is found in most tissues expressed at a modest or low level and it does not appear to make much contribution to drug clearance. CYP1B1 is the only member of its sub-family discovered so far and the gene that codes for it is found on chromosome 2. It is inducible through the same pathway as CYP1A1/2 and it can form reactive and carcinogenic species from endogenous oestrogens as well as from PAHs and it also metabolizes the anti-oestrogenic drug tamoxifen. Recent research has uncovered several endogenous roles for CYP1B1, which paradoxically may even include suppression of tumorigenesis, if work in mice is confirmed in man. It is now established that CYP1B1 is vital in eye development and it catalyzes the production of an arachidonic acid metabolite which maintains the transparency of the cornea, as well as the regulation of its aqueous humour. Indeed, mutations in this CYP are linked with congenital glaucoma, where intra- ocular pressure is excessive and can lead to early blindness. CYP1B1 ’s role in oestrogen metabolism is linked strongly with its overexpression in malignant breast tissue. Indeed, CYP1B1 is overexpressed in tumours of so many differing tissues (particularly the lung and pancreas) that it is being considered as a therapeutic target, as the effects of anticancer prodrugs which are activated by this CYP would be confined to the tumours, as the CYP is poorly expressed in bystander tissues. Interestingly, the messenger RNA which codes for CYP1B1 has a site which binds a microRNA known as MIRN27B, which is thought to inhibit the amount of active CYP produced. In patients with breast cancer, a study has shown that these patients possessed low levels of MIRN27B, which suggests that defective post-transcriptional regulation of CYP1B1 by MIRN27B was a major factor in the development of their malignancy.
CYP2 series
Around 18-30 per cent of human CYPs are in this series, making it the largest single group of CYPs in man. They appear to have evolved to oxidize various sex hormones, so their expression levels can differ between the sexes. As with many other CYPs, they are flexible enough to recognize many potential xenobiotics and they are thought to oxidize as much as half of all administered drugs.
CYP2A6
This CYP was originally of interest as it is partly responsible (with a cytosolic aldehyde oxidase) for the metabolism of nicotine to its much less pharmacologically active metabolite, cotinine. CYP2A6 is also known for its small binding site area in relation to other CYPs. More recently, studies of the polymorphisms associated with this CYP have indicated that low expression leads to reduced smoking behaviour and it is much easier for these individuals to stop the habit. CYP2A6 comprises up to 10 per cent of total liver CYP content and it uniquely clears coumarin to 7-hydroxycoumarin, which has been used as the major marker for this CYP for many years. Methoxsalen (an antipsoriatic agent) is a potent mechanism-based inhibitor of 2A6, as is grapefruit juice, although it is also weakly inhibited by imidazoles (e.g. keto-conazole). Methoxsalen will inhibit CYP2A6 in man and it prolongs the plasma survival of nicotine, so reducing smoking. Interestingly, CYP2A6 also has toxicological significance, in that it oxidizes carcinogens and mutagens, such as aflatoxins, 1, 3 butadiene and nitrosamines, which are all discussed. It tends to have a small role in the metabolism of a number of drugs (pilocarpine, letrozole and valproate), but it is often difficult to determine how large a contribution CYP2A6 is making in the metabolism of these substrates. This CYP is clinically inducible by anticonvulsants such as phenobarbi-tone and the antibacterial rifampicin, amongst others.
CYP2B6
This originates from a gene found on chromosome 19; the 2B series have been extensively investigated in animals, but CYP2B6 is the only 2B form found in man. This isoform is found in all human livers and comprises around 3-10 per cent of total hepatic CYPs. Its level of expression varies by more than 100,fold and it may be subject to sex differences, being more common in women than men. CYP2B6 may be the most polymorphic of all CYPs in man.It is thought to be implicated in the metabolism of more than 70 xenobiotics, including amfebutamone (bupropion), mephenytoin, some coumarins, cyclophosphamide and its relatives, the antimalarial artemisinine, selegiline (l- deprenyl), as well as methadone and ketamine. Three agents have been shown to inhibit this isoform, the antiplatelet drugs clopidogrel and ticlopidine and the antineoplastic drug thiotepa. It is inducible by rifampicin, phenobarbitone, the anti-HIV reverse-,ranscriptase inhibitor efavirenz and also the DDT substitute pesticide methoxychlor. This pesticide acts as an endocrine disruptor when it is oxidized to pro-oestrogenic metabolites by a number of human CYPs including CYP2B6 itself. CYP2B6 prefers non- planar neutral or weak bases which accept hydrogen bonding. It tends to hydroxylate at highly specific areas of molecules, particularly close to methoxy groups, which suggests that it may have a biosynthetic role in the assembly of specific endogenous molecules.
CYP2C8
This polymorphic CYP originates on chromosome 10 and is one of four CYP2C isoforms (the others are CYP2C9, 18 and 19) which comprise about 18 per cent of total CYP content which share 80 per cent amino acid sequence identity. CYP2C8 tends to bind and metabolize relatively large molecules which are also weak acids. It is known to clear the anticancer agent taxol as well as verapamil, cerivastatin (now withdrawn), amodiaquine and rosiglitazone. Although CYP2C8 is similar in structure to CYP2C9, it differs catalytically concerning several drugs, metabolizing tolbutamide more slowly than 2C9, but clearing trans-retinoic acid more efficiently. Warfarin is cleared by CYP2C9 to a 4-hydroxy metabolite, whilst 2C8 forms a 5- hydroxy derivative. Ibuprofen is mainly cleared by CYP2C9, whilst 2C8 plays a minor role. The different binding orientations of substrates such as diclofenac that both 2C9 and 2C8 metabolize are related to differences between the hydrophobicity and geometry of the respective binding sites. CYP2C8 can be inhibited by quercetin, the glitazone drugs, gemfibrozil and diazepam in high concentrations, as well as the leukotriene receptor antagonist montelukast and the antibacterial trimethoprim. The antiretroviral agents efavirenz and saquinavir are also inhibitors of this isoform. It is inducible by rifampicin and phenobarbitone.
CYP2C9
CYP2C9 has been particularly heavily studied in terms of its structure and function. It has evolved to process relatively small, acidic and lipophilic molecules that accept hydrogen bonds. It is thought that the structure of the access channel area may be responsible for this enzyme’s preference for acidic molecules. There are a large number of substrates for this CYP, which include tolbutamide, dapsone, phenytoin, valproate, rosuvastatin and warfarin. Substrates of CYP2C9 often have relatively narrow TIs, so polymorphisms and inhibition of the CYP can have serious clinical consequences. As described above in section 3.4.4, the active site is flexible, extensive and capable of great binding ‘plasticity’. Indeed, initial binding is not always productive, in the case of warfarin. This CYP is a particular focus for many analytical and theoretical studies to uncover CYP mechanisms of action, such as how substrate binding affects REDOX electron supply, subsequent substrate binding and promotion of product egress. Sulphafenazole is a potent inhibitor of this polymorphic enzyme and other inhibitors include amiodarone and fluconazole. It is inducible by the usual suspects (rifampicin and phenobarbitone) as well as the steroids prednisone and norethindrone.
CYP2C18
This CYP does not have any real role in either the metabolism of drugs or environmental toxins, as its mRNA is found in the liver in plentiful amounts, yet very little actual protein appears. Studies with the CYP when it is expressed have shown that it can clear some CYP2C substrates such as tolbutamide and ifosfamide, as well as some steroids. It is polymorphic, but there is relatively little literature on it and it does not appear to be linked with any disease states or conditions.
CYP2C19
This is also inducible and differs by only around 10 per cent of its amino acids from CYP2C9, but it does not oxidize acidic molecules, indicating that the active sites and access channels are subtly different. CYP2C19 prefers weak bases such as amides which have at least two hydrogen bond acceptors. It metabolizes omeprazole and its related proton pump inhibiting anti-ulcer agents, as well as S-mephenytoin and diazepam. CYP2C19 is inducible (rifampicin and carbamazepine) and polymorphisms in this gene exist and there is a higher incidence of poor metabolizer phenotypes in Chinese/Japanese than Caucasians. This means that parent drug can accumulate in poor metabolizers, sometimes advantageously in the case of ulcer therapy.Tranylcypromine acts as a potent inhibitor of 2C19, as well as the antiplatelet drugs (clopidogrel, ticlopidine) and fluvoxamine. Paradoxically, clopidogrel’s clinical anti-platelet response is linked strongly with CYP2C19 status.
CYP2D6
The gene which codes for this isoform is on chromosome 22; CYP2D6 processes basic drugs which feature a nitrogen atom which can be protonated, so it is responsible for myriad N-dealkylation reactions. Indeed, this CYP is responsible for more than 70 different drug oxidations and is particularly noteworthy as it is non-mducible, which is very unusual for human CYP isoforms. Famously, it was the first CYP to reveal the clinical effects of a genetic polymorphism,with around 7-10 per cent of Caucasians expressing poorly or even non-functioning enzyme. As with CYP2C9 substrates, in cases of polymorphism or enzyme inhibition, serious clinical problems can arise if CYP2D6 is the main route of clearance of a low TI drug. As many as 15 per cent of prescribed drugs are cleared by this isoform, including:
• antiarrhythmics: (flecainide, mexiletine);
• TCAs, SSRI and related antidepressants: (amitriptyline, paroxetine, venlafaxine, fluoxetine);
• antipsychotics: (chlorpromazine, haloperidol);
• beta-blockers: (labetalol, timolol, propanolol, pindolol, metoprolol);
• analgesics: (codeine, tramadol, oxycodone, hydrocodone, meperidine [pethidine]).
It is important to realize that the clinical risks of polymorphisms are difficult to assess conclusively and to some extent remain controversial.CYP2D6 also forms the metabolites necessary for the optimal anti-oestrogenic actions of tamoxifen. Quinidine, fluoxetine, propoxyphene, celecoxib and paroxetine inhibit this enzyme. Relatively little is found in the gut, and it comprises about 2-4 per cent of the CYPs in human liver.
CYP2E1
This comprises around 7 per cent of human liver P450 and is the only human member of the CYP2E subfamily. It is found in a number of extra-hepatic sites, such as the lung, kidney and lymphocytes. Curiously, CYP2E1 has been detected in cellular areas besides the endoplasmic reticulum, such as the Golgi apparatus and the plasma membrane. CYP2E is also found in mitochondria, although it is not clear why it is there but it is functional and uses adrenodoxin reductase to supply it with reducing power, as this variant does not use NADPH.
Generally, this CYP is also unusual in that it oxidizes small, often water soluble heterocyclic agents, ranging from pyridine through to ethanol, acetone and other small ketones such as methyl ethyl ketone and methyl isobutyl ketone (MEK and MIBK). Ethanol and acetone are strong inducers of this isoform and CYP2E1 is found in 5-10 fold greater quantities than normal in heavy drinkers, compared with those who imbibe moderately or lightly. Indeed, this ethanol-mediated induction leads to much increased vulnerability to CYP2E1-mediated drug and toxin damage, most notably paracetamol-induced liver failure in alcoholics.The muscle relaxant drug chlorzoxazone is cleared by this CYP and is a marker for CYP2E1 capability in man.
Many of the substrates of CYP2E1 are implicated in toxicity and/or carcinogenicity, as the metabolites formed can be highly reactive. It also seems that CYP2E1 produces large amounts of reactive oxygen species and it has been suggested that oxygen is its main substrate. This process underlines the significant role CYP2E1 has in oxidative stress in alcoholics. CYP2E1 is also linked with hepatotoxicity due to trichloroethylene, which was used in manufacturing as a degreaser and also in dry cleaning. It is also partly responsible for the oxidation of paracetamol and it is thought to convert acrylamide into its carcinogenic epoxide glycidamide. Several dietary N^itrosamines are activated by CYP2E1, as is the tobacco procarcinogen NNK (4-[methylnitrosamine]1-[3-pyridyl]-1-butanone). This is one of a number of CYP isoforms that may be related to smoking ~ induced cancers (see CYP1A1/2 above). Many sulphur-containing agents block this enzyme, such as carbon disulphide, diethyl dithiocarbamate and the ethanol abstinence-mducing drug antabuse.There is evidence in mice that CYP2E1 mRNA is tightly regulated around 24 circadian rhythms by hepatic nuclear factor-1a (HNF-1a) and molecular clock genes such as CRY- 1. It is probable that a similar system may exist in man and regulates more than one CYP.
CYP3A series
CYP3A4, 3A5, 3A7 and 3A43
All the CYP3A protein gene loci are found on chromosome 7. CYP3A5 exhibits only about a 10 per cent difference in its sequence homology with CYP3A4 and the two isoforms are also difficult to distinguish catalytically, as they metabolize mostly the same substrates at similar rates. CYP3A5 appears to be in the minority, as only about a fifth of human livers express it, making CYP3A4 the major human biotransformational CYP by some margin. In contrast, CYP3A5 is found in greater quantity than CYP3A4 in human lung. Overall, the two main CYP3A isoforms are responsible for the metabolism of more than 120 drugs and they comprise more than half of our hepatic CYP content. Indeed, the colour of a healthy liver with normal blood flow is actually partly due to these enzymes. CYP3A isoforms are also found in our intestinal walls in considerable quantity. The major endogenous function of the CYP3As is to metabolize steroids, but their active sites are so large and flexible that a vast array of different molecules can undergo at least some metabolism by these enzymes. Crystallographic studies have shown the three-dimensional structure of human CYP3A4 to resemble most closely bacterial CYPBM-3, but there are similarities with other bacterial CYPs that can metabolize erythromycin. It is clear that human CYP3A isoforms have much larger active sites compared with bacterial enzymes, which underlines their evolution to oxidize such a wide variety of exogenous substrates. As discussed above, like CYP2C9, CYP3A4 has a large access channel and hugely flexible active site with many points of potential hydrophobic binding. Binding studies with steroids and erythromycin again show the similarities with other CYP enzymes across various species.
Studying CYP3A4 can be difficult, as its expression is linked closely with the efflux pump p-glycoprotein and many drugs are substrates for the CYP and p-glycoprotein, so it is difficult to determine their respective contributions to drug clearance. Drugs used as ‘probes’ for CYP3A include the short-acting benzodiazepine midazolam, as well as erythromycin and alfentanil. The 6ß-hydroxylation of endogenous cortisol can also be used reliably to measure the effects of induction and inhibition of CYP3A activity. Total gut and hepatic CYP3A activity can be differentiated from just hepatic activity by using oral and intravenous midazolam administration respectively. Although some reactions are specific to CYP3A5, such as the demethylation of the anti-rejection drug tacrolimus in vitro’ it is usually very difficult in vivo to determine the relative contributions of CYP3A4 and CYP3A5.
CYP3A4 and CYP3A5 are inducible through several agents, such as anticonvulsants.Interestingly, with some inducers, the tissue concerned, as well as the isoform involved may be crucial to the inductive effect. In vitro studies with human cell models suggest that rifampicin and phenobarbitone preferentially induce CYP3A4, with little or no effect on CYP3A5. Conversely, phenobarbitone is a potent inducer of lung CYP3A5, with a minor effect on CYP3A4.
Inhibitors of the CYP3A isoforms include verapamil, the azole antifungals (ketocona-zole, itraconazole, fluconazole, voriconazole), as well as the macrolides (erythromycin, clarithomycin, troleandomycin) and various citrus juices, most notably grapefruit. Fascinatingly, verapamil will not inhibit CYP3A5 if cytochrome b5 is absent, which underlines the complex relationship between CYP REDOX-partners and substrate/inhibitor CYP binding (see end of section 3.4.7). Ketoconazole is thought to inhibit CYP3A4 more strongly than CYP3A5, whilst troleandomycin inhibits both isoforms to a similar degree, but through different mechanisms. Hence, although there are clearly differences between CYP3A4 and 3A5 in terms of their catalytic behaviour as brought out by the effects of various inhibitors and inducers, the net clinical impact of these differences is probably marginal at best.
Of the other CYPs in the 3A sub-family, CYP3A7 is mainly foetal, where it hydroxy-lates retinoic acid and steroids. It has been found in adults, mainly extrahepatically. CYP3A43 is a relatively recent discovery and it is found in the testis and prostate, although its level of expression is linked with a risk of cancer in these organs. Neither CYPs 3A7 nor 3A43 are involved with biotransformations on the scale of CYP3A4 and CYP3A5.
CYP3A4 binding characteristics-cooperativity
As mentioned previously, CYP3A4 and 3A5 are the most flexible CYP isoforms in terms of their ability to bind a vast range of different sized substrates, although this has been bought at the price of reduced stability of the structure of these proteins compared with other CYPs. Before CYP crystal structures were available, the active sites were studied through a combination of mutagenesis and binding studies. Using bacterial or insect cell expression systems, human genes that expressed CYP3A4 were damaged by the use of mutagens in areas coding for specific amino acids that corresponded to the active site of the enzyme. This would, of course, change the binding characteristics of the damaged enzyme. The information gained from these and other studies show that the CYP3A isoforms can bind up to three substrate molecules at a time, in a similar manner to CYP2C9. Clearly, in this area, our basic perception of enzymes just binding one substrate at a time is far too simplistic. CYPs have evolved the capacity to bind multiple substrates probably for efficiency, as it utilizes resources (reducing power) in the most rapid and economical way, in the same way an assembly line is more efficient than hand building cars. An added bonus, as we have seen, is that the space and flexibility in CYP3A4 and CYP2C9 allow the option of processing of very large molecules as well as a number of smaller ones.
Regarding the processing of multiple small molecules, if the substrates are the same, the process of one molecule being metabolized appears to allosterically adjust the mobility of the molecule on its binding site, making it easier or sometimes more difficult, for it to be oriented for oxidation, thus modulating the process of CYP function. This is termed a ‘homotropic’ effect. With CYP3A isoforms, the active site is large as we have seen and testosterone, for example, is a small molecule. When a single testosterone molecule binds there is no actual catalytic activity in the isoform. Indeed, the CYP is not even coupled to its REDOX partner and in effect, nothing happens. Once a second testosterone molecule binds, the CYP is coupled with its REDOX partner and is now ‘m gear’ and operational and ready to process at maximum capacity as the testosterone molecules displace bound water molecules in the CYP and cause each other to be ‘pushed’ towards the haem iron to be oxidized, rather like bullets packed into a magazine as they feed into a machine gun.
These multi-site bindings can be yet more complex; studies with flavonoids have shown that they can bind at one place in the active site and at the same time stimulate the metabolism of a different type of molecule (PAHs) in another area of the active site. This simultaneous binding at two distinct but adjacent areas of the same broad site also is thought to be responsible for the inhibitory effects of one compound on the metabolism of another, which is termed a ‘heterotropic’ effect. Testosterone metabolism is partially competitively inhibited by erythromycin. Whilst in another topic it will be explained how the amount of CYPs present in the liver is a response to substrate pressure ,it is now clear that there is a high degree of sophistication in control of function of all CYPs in all tissues. This is especially important in the liver, where fine – tuning of steroid levels is necessary in response to changes in menstrual cycles or pregnancy in women, as well as spermatogenesis formation in men, where various steroid molecules are required to maintain different levels relative to each other at specific times in the cycles. This allosterically based process of multi-site internal regulation of CYP function is just one of the mechanisms whereby hormone metabolism is controlled. As the presence and binding of one CYP substrate could markedly influence the clearance of another, it is clear how xenobiotics can and do exert very complex disruptive effects on hormone regulatory processes. In a sense, this is a potential ‘Achilles Heel’ of CYP regulation and function.
As has been mentioned, the range of substrates that can be oxidized by CYP3A4 runs from bulky molecules such as cyclosporine A (molecular weight 1202), to small phenolics such as paracetamol. That a molecule of fungal origin such as cyclosporine can be easily and rapidly metabolized by CYP3A4, underlines the evolution of these enzymes to cope with the possibility of the ingestion of exogenous toxin molecules in the diet of humans. Other substrates include: codeine (narcotic), diazepam (tranquillizer), erythromycin (antibiotic), lidocaine (anaesthetic), lovastatin (HMGCoA reductase inhibitor, a cholesterollowering drug), taxol (cancer drug), warfarin (anticoagulant).
Azole antifungal agents, such as ketoconazole and fluconazole, as well as anti’HIV agents such as ritonavir, inhibit CYP3A4. Due to the importance of this enzyme in the endogenous regulation of steroid metabolism, any inhibition can have serious consequences in the form of disruption of hormone control and more immediately, marked changes to the clearance of drugs metabolized by this CYP.
