The Action Of Drugs On Cytochromes

Published Date: 02 Nov 2017

Abstract

Cytochrome P450 are considered to be the major enzymes which are responsible for the biotransformation of drugs. By now, numerous researches have been carried to exploit the mechanism of these enzymes metabolizing target drugs. The aim of this review is to summarize the general knowledge of cytochrome P450 and the molecule mechanism in various kinds of drug metabolism, which can result in drug activation�� toxicity and influence the pharmaceutical effect. The catalytic mechanism of CYP450 varies with the characteristics of target drugs. In this review, several typical mechanisms which are well-studied hitherto are introduced. Ritonavir, an anti-HIV drug, is the substrate as well as the most effective inhibitor of cytochrome P450 3A4. It binds to CYP 3A4 irreversibly and leads the suicide inhibition, causing the inactivation of CYP3A4 and high concentration of other anti-HIV drugs metabolized by CYP 3A4 in plasma. Antimycotics is involved in the drug-drug interaction, affecting the pharmacology efficiency of the co-administered drugs. For instance, the combination of midazolam and antimycotics can result in hypnosis instead of sedation in clinics. Paradoxically, instead of detoxication, CYP 450 can increase the risk of toxication of some drugs. Paracetamol is one of the most widely used over the counter drugs, but its toxicity is one of the most common causes of poisoning in the world. GST and CYP 450 are considered to be responsible for the catalysis of paracetamol. Among these enzymes, CYP 2E1 is the principal enzyme initiating the cascade metabolism of the toxicity of Paracetamol. CYP2E1 is essential in the cleavage of xenobiotics, however, high level of CYP2E1 can induce the overproduction of peroxide, damaging the macromolecules.

Introduction

Cytochrome P450 are a group of haemoprotein which catalyze a series of oxidation of chemical substances. They are widely discovered in animals, plants, fungi, bacteria and archaea.[1] The human P450 superfamily is consisted of 18 families, 42 subfamilies and 57 individual genes. P in P450 represents the letter in word pigment, 450 reflects the maximum wavelength at which the enzymes absorb when they are in the reduced states and complexed with CO. The substrates of these enzymes can be metabolic intermediates (lipids, steroidal, hormones), xenobiotics, drugs, and other toxic chemicals. It is evident that the functions of P450 evolved during the course of the biological development from prokaryotic to eukaryotic. Being a haemoprotein, the haem group is usually used to bind the dioxygen. However, in P450, dioxygen is not only bound, but activated via the thiolate ligand.[2] The Fe2+/Fe3+ redox potential is largely influenced by the binding substrate, i.e. the redox potential changes need the presence of redox partners. Some drugs are designed on the basis of this mechanism. For example, in the ideal system of CYP 101, the substrate camphor binds the CYP 101, making its redox potential lie between those of the NADPH, FAD and dioxygen. In this case, the redox reaction will not happen without the existence of camphor. [3]

Some residues on the surface of the CYP 450 are considered to interact with their redox partners. For example, lysine, situated between the K and K�� helices, is thought to be as a site for ion-paired contact with flavoprotein reductase and for ferredoxin reductase in certain bacterial P450.[4]. The dioxygen is an important substance that the haem core in cytochrome P450 binds. As stated above, the binding of dioxygen can facilitate the change of redox potential. Oxygen is an essential substance in biological reaction, but it is inert because it is in a triplet state while other substances are usually in a singlet state. The haem core can bind the dioxygen in the form of O2- with an electron transferred from the Fe2+ to the oxygen. The process changes the redox potential of cytochrome P450 to make it less negative, thus facilitate the binding of its redox partner. The binding substrates vary with the target enzymes to which they bind. The substrate selectivity is a new research area for the development of chemical drugs. However, this research is complex for different enzymes exhibit distinct, but overlapping selectivity.

It is well-known that the lipophilic character has an obvious impact on the activity of CYP 450. It is also the mechanism based when measure the structure which undergo the cytochrome P450-dependent metabolism because 95% of the substrates are lipophilic positive.[5]

Cytochrome P4503A4 and ritonavir

Cytochrome P450 3A has four well-known genes, CYP 3A4, CYP 3A5, CYP3A7, CYP 3A43. CYP 3A4 shares approximately 90% of the total cDNA with CYP 3A5. CYP 3A is most abundantly expressed in human��s liver. They also exist in extra-hepatic tissues, such as in intestine and kidney. CYP 3A4 is one of the most important enzymes in the family of cytochrome P450, which has a broad capacity for oxidation and can accommodate a variety of structurally different substrates. In fact, CYP 3A (mainly CYP 3A4, CYP 3A5) is 40% and 80% of total P450 content in human liver and intestine respectively. [6] Most of chemicals in biology are due to be metabolized by cytochrome P450, forming different intermediates. Compared with the other P450s, CYP 3A is obviously the most important and essential drug-metabolizing enzymes in human body. Accumulating evidence has shown that CYP3A mediates the biotransformation of approximately half of all marketed drugs [7]. Interactions between drug and CYP 3A may occur via metabolic inhibition or metabolic inductions. The cases vary with drugs in clinics. One kind of intermediates is inhibitor, which is a vital factor that should be considered when developing a new drug. The inhibition can be reversible or irreversible. In the irreversible process, drugs are usually metabolized by specific cytochrome P450 enzymes to become inhibitors. Then the inhibitors bind to the active sites of CYP 450 causing a long time inactivation. This process is called a ��suicide inhibition�� or mechanism-based inactivation[8]. Classic inhibitors include chloroform, bromo-dichloromethane, etc. [9] The mechanism-based inactivation of cytochrome P450 is important in the development of related drugs, which can be achieved through a modifying process, which modifies the haem or the enzyme or both.

As CYP 3A4 is the most abundant kind of enzymes in the family of cytochrome P450 and responsible for the metabolism of over 50% of marketed drugs [10]��the study of CYP 3A has been heightened in recent years. Ritonavir, a most widely used anti-HIV drug, is CYP 3A4-dependent. Ritonavir is the substrate of CYP 3A4 as well as the most effective inhibitor of CYP 3A4 known to date. As a substrate, it can bind CYP 3A4 to inhibit the activity of this enzyme, which is supposed to metabolite the other anti-HIV drugs in plasma. Thus coadministration of other kinds of anti-HIV drugs with ritonavir can enhance the clinical efficiency.

The metabolism of ritonavir mainly contains two parts, the N-demethylation and hydroxylation of the isopropyl side chain; the oxidation and cleavage of the terminal isopropyl group. The oxidation step converts the drug into a reactive intermediate which can bind to the heme or the active sites of the protein and inhibits the biological activity of CYP 3A4. [11][12]

The binding mechanism of ritonavir to CYP 3A4 is measured by monitoring absorbance changes at 426 and 442 nm, respectively. [13]The association of CYP 3A4 and ritonavir is influenced by both the interaction of ligands-protein and protein-protein. As shown in fig.1, ritonavir is a long chain molecule with a thiazole and isopropyl-thiazole at each end. When ritonavir docks the CYP3A4, it could be with thiazole end or isopropyl-thiazole end toward the haem. With the fact that CYP3A4 can metabolite isopropyl-thiazole,[14] the drug has to undergo a positional/conformational rearrangement if docking with isopropyl-thiazole heading the heme core, otherwise, it will be disassociated. In this case, a new molecule needs to dock to the active site in an opposite orientation, which leads to a slow phase of CYP 3A4 binding reaction. [13]

Fig 1.1 The structure of ritonavir with a isopropyl-thiazole and a thiazole at each end of the molecule.

Fig 1.2 The crystal structure of cytochrome P450 3A4��ritonavir complex. The cavity inside of the molecule represents the active site of CYP 450. The heme is red and the ritonavir is green.

It is corroborated that ritonavir is a type ��binding ligand. When ritonavir docks CYP 3A4, it binds the heme more tightly than the coordinated water molecules.[15] Thus reduces the redox potential of CYP 3A4, making the electron transfer from cytochrome P450 reductases thermodynamically unfavourable.[16] As a result, the binding of ritonavir precludes the reduction of CYP3A4 by cytochrome P450 reductase. Therefore, the activity of CYP3A4 is inhibited. This reaction, as stated above, is called the mechanism-based inhibition. Under this condition, no bioactive metabolites are produced. However, there is an exception when ritonavir heads into the active site of CYP 3A4 in a wrong orientation. In this case, conformational rearrangement is needed. In fact, the conformational rearrangement is so slow that it facilitates two electrons transferred from the cytochrome P450 reductases to CYP 3A4. [13]

Cytochrome P450 involved in the metabolism of anaesthetic drugs

The drug interactions exist between the substrates for CYP 450 and the inhibitors or inducers of these enzymes. Cytochrome P450 can affect the efficiency of drugs in this way.

Cytochrome P450 is involved in the metabolism of anaesthetic drugs, which does not only metabolize drugs in vivo, but affect the clinical efficiency of the co-administrated drugs. The oral anesthetic drugs need to bypass a first-pass metabolism in the intestinal epithelium and liver before they are transported by blood to other parts of the body. In the first-metabolism process, drugs are metabolized by various enzymes existing in the intestine, one of the most important enzymes that can affect the clinical efficiency is cytochrome P450 3A. As stated above, some substances are inhibitors or inducers of cytochrome P450. The effect of CYP 3A on drugs may be more significant when drugs are co-administrated; especially one of the co-administrated drugs is an inhibitor or inducer of cytochrome P450. Under this condition, the plasma concentration of anaesthetic may turn out much higher or lower than expected, thus the clinical manifestation of administrated anaesthesia will be hard to predict. For example, if antimycotics is given in combination with midazolam, which is a 1,4-benzodiazepine used as a short-acting hypnotic agent for pre-medication and for sedation during local anaesthesia[17], antimycotics acts as an inhibitor of CYP3A. The concentration of midazolam will get higher than expected, thus causing hypnosis instead of sedation in clinics. Not only drugs can act as inhibitors of cytochrome P450, some organic substances can also play the same role. Trails have proved that grapefruit can also inhibit the activity of CYP 3A, as the plasma concentration of midazolam increases slightly after drinking grapefruit juice.

The effect of CYP 3A inducers carbamazepine, phenytoin and rifampicin (rifampin) on midazolam can be significant.[17] The peak of plasma concentration decreases more than 90% when midazolam administrated orally in combination with either of the drugs mentioned above. [17]

In conclusion, the efficiency of midazolam can be affected by inducers or inhibitors of CYP 3A, both pharmacokinetically and pharmacodynamically, causing the unpredictable effects. Because of the sensibility to the bioactivity of CYP 3A, midazolam is used as a probe drug to test the activity of CYP 3A in the liver and intestine. Other oral anaesthesia drugs have the similar drug interactions with midazolam, such as triazolam, diazepam, alfentanil, fentanyl, methadone, etc. [17]

The role CYP 2A plays in the metabolism of N-nitrosamines

As a subfamily of CYP 450, CYP 2A has relatively limited selections on substrates and minor hepatic form in quantity terms. Thus, it plays a minor role in the metabolism of drugs in vivo, especially compared to other subfamilies of cytochrome P450, such as CYP3A4, CYP2E1. By now, the most well-studied species are CYP 2A6��CYP 2A13. CYP 2A 6 is mainly expressed in liver, where it represents less than 1% to 13% of the total cytochrome P450. Minor amounts of CYP2A6 are also found in the nasal mucosa, oesophagus, and lung.[ 18] [19]

Smoking can cause a series of related diseases, which makes it a big threat of health. But 1/3 population of the world have a smoking age over 15 years. Statistics have indicated that people who smoke are more likely to have cancer, respiratory and cardiovascular diseases, gastrointestinal disorders, as well as many other medical complications [20]. Nicotine is the main reason for establishing and maintaining tobacco dependence. Smokers can detect the consumption of nicotine in their brain to maintain the steady concentration. Factors influencing nicotine plasma levels, by either intake or removal, affect smoking behaviour.[21] Smokers with CYP 2A6 deficiencies metabolite nicotine slower than those who have the normal level of CYP 2A6, and they are twice less likely to be adult smokers. Even though they have established the nicotine dependence, they are likely to smoke 7-10 cigarettes less the ��normal�� smokers.[21] In the metabolism in vivo, 80% of nicotine is transferred into the inactive cotinine. Two steps are involved in this process. Firstly, nicotine is oxidized to nicotine iminium ion, and then to cotinine catalyzed by the cytosolic aldehyde oxidase. This process is CYP 2A6-dependent, also the rate determining step. Subsequently, nicotine iminium ion is catalyzed to cotinine. CYP 2A13 also takes part in this process, but when the concentration of nicotine is very low (��50��M), CYP2A6 is considered the sole enzyme which is effective in the metabolism of nicotine. [21]

Fig.2 The graph shows the mechanism of the CYP 2A6-catalyzed reaction in details. Nicotine is first catalyzed by CYP2A6 to nicotine iminium ion, then transferred into cotinine by the catalyst aldehyde oxidase. The first step is the slowest step in this process, and also the rate determining step.[22]

Cytochrome P450- and peroxidase-mediated oxidation of anticancer alkaloid ellipticine dictates its anti-tumor efficiency

In most cases, the pharmaceutical efficiency of a prodrug is dependent on cytochrome P450 as well as other species of enzymes in vivo. For example, ellipticine is a kind of anticancer alkaloids which was firstly isolated from the leaves of the evergreen tree Ochrosia elliptica,[23]. Experimentally, ellipticine has remarkable curative effects on anti-HIV and anti-cancer. The target anti-cancer line is leukemias (L1210, P388, HL-60, and CCRF-CEM cell lines), lymphosarcomas, B16 melanoma, colon cancer SW480 cell line, Lewis lung carcinoma, human non-small-cell-lung-cancer, hepatocellular carcinoma HepG2 cells, with effective concentration ranging from 10-10 to 10-6 M. (24-33)

Cytochrome P450 are vital enzymes involved in the oxidation of ellipticine, which facilitate the production of species binding to DNA and detoxication. There are five metabolites formed in this process, which are 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxy-ellipticine, and ellipticine N2-oxide. [34][35][36] Of all the cytochrome P450 involved in this process, CYP 1A1,1A2, 1B1, 3A4 ,2C9 2D6 [23] are the major enzymes responsible for the production of these metabolites. As these metabolites end up excreting from organisms in vivo, the enzymes are also considered involved in the detoxication of ellipticine. Among these enzymes, CYP 3A4 catalyzes ellipticine into 13-hydroxy-ellipticine, and ellipticine N2-oxide. Because CYP 3A4 is the most abundant species of cytochrome P450 in human liver, converting the ellipticine into 13-hydroxy-ellipticine, and ellipticine N2-oxide is also considered to be the main metabolic pathway of this anticancer drug.

Fig.3 This graph shows the dock of ellipticine in CYP 3A4, the binding ligands to the substrate, ellipticine, are side chains of Thr308, Glu307, Ile368, and Leu372. [23]

The main mechanism that CYP 3A4 metabolize ellipticine is that it oxidizes this anticancer drug into 13-hydroxy-ellipticine, and ellipticine N2-oxide, which can bind to the DNA, forming DNA adducts.

The metabolism of ellipticine is mainly divided into two pathways, the CYPs catalyzed pathway and the peroxidase catalyzed pathway. As stated above, CYP 3A4 as well as other enzymes, such as CYP2D1 and CYP1A, firstly metabolize ellipticine to either 13-hydroxy-ellipticine or ellipticine N2-oxide, which is converted into 12-hydroxy-ellipticine by Polonowski rearrangement . Then ellipticine-13-ylium and ellipticine-12-ylium are generated after the spontaneous cleavage of 13-hydroxy-ellipticine and 12-hydroxy-ellipticine, resulting in the formation of dG adduct 1 and dG adduct 2 respectively. [23] The oxidation of ellipticine by the peroxidases is another way to detoxicate or activate ellipticine. In this process, ellipticine is metabolized by peroxidase to ellipticine methylene-imine which binds DNA generating the typical DNA adducts. [23]

Fig.4 The graph above indicates two metabolism pathways of ellipticine briefly, which are the CYPs catalyzed pathway and the peroxidase catalyzed pathway [23]

Except a limited number of cytochrome P450 are responsible for the biosynthesis of steroids and bile acids, most of Cytochrome P450 are involved in the metabolism of xenobiotics in vivo. Paradoxically, rather than eliminate xenobiotics, some subfamilies of cytochrome P450 induce toxicity in cells, causing cell death or inhibiting cell apoptosis.

There are considered to have two phases in the oxidation and conjugation of xenobiotics, known as phase 1 and phase 2. The phase 1 enzymes include P450, the flavin-containing monoxygenases (FMO) and epoxide hydrolases (EH). The phase 2 enzymes include the glutathione S-transferases (GST), UDPglucuronosyltransferases

(UGT), N-acetyltransferases (NAT) and sulfotransferases (SULT). [23]

The catalytic activity of CYP2E1 is sensitive to some chemicals, such as the exposure to benzene can inhibit the activity of CYP2E1, this offers an reasonable explanation to why get access to benzene can increase the risk of cancer.

Paracetamol, also called acetaminophen (AP) in North America, is a widely used analgesic drug over the counter. But its toxicity is one of the most common causes of poisoning in the world. One of the serious paracetamol toxicity symptoms is acute liver failure. [37]GST and CYP 450 are considered to be responsible for the catalysis of paracetamol, Among these enzymes, CYP 2E1 is the principal enzyme initiating the cascade metabolism of the toxicity of AP.

Fig.4 This graph indicates briefly the metabolism of toxicity by CYP 2E1 and GST. AP is catalyzed to a mediate-metabolite which is the substrate of GST and metabolized later. Higher lever of CYP 2E1 or lower level of GST than the normal one can lead the toxic metabolite binding to the macromolecules, such as DNA or protein, resulting in cell death. [38]

Paradoxically, the presence of CYP 2E1 is essential for the elimination of toxicity, high level of CYP2E1 may induce the toxicity.

The role of P450s in the metabolism of acetaminophen in various P450 null mouse model. (�� mice lacking CYP 2E1; �� mice lacking CYP 1A2; �� wild type mice; �� lacking both CYP 1A2 and CYP 2E1 ) [38]

CYP 2E1 null mouse model is used to access the chemicals which are of concern for the human risk, including the susceptibility to AP. From the testing trails, the CYP ��2E1 is responsible for the cascade of metabolism of AP. Meanwhile. CYP2E1 null mouse have high tolerance to the administrated AP. This proves that CYP 2E1 can increase the risk of acetaminophen toxicity.

In the other hand, CYP 2E1 are required for the oxidation stress in vivo. ��Oxidation stress is the result of an increase in intracellular prooxidant species such as H2O2, hydroxyl radicals (?OH) and superoxide anion radical High intracellular levels of these molecules, collectively called reactive oxygen species (ROS)�� (@) ROS can cause a series of diseases, for instance, cancer, obesity, Parkinson's disease, Alzheimer's disease,[39] atherosclerosis, heart failure,[40] and myocardial infarction, [41] the oxidation stress is also a principle reason causing aging.

Alcohol can be an efficient inducer of CYP2E1, which gives a reasonable explanation to the increasing risk of acetaminophen toxicity when alcohol is administrated in combination of acetaminophen.

Experimentally, CYP2E1 is considered to play an important role in the metabolism of numerous low Mr into intermediates which can bind to some biomacromolecules in vivo, such as DNA, RNA and protein, causing damage to these molecules, thus resulting in the diseases stated above. In this process, CYP 2E1 dissociates a H+ from NADPH and reacts with O2 leading to the production of superoxide. As CYP 2E1 uncoupling the mitochondria and breaking the catalytic cycle, superoxide is released to the plasma. Similarly, ROS produced by CYP2E1 can react with polyunsaturated fatty acids (PUFA) forming lipid peroxides which can bind to other macromolecules, resulting in cell death. [23]

This graph shows pathway of the ethanol-induced CYP2E1 cytotoxicity. Ethanol can induce the expression of CYP2E1, which uses the H+ from NADPH to reduce the O2 into peroxides and superoxides, causing the damage to biomacromolecules. .

By now, therapeutic intervention to decrease the toxicity caused by paracetamol is to remove it from body Active charcoal is usually used to absorb the drug when it is overdosed. Another way to decrease the toxicity is to replace glutathione. In clinic, paracetamol is administrated in combination with methionine, which is converted into glutathione in the liver.

Conclusion

Cytochrome P450 is one of the most important enzymes responsible for the catalysis and metabolism of xenobiotics and drugs, facilitate the removal of foreign chemicals from the body. In recent years, numerous researches on cytochrome P450 have been carried in order to exploit the molecule mechanism of drug metabolism. On basis of these mechanisms, some new drugs are designed mainly used in antivirus and anticancer therapy. Paradoxically, instead of detoxication, some species of cytochrome P450 may increase the risk of toxication in vivo, causing damage to biomacromolecules, for instance, high level of CYP2E1 can induce the toxicity of paracetamol, forming DNA adduct. Or affect the clinical efficiency of the co-administrated drugs, such as leading to hypnosis instead of sedation when antimycotics is given in combination with midazolam in clinics. So it is rather important to investigate interactions between CYP 450 and drugs. However, investigation on the role cytochrome P450 play is no easy a task.

In human body present large numbers of enzymes , whose biochemical roles and substrate selectivity is overlapped. Some drugs target on a sole specie of enzyme. However, sometimes , a drug can be a substrate of several enzymes in vivo. Thus it could be extremely tough to determine the species and functions of the enzymes involved in catalytic reaction. In this case, advanced technologies and experimental approaches are needed in the research of structures and metabolic functions of cytochrome P450, such as obtaining the docked structures by X-ray crystallography, determining the binding ligands by NMR and telling the function of a particular p450 enzyme using the P450 null mice model. Meanwhile, several new methods are developed to investigate the mechanism of cytochrome P450, for instance, random acceleration molecular dynamics(RAMD), which provides ��an unbiased procedure to probe for ligand egress routes on an accelerated timescales��[42]