CYP3A4

CYP3A4 is a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases that catalyze many reactions involved in drug metabolism and synthesis of steroids (including cholesterol), and other lipids. The CYP3A4 protein localizes to the endoplasmic reticulum, and its expression is induced by glucocorticoids and some pharmacological agents. substrates include acetaminophen (paracetamol), codeine, ciclosporin (cyclosporin), diazepam, erythromycin, and chloroquine. Most drugs undergo deactivation by CYP3A4, either directly or by facilitated excretion from the body. Also, many substances are bioactivated by CYP3A4 to form their active compounds, and many protoxins are toxicated into their toxic forms (see table below for examples). CYP3A4 also possesses epoxygenase activity in that it metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs), i.e. (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acids. EETs have a wide range of activities including the promotion of certain types of cancers (see epoxyeicosatetraenoic acid). CYP3A4 promotes the growth of various types of human cancer cell lines in culture by producing (±)-14,15-epoxyeicosatrienoic acids, which stimulate these cells to grow. The CYP3A4 enzyme is also reported to have fatty acid monooxgenase activity for metabolizing arachidonic acid to 20-Hydroxyeicosatetraenoic acid (20-HETE). 20-HETE has a wide range of activities that include growth stimulation in breast and other types of cancers (see 12-hydroxyeicosatetraenoic acid). == Evolution ==

The CYP3A4 gene exhibits a much more complicated upstream regulatory region in comparison with its paralogs. This increased complexity renders the CYP3A4 gene more sensitive to endogenous and exogenous pregnane X receptor (PXR) and constitutive androstane receptor (CAR) ligands, instead of relying on gene variants for wider specificity. This change in consequence contributes to an increased human defense against cholestasis. == Tissue distribution ==

Fetuses do not express CYP3A4 in their liver tissue, but rather CYP3A7 (), which acts on a similar range of substrates. CYP3A4 increases to approximately 40% of adult levels in the fourth month of life and 72% at 12 months. Although CYP3A4 is predominantly found in the liver, it is also present in other organs and tissues of the body, where it may play an important role in metabolism. CP3A4 is the major CYP enzyme in the intestine. CYP3A4 in the intestine plays an important role in the metabolism of certain drugs. Often this allows prodrugs to be activated and absorbed, as in the case of the histamine H1-receptor antagonist terfenadine. CYP3A4 has also been identified in the brain, but its role in the central nervous system is unknown. == Mechanisms ==

Cytochrome P450 enzymes perform an assortment of modifications on a variety of ligands, utilizing its large active site and its ability to bind more than one substrate at a time to perform complicated chemical alterations in the metabolism of endogenous and exogenous compounds. These include hydroxylation, epoxidation of olefins, aromatic oxidation, heteroatom oxidations, N- and O- dealkylation reactions, aldehyde oxidations, dehydrogenation reactions, and aromatase activity. Hydroxylation of an sp3 C-H bond is one of the ways in which CYP3A4 (and cytochrome P450 oxygenases) affects its ligand. In fact, hydroxylation is sometimes followed by dehydrogenation, leading to more complex metabolites. An example of a molecule that undergoes more than one reaction due to CYP3A4 includes tamoxifen, which is hydroxylated to 4-hydroxy-tamoxifen and then dehydrated to 4-hydroxy-tamoxifen quinone methide. Two mechanisms have been proposed as the primary pathway of hydroxylation in P450 enzymes. The first pathway suggested is a cage-controlled radical method ("oxygen rebound"), and the second involves a concerted mechanism that does not utilize a radical intermediate but instead acts very quickly via a "radical clock". == Inhibition through fruit ingestion ==

In 1998, various researchers showed that grapefruit juice, and grapefruit in general, is a potent inhibitor of CYP3A4, which can affect the metabolism of a variety of drugs, increasing their bioavailability. In some cases, this can lead to a fatal interaction with drugs like astemizole or terfenadine. In addition to grapefruit, other fruits have similar effects. Noni (Morinda citrifolia), for example, is a dietary supplement typically consumed as a juice and also inhibits CYP3A4. Pomegranate juice has shown some inhibition in limited studies, but has not yet demonstrated the effect in humans. == Variability ==

While over 28 single nucleotide polymorphisms (SNPs) have been identified in the CYP3A4 gene, it has been found that this does not translate into significant interindividual variability . It can be supposed that this may be due to the induction of CYP3A4 on exposure to substrates. CYP3A4 alleles that have been reported to have minimal function compared to wild-type include CYP3A4*6 (an A17776 insertion) and CYP3A4*17 (F189S). Both of these SNPs led to decreased catalytic activity with certain ligands, including testosterone and nifedipine in comparison to wild-type metabolism. By contrast, CYP3A4*1G allele has more potent enzymatic activity compared to CYP3A4*1A (the wild-type allele). Variability in CYP3A4 function can be determined noninvasively by the erythromycin breath test (ERMBT). The ERMBT estimates CYP3A4 activity by measuring the radiolabelled carbon dioxide exhaled after an intravenous dose of (14C-N-methyl)-erythromycin. == Induction ==

CYP3A4 is induced by a wide variety of ligands. These ligands bind to the pregnane X receptor (PXR). The activated PXR complex forms a heterodimer with the retinoid X receptor (RXR), which binds to the XREM region of the CYP3A4 gene. XREM is a regulatory region of the CYP3A4 gene, and binding causes a cooperative interaction with proximal promoter regions of the gene, resulting in increased transcription and expression of CYP3A4. Activation of the PXR/RXR heterodimer initiates transcription of the CYP3A4 promoter region and gene. Ligand binding increases when in the presence of CYP3A4 ligands, such as in the presence of aflatoxin B1, M1, and G1. Indeed, due to the enzyme's large and malleable active site, it is possible for the enzyme to bind multiple ligands at once, leading to potentially detrimental side effects. Induction of CYP3A4 has been shown to vary in humans depending on sex. Evidence shows an increased drug clearance by CYP3A4 in women, even when accounting for differences in body weight. A study by Wolbold et al. (2003) found that the median CYP3A4 levels measured from surgically removed liver samples of a random sample of women exceeded CYP3A4 levels in the livers of men by 129%. CYP3A4 mRNA transcripts were found in similar proportions, suggesting a pre-translational mechanism for the up-regulation of CYP3A4 in women. The exact cause of this elevated level of enzyme in women is still under speculation, however studies have elucidated other mechanisms (such as CYP3A5 or CYP3A7 compensation for lowered levels of CYP3A4) that affect drug clearance in both men and women. CYP3A4 substrate activation varies amongst different animal species. Certain ligands activate human PXR, which promotes CYP3A4 transcription, while showing no activation in other species. For instance, mouse PXR is not activated by rifampicin and human PXR is not activated by pregnenolone 16α-carbonitrile In order to facilitate study of CYP3A4 functional pathways in vivo, mouse strains have been developed using transgenes in order to produce null/human CYP3A4 and PXR crosses. Although humanized hCYP3A4 mice successfully expressed the enzyme in their intestinal tract, low levels of hCYP3A4 were found in the liver. Due to the enzyme's extensive presence in the intestinal mucosa, the enzyme has shown sensitivity to starvation symptoms and is upregulated in defense of adverse effects. Indeed, in fatheaded minnows, unfed female fish were shown to have increased PXR and CYP3A4 expression, and displayed a more pronounced response to xenobiotic factors after exposure after several days of starvation. By studying animal models and keeping in mind the innate differences in CYP3A4 activation, investigators can better predict drug metabolism and side effects in human CYP3A4 pathways. ==Turnover==

Estimates of the turnover rate of human CYP3A4 vary widely. For hepatic CYP3A4, in vivo methods yield estimates of the enzyme half-life mainly in the range of 70 to 140 hours, whereas in vitro methods give estimates from 26 to 79 hours. ==Technology==

Due to membrane-bound CYP3A4's natural propensity to conglomerate, it has historically been difficult to study drug binding in both solution and on surfaces. Co-crystallization is difficult since the substrates tend to have a low KD (between 5–150 μM) and low solubility in aqueous solutions. A successful strategy in isolating the bound enzyme is the functional stabilization of monomeric CYP3A4 on silver nanoparticles produced from nanosphere lithography and analyzed via localized surface plasmon resonance spectroscopy (LSPR). These analyses can be used as a high-sensitivity assay of drug binding, and may become integral in further high-throughput assays utilized in initial drug discovery testing. In addition to LSPR, CYP3A4-Nanodisc complexes have been found helpful in other applications including solid-state NMR, redox potentiometry, and steady-state enzyme kinetics. • tacrolimus, • many chemotherapeutics: • docetaxel, • etoposide, • SSRI antidepressants : • citalopram • opioids (mainly analgesics): • alfentanil, (analgesic, addiction maintenance treatment), • codeine (partial involvement, not the bioactivation factor), • methadone • some hypnotics: • zopiclone,), • felodipine), • nifedipine), • verapamil), • amlodipine), • lercanidipine, • kinins • glucocorticoids: • budesonide, • dexamethasone, • some H1-receptor antagonists (H1 antihistamines): • ketotifen, • terfenadine, • chlorphenamine; • delavirdine, (antihelminthic) • cisapride, (anticoagulant) • clopidogrel becoming bioactivated (antiplatelet), • 2-oxo-clopidogrel, • montelukast (leukotriene receptor antagonist), • vilaprisan (selective progesterone receptor modulator), • certain angiotensin II receptor blockers: • losartan (sensitive substrates) • irbesartan. The inhibitors of CYP3A4 are the following substances. Strong inhibitors • boceprevir, • protease inhibitors: • ritonavir, (although FDA lists it as a moderate inhibitor, and inhibitor of P-glycoprotein, defined as those increasing the AUC of digoxin to ≥1.25-fold); • some azole antifungals: • ketoconazole, • voriconazole; • dillapiole (compound present in dill plants), • apigenin (compound present in plants such as celery, parsley, and chamomile) • Artemisia annua Moderate inhibitors • amiodarone (class III antiarrhythmic), (dietary flavonoid), • tofisopam, • bergamottin Weak inhibitors • berberine (an alkaloid found in plants such as berberis or goldenseal), • buprenorphine (analgesic), • cafestol (in unfiltered coffee) • cilostazol, • amlodipine, • cannabidiol, • dithiocarbamate • mifepristone • delavirdine; • milk thistle, • niacin (nicotinic acid) and its form – niacinamide (nicotinamide), collectively called as Vitamin B3, • ginkgo biloba, • sesamin (a lignan constituent in sesame seeds and oil), • piperine, • isoniazid, • serenoa, • phenelzine. Inducers Strong and moderate CYP3A4 inducers are drugs that decrease the AUC of sensitive substrates of a given pathway where CYP3A4 is involved by ≥80 percent and ≥50 to <80 percent, respectively. The inducers of CYP3A4 are the following substances. Strong inducers • carbamazepine, • antiandrogens: • enzalutamide, • apalutamide; • primidone • phenytoin (anticonvulsant), • rifampin. • barbiturates: • brigatinib, • clobazam, • dabrafenib, • elagolix, • eslicarbazepine, • letermovir, • lorlatinib, • oritavancin, • perampanel, • telotristat. ==Interactive pathway map==