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Cytochrome p450 (CYP 450) is the name of a large family of universal enzymes in the human body responsible for the metabolism of most drugs and other foreign organic compounds (xenobiotics).

The metabolism of many classes of drugs (antihistamines, retroviral protease inhibitors, benzodiazepines, calcium channel blockers, etc.) occurs with the participation of cytochromes.

In addition, cytochromes provide various physiological processes, including the biosynthesis of steroids and cholesterol, the metabolism of fatty acids and the provision of calcium metabolism (hydroxylation of vitamin D3, which is the first step in the formation of calcitriol).

History of cytochrome p450

Cytochrome P450 was discovered in the late 1950s by M. Klingenberg and D. Garfinkel. The term "cytochrome" (cito - cell; with hromos - color) appeared in 1962 as a temporary name for a colored substance found in cells.

As it turned out, various types of cytochrome P450 are widely distributed in the cells of microorganisms, plants, and mammals. These enzymes are absent only in anaerobic bacteria.

Scientists assume that all the genes encoding different types of CYP450 originated from a single precursor gene that existed two billion years ago. The function of this "original" gene was to utilize energy. At the moment, more than 1000 different types of cytochrome CYP 450 have been found in nature.

Variety of cytochromes

To date, about 55 different types of cytochromes have been found in mammals, and more than 100 in plants.

Thanks to the success of genetic engineering, it was possible to establish that the enzymes of the cytochrome family perform various functions, which determines their division into three main classes:

• involved in the metabolism of drugs and xenobiotics;
• involved in the synthesis of steroids;
• participating in other important endogenous processes occurring in the body.

Classification of cytochromes

All cytochromes and the genes encoding their synthesis are named according to the following guidelines:

• the name of the cytochrome must indicate the root CYP;
• the name of the gene encoding the synthesis of the corresponding cytochrome also contains CYP , but written in italics;
• cytochromes are divided into families (denoted by numbers), subfamilies (denoted by letters) and isoforms (denoted by numbers reflecting the number of the coding gene).

For example, CYP 2 D 6 belongs to the 2nd family, subfamily D, encoded by gene 6. The name of the gene itself looks like CYP 2 D 6.

Basic cytochromes

Despite the diversity of cytochromes in the human body, drug metabolism occurs with the participation of a predominantly limited amount of CYP 450. The most common representatives of this group are: CYP 1A2, CYP 2C9, CYP 2C19, CYP 2 D 6, CYP 2E1, CYP 3A4.

These enzymes catalyze a wide range of metabolic reactions:

• one cytochrome can metabolize several drugs with different chemical structures;
• the same drug may be affected by different CYP 450s in different organs and systems of the human body.

Dual nature of cytochromes P450

In most cases, fat-soluble drugs and other chemicals are converted into water-soluble metabolites that are more easily excreted from the body. The introduction of hydroxyl groups (due to cytochrome P450) increases the polarity of the molecules and their solubility, which also contributes to their removal from the body. Almost all xenobiotics entering the liver are oxidized by some isoform of cytochrome p450.

However, the same enzymes that catalyze "purification" processes can activate inert chemical molecules to a highly reactive state. Such messenger molecules can interact with proteins and DNA.

Thus, the impact of cytochromes p450 can occur through one of two competitive pathways: metabolic detoxification or activation.

Variability in the action of cytochromes

Each person is characterized by their own metabolism of medicinal substances, which differs from that of other people. Individual characteristics depend on genetic factors, the patient's age, gender, health status, diet, concomitant pharmacotherapy, etc.

The genetic variability of drug metabolism was discovered by chance: standard doses of drugs unexpectedly caused non-standard reactions in different individuals.

Enzyme activity can be of two (sometimes three) main types: intense and weak (medium), respectively, the metabolism of drugs can occur quickly and slowly.

Cytochromes and drug metabolism

Cytochrome CYP 1A2 involved in the metabolism of many drugs, including aminophylline and caffeine. The activity of this enzyme increases under the influence of chemicals that enter the human body during smoking.

Cytochrome CYP 2A6 plays an important role in the metabolism of coumarin (an indirect anticoagulant) and nicotine.

Cytochrome CYP 2S9 involved in the metabolism of phenytoin, tolbutamide, warfarin. If at least one amino acid changes in the structure of the gene encoding the synthesis of a given cytochrome, then its enzymatic activity is disturbed. Enzyme deficiency of this cytochrome causes an innate predisposition to phenytoin intoxication and to complications as a result of warfarin therapy.

Cytochrome CYP 2S19 participates in the metabolism of omeprazole, diazepam, imipramine. However, the clinical significance of this enzyme polymorphism remains controversial. The effective doses of many drugs metabolized by CYP 2C9 are so far from toxic that potential deviations in CYP 2C9 cytochrome activity do not play a significant role.

Cytochrome CYP 2 D 6 is an example of genotypic differences among different ethnic groups. In the 70s of the last century, the pharmacokinetics of the antihypertensive drug debrisoquine and the antiarrhythmic spartein were studied. The following results were obtained: with a general trend towards ultra-fast metabolism of debrisoquine, among Caucasians, slow metabolism was observed in 5-10% of cases, among the Japanese this figure was less than 1%.

Drugs metabolized by CYP2D6 (b-blockers, antiarrhythmics, psychoanaleptics, antidepressants and narcotic analgesics) have a narrow therapeutic index, i.e. there is little difference between the dose required to achieve a therapeutic effect and the toxic dose. In such a situation, individual deviations in drug metabolism can play a dramatic role: increasing the concentration of the latter to a toxic level, or reducing it to the point of losing effectiveness.

The history of the use of perhexilin (Australia) has clearly demonstrated the great importance of CYP2D6 polymorphism. After the first experience of prescribing, the drug was withdrawn from the arsenal of drugs for the treatment of angina pectoris due to high hepato- and nephrotoxicity. But now perhexilin is being used again and is recognized as highly effective because it is only toxic to patients with poor CYP2D6 metabolism. The safety of prescribing perhexilin is ensured by a preliminary determination of the individual level of this cytochrome.

Cytochrome CYP 3A4 presumably metabolizes about 60% of all drugs. This is the main cytochrome of the liver and intestines (of the total number of cytochromes, it is 60%). Its activity may increase under the influence of rifampicin, phenobarbital, macrolides and steroids.

Inhibition of drug metabolism

Inhibition of drug metabolism is the most common cause of clinically significant drug interactions resulting in an undesirable increase in drug concentrations in the blood. This most often occurs when two different drugs compete for the ability to bind to the same enzyme. A drug that “loses” in this competitive “struggle” loses its ability to be adequately metabolized and accumulates excessively in the body. It is encouraging that there are not many drugs that have the characteristics of a pronounced inhibitor. Typical inhibitors are cimetidine, erythromycin, ketoconazole and quinidine. Among the newer drugs, selective serotonin reuptake inhibitors and protease inhibitors have potential inhibitory properties.

The rate of inhibition depends on the pharmacokinetic properties of the "conflicting" drugs. If both the inhibitor and the drug-substrate have a short half-life (for example, cimetidine and the inhibitor of its metabolism - theophylline), the interaction will be maximum on the 2-4th day. The same amount of time will be required for the termination of the interaction effect.

In the case of simultaneous use of warfarin and amiodarone, it will take 1 month or more to stop the inhibitory effect, which is associated with a long half-life of the latter.

Despite the fact that the inhibition of cytochrome-mediated metabolism is a big problem, conditions are sometimes created in clinical practice that allow the targeted use of this phenomenon. The antiviral drug saquinavir has a very low bioavailability due to its extensive metabolism by CYP 3A4. The bioavailability of the drug when taken orally is only 4%. Simultaneous administration of the related drug ritinavir, which suppresses the activity of cytochrome, leads to a 50-fold increase in the plasma concentration of saquinavir, which makes it possible to achieve a therapeutic effect.

Induction of drug metabolism

Metabolic induction occurs when a drug stimulates the synthesis of enzymes involved in the metabolism of another drug (or reduces the natural breakdown of these enzymes).

The best known cytochrome inducer is rifampicin, which increases CYP 3A4 and CYP 2C levels in the liver, resulting in increased metabolism of a number of drugs (table).

It is quite reasonable to assume that cytochrome inducers reduce the effectiveness of drug substrates. However, there is another side to this phenomenon. Sudden withdrawal of an inducer drug (or cessation of environmental exposure to an inducer) can unexpectedly lead to a large increase in the plasma concentration of a drug that has previously been extensively metabolized. An example is when smokers, who are accustomed to the constant use of coffee, decide to suddenly stop smoking, as a result of which CYP 1A2 activity decreases, and the concentration of caffeine in the blood plasma increases. This can exacerbate the severity of the withdrawal syndrome: headache and agitation.

Interaction of cytochromes with food

As a result of a study conducted in 1991, it was found that one glass of grapefruit juice causes a three-fold increase in the plasma level of felodipine. However, other juices did not cause a similar effect. It is assumed that the components of grapefruit - flavonoids or furanocoumarin - inhibit the metabolism of felodepine in the intestine, mediated by cytochrome CYP 3A4.

Pharmacogenomics and its promising areas

The science that studies the genetically determined response of the body to drugs has recently been called pharmacogenomics. The development of this science will make it possible to accurately predict the individual response of the body to a particular treatment, as well as to identify patients with a high risk of developing toxic reactions.

Table. The main types of cytochromes p450 in humans

Cytochrome	Substrates that are affected	Inhibitor	Inductor
	Amitriptyline, caffeine, clomipramine, imipramine, clozapine, mexiletine, estradiol, paracetamol, propranolol, tacrine, theophylline, R-warfarin	Cimetidine, fluvoxamine, fluoroquinolone antibiotics (ciprofloxacin, norfloxacin), grapefruit juice	Omeprazole, phenobarbital, phenytoin, polycyclic aromatic hydrocarbons (eg barbecue), cigarette smoking
	Diclofenac, indomethacin, losartan, naproxen, phenytoin, piroxicam, tolbutamide, S-warfarin	amiodarone, chloramphenicol, cimetidine, fluconazole, fluoxetine, isoniazid, omeprazole, sertraline, sulfinpyrazone	Rifampicin
	Clomipramine, clozapine, diazepam, imipramine, lansoprazole, omeprazole, phenytoin, propranolol	fluoxetine, fluvoxamine, isoniazid, omeprazole, sertraline	Rifampicin
	Amitriptyline, chlorpromazine, clomipramine, clozapine, codeine, desipramine, dextromethorphan, doxepin, fluoxetine, haloperidol, imipramine, labetalol, methadone, metoprolol, procainamide, promethazine, propafenone, propranolol, thioridazine, timolol	Amiodarone, cimetidine, haloperidol, mibefradil, quinidine, propafenone, all serotonin reuptake inhibitors
	Caffeine, ethanol, paracetamol, theophylline	Cimetidine, disulfiram	Ethanol, isoniazid
	Amiodarone, amitriptyline, atorvastatin, buprenorphine, carbamazepine, clarithromycin, clomipramine, clonazepam, cocaine, cortisol, cyclophosphamide, cyclosporine, dexamethasone, digitoxin, diltiazem, diazepam, doxorubicin, erythromycin, felodipine, fentanyl, imipramine, ketoconazole, loratadine, nifedipine, estradiol, omeprazole, propafenone, quinidine, simvastatin, theophylline, verapamil, vincristine, warfarin	Amiodarone, cannabinoids, cimetidine, clarithromycin, clotrimazole, diltiazem, erythromycin, grapefruit juice, ketoconazole, metronidazole, miconazole	Carbamazepine, glucocorticoids, phenytoin, rifampicin, sulfadimidine

Cytochromes P450. Structure and function

Among the enzymes of the 1st phase, the cytochrome P450 system (P450 or CYP) occupies a leading position in terms of catalytic activity against a huge number of xenobiotics. The highest concentration of cytochrome P450 is found in the endoplasmic reticulum of hepatocytes (microsomes). Hepatic microsomal cytochromes P450 play an important role in determining the intensity and time of action of foreign compounds and a key one in the detoxification of xenobiotics, as well as in their activation to toxic and/or carcinogenic metabolites. Cytochrome P450-dependent monooxygenases are a multienzymatic electron transport system. All cytochromes P450 are heme-containing proteins. Heme iron is usually in an oxidized state (Fe3+). Recovering to the Fe2+ state, cytochrome P450 is able to bind ligands such as oxygen or carbon monoxide. The complex of reduced cytochrome P450 with CO has an absorption maximum of 450 nm, which was the basis for

the names of these enzymes. The main reaction catalyzed by cytochromes P450 is monooxygenase, in which one oxygen atom interacts with the substrate (RH), and the other is reduced to H2O. NADPH is involved as a reducing agent in the reaction:

RH (substrate) + O2 + NADPH + H+ --> ROH (product) + H2O + NADP+

The mechanism by which cytochrome receives an electron from NADPH depends on the intracellular localization of cytochrome P450. In the ER, where most of the hemoproteins involved in the biotransformation of xenobiotics are located, the electron is transferred through a flavoprotein called NADPH-P450 reductase. One reductase molecule can deliver electrons to several different P450 molecules. In mitochondria, where itochromes P450 are located, which are involved in the biosynthesis of steroid hormones and vitamin D metabolism, the electron is transferred using 2 proteins: ferrodoxin or ferrodoxin reductase.

On fig. 1 shows the catalytic cycle of cytochrome P450. The 1st part of the cycle consists in the activation of oxygen, the 2nd - in the oxidation of the substrate. The scheme of action of the microsomal monooxygenase system was first described by Estabrook et al., and has now been confirmed by many researchers. This scheme is as follows: the first stage consists in the interaction of the substrate with the oxidized form of P450. When P450 binds to substrates

the transition of heme iron from a low-spin state to a high-spin state occurs. The second stage consists in the reduction of the resulting enzyme-substrate complex with the first electron that comes from the NADPH-specific transfer chain from NADPH through

flavoprotein I (NADPH-cytochrome P450 reductase). The third stage consists in the formation of a triple complex: reduced cytochrome P450-substrate-oxygen. Fourth stage

represents the reduction of a ternary complex by a second electron, which, as

thought to come from a NADH-specific electron transport chain composed of NADH-

cytochrome b5 reductase or flavoprotein II and cytochrome b5. The fifth stage consists of several processes, including intramolecular transformations of the reduced ternary complex and its decomposition with the formation of a hydroxylated product and water. At this stage, cytochrome P450 goes into the original oxidized form.

Cytochromes P450 catalyze the following types of reactions: hydroxylation of an aliphatic or aromatic carbon atom; double bond epoxidation;

atom oxidation (S, N, I) or N-hydroxylation; transfer of an oxidized group;

destruction of the ether connection; dehydrogenation. Some of the reactions catalyzed

cytochrome P450 are presented in fig. 2 and 3. Several classes of reagents are good

the last carbon in the chain is hydroxylated, the so-called omega-hydroxylation. So

but there is internal hydroxylation in several positions (positions -1, - 2).

This leads to many different product options, even with a simple alkane like hexane. Note that cyclic hydrocarbons also undergo hydroxylation. In the hydroxylation reaction, a hemiacetal is first formed, which then turns into an alcohol and an aldehyde. When alkenes are oxidized by cytochrome P450, diatomic oxides are formed. They differ in their stability and can be highly reactive. For example, vinyl chloride is metabolically converted to oxide, which then turns into chloroacetaldehyde, a mutagen that acts directly on DNA. These studies led to a ban on the use of vinyl chloride in sprayers. The vinyl group of the sterol (vinylbenzene) is known for its carcinogenic properties, but the human body is able to neutralize it by converting the oxide to diol using the enzyme epoxyhydrolase. But epoxyhydrolase does not always help. For example, cytochrome P450 synthesizes Aflotoxin B1 epoxide in vivo. This compound is a highly reactive electrophile, is not stable, and quickly forms an adduct with DNA. In addition, the diol formed from epoxide is also unstable and highly reactive. Oxidation of aromatic compounds with cytochrome P450 also gives epoxides, but they quickly turn into phenol. As a result of the hydroxylation of benzene, the resulting phenol can be hydroxylated again, turning into catechol or hydroquinone. Note that catechol and hydroquinone can react with oxygen, inhibiting similar reactions with quinones and superoxides, which are toxins. Such a well-known compound as 2,3,7,8-tetrachlorodibenzenedioxine (TCDD) is not subject to hydroxylation and is stable (half-life in the human body is a year or more).

Cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9). A1075C (Ile359Leu) mutation detection

The name of the geneCYP2C9

Localization of a gene on a chromosome– 10q23.33

• *1/*1
• *1/*3
• *3/*3

Occurrence in the population

allele CYP2C9*3 found in Europeans with a frequency of 6%.

Association of the marker with drug metabolism

It is being studied to identify the physiological effectiveness of the use of drugs: oral anticoagulants from the class of coumarins (warfarin), sulfonylurea derivatives, non-narcotic analgesics (tenoxicam, flurbiprofen, lornoxicam, piroxicam), losartan and irbesartan (angiotensin II receptor blockers).

General information about the study

For the prevention and treatment of thromboembolic complications, the drug warfarin ("Coumadin") is most commonly used. It is prescribed for long-term use in a series of cases associated with increased blood clotting, as well as in the postoperative period in order to prevent the formation of blood clots due to surgical intervention. It is often practiced to prescribe the drug to people who have had strokes, myocardial infarction.

To achieve the effect of drugs, their bioactivation in the body (transformation into an active form) in the liver cells (hepatocytes) by the cytochrome P450 (CYP) enzyme system is necessary. The genes encoding these enzymes are polymorphic, and alleles encoding the formation of enzymes with reduced or absent function are often found.

The activity of cytochromes, in addition to the structural features of the genes encoding them, is influenced by factors such as age, body weight, lifestyle, bad habits, dietary habits, concomitant diseases, and medication. These factors are responsible for the formation of the individual characteristics of the work of P450 enzymes and determine the nature of the metabolism of most drugs. The main enzyme for the biotransformation of indirect anticoagulants is the cytochrome P450 isoenzyme CYP2C9.

Gene CYP2C9 localized on the 10th chromosome in the region 10q23.33. There are variants of a gene (alleles) CYP2C9 encoding the formation of an enzyme with reduced or absent function. The gene variant carrying a point substitution of adenine for cytosine at position 1075 (A1075C) leads to a decrease in the metabolic activity of the enzyme and is designated as CYP2C9*3. A single nucleotide substitution entails the replacement of the amino acid isoleucine with leucine (Ile359Leu) in the CYP2C9 enzyme. Thus, an enzyme with an altered function is synthesized, the activity of which is less than 5% of the activity of the enzyme *1. The main (unchanged) variant of a gene is designated as CYP2C9*1.

The most common genotype, which determines the normal metabolism of warfarin and is designated as CYP2C9 *1/*1.

genetic marker CYP2C9*3(genotypes *3/*3 and *3/*1) is associated with a change in the functional activity of the cytochrome P450 enzyme, which reduces the rate of warfarin excretion from the body. The presence of the *3 allele in a patient leads to a significant decrease in the activity of the cytochrome isoenzyme, which increases the anticoagulant effect of the drugs up to 7 times and can cause complications such as extensive internal bleeding and episodes of excessive hypocoagulation.

Polunina T.E.

Oksana Mikhailovna Drapkina

We continue our program. Our lectures and discussions on gynecology are coming to an end, we have fully entered the regulations, so we will try not to go out of it. Professor Polunina Tatyana Evgenievna opens the section of gastroenterology. Lectures "The role of the cytochrome P450 family in the pathogenesis and treatment of non-alcoholic fatty liver disease."

Tatyana Evgenievna Polunina, professor, doctor of medical sciences:

- Cytochromes P450 (CYP 450) - this is the name of a large family of universal enzymes in the human body. Cytochromes P450 play an important role in the oxidation of numerous compounds such as endogenous compounds (steroids, bile acids, fatty acids, prostaglandins, leukotrienes, biogenic amines) as well as exogenous compounds (drugs, industrial pollution products, pesticides, carcinogens and mutagens), the latter are called xenobiotics.

In this slide you can see where the cytochromes P450 are located. They are located in the hepatocyte, in the cytosol. The endoplasmic reticulum is the basis for location. And, in particular, the lipid membrane, which contains a bilayer of phospholipids, has several connected structures on it. This is a cytochrome that includes iron protein, nicotinamide adenine dinucleotide and oxidoreductase, which will be included in the complex of drug metabolism and the above xenobiotics.

The most common representatives of this group that clinicians turn to are cytochromes P452 AC, P450 2D, P450 2E1, P450 3A4. These enzymes catalyze a wide range of metabolic reactions and a single cytochrome can metabolize several drugs that have different chemical structures. The same drug has different effects in cytochrome P450 and in different organs. And here, in particular, the most important cytochrome that we pay attention to is cytochrome P450 2E - the most important isoenzyme of cytochrome P450, it breaks down low-density lipoproteins.

Currently, methods have been developed not only for phenotyping, which are based on the substrate specificity of certain cytochrome P450 isoenzymes, but also the activity of a particular enzyme and metabolism is determined by the pharmacokinetics of the marker substrate and changes in the concentrations of the unchanged substance and its metabolite. But the determination of cytochrome P450 isoenzymes by identifying the genes of the corresponding isoenzymes is carried out using a polymerase chain reaction. This is called cytochrome P450 isoenzyme genotyping.

On this slide, we see that in the hepatocyte, the place where the endoplasmic reticulum, P450 cytochromes, of which there are more than 50, and drugs that are broken down in a certain cytochrome, is located, in some cases it combines with the cytochrome and forms a vesicle that damages the hepatocyte, causing while stress and cytokines; leads to the activation of the tumor necrotic factor and, in particular, is a trigger factor for the launch of caspases, which manifests itself with catalytic processes.

Non-alcoholic fatty liver disease, which was subsequently identified as a nosological entity, has been referred to as non-alcoholic fatty liver disease (NAFLD) since 1980, finding changes in the liver of non-alcohol-abusing patients that are similar to those of alcohol damage.

The natural course of non-alcoholic fatty liver disease includes steatosis as an initial stage, which, without progressing, can be asymptomatic, and steatohepatitis, which is accompanied by terrible autonomic manifestations, cytolysis syndrome and dyspeptic manifestations. With the development of fibrosis, a rather serious problem arises - cirrhosis of the liver, and portal hypertension and carcinoma develop in the future.

I would like to draw your attention to the fact that back in 1894, Kiernan proposed a certain architectonics of the liver, which consists of a beam structure. On the periphery of the beams, which consist of polygonal hepatocytes, there is a triad: the bile duct, portal vein and artery. This slide represents a normal healthy liver and hepatocyte fatty infiltration. Liver steatosis, which is one of the first phases of the development of non-alcoholic fatty liver disease, is presented in morphological form in this diagram.

The next option for the development of the inflammatory process, which leads to fibrous tissue, its spread through the liver, we see steatohepatitis and later cirrhosis of the liver with the development of portal hypertension. Most often, this is micronodular cirrhosis of the liver, which has already been clearly established in the stages of development of non-alcoholic fatty liver disease, it is accompanied by portal hypertension, varicose veins of the esophagus, stomach, complications that are typical for liver cirrhosis, and death.

With non-alcoholic steatohepatitis, the most common moments that are most often associated as concomitant diseases develop: diabetes mellitus, obesity. In patients, non-alcoholic steatohepatitis develops up to 75%, and if diabetes mellitus and obesity are combined, then this is already 90% of patients with non-alcoholic fatty liver disease.

The liver is undoubtedly the main target organ affected in the metabolic syndrome. Insulin resistance is a key feature that is the basis for the accumulation of lipids inside hepatocytes, fatty liver, non-alcoholic steatohepatitis and liver cirrhosis.

I would like to draw attention to the fact that the metabolic syndrome includes not only impaired glucose tolerance, but also dyslipidemia, abdominal-visceral obesity, insulin resistance and hyperinsulinemia, arterial hypertension, early atherosclerosis, impaired hemostasis, hyperuricemia, hyperandrogenism. I would like to say that non-alcoholic fatty liver disease, steatosis, is part of the metabolic syndrome and is currently a quintet, which was previously called the "death quartet".

The risk factors presented on this slide sometimes change in different countries, in particular, the American positions and the European positions differ slightly. But, nevertheless, waist circumference, the level of triglycerides, lipoproteins, blood pressure, in particular 130/85, glucose levels are those indicators that must be monitored in a patient with metabolic syndrome.

Diseases associated with lipid metabolism are: non-alcoholic fatty liver disease, type 2 diabetes mellitus, ischemic liver disease, hypertension.

In the scheme of pathogenesis, insulin resistance of adipose tissue is of particular importance. An increase in lipogenesis, that is, an increase in the level of fatty acids, an increase in the synthesis of triglycerides and lipotoxicity lead to the development of insulin resistance, and this leads to metabolic dysfunctions, stress of the endoplasmic reticulum, in which fatty acids and in particular lipoproteins are also metabolized, and to the activation of inflammation . These are Kupffer cells and stellate cells, which further lead not only to the fact that the level of very low density lipids rises, undoubtedly, this leads to the development of steatohepatitis with fibrosis, and we get the activity of the process that moves towards cirrhosis of the liver.

At the level of the hepatocyte, fatty acids that undergo esterification into triglycerides and are exported as low-density lipoproteins, this is the situation in the normal hepatocyte, which is associated with oxidation in mitochondria, peroxisomes and microsomes.

Undoubtedly, in the mechanism of insulin resistance, which is presented here, the key role belongs to tumor necrotic factor, free radicals, leptin, fatty acids and increased lipolysis, which leads to the absorption of fatty acids, to the disruption of β-oxidation of fatty acids in mitochondria and also to the accumulation of fatty acids in hepatocyte.

Induction of cytochromes P450 4A11 and P450 2E1 leads to lipid peroxidation, which undoubtedly leads to the activation of moments associated with the accumulation of triglycerides. Hyperinsulinemia is a key factor that leads to insulin resistance. It also leads to an increase in glycolysis, the synthesis of fatty acids and the accumulation of triglycerides in hepatocytes.

The next slide shows the mechanism of interaction between microsomal oxidation and mitochondrial β-oxidation. Note that mitochondrial Ω-oxidation and mitochondrial β-oxidation lead to the activation of so-called peroxisomal β-oxidation receptors and in particular receptors activated in peroxisome proliferation. This leads to the expression of the accumulation of a certain protein and, accordingly, acetyl-coenzyme A, which accumulates and triggers the mechanism, leads to an overload of dicarboxylic fatty acids.

In the next slide, you see that steatohepatitis and fibrosis are formed against the backdrop of mitochondrial reactive oxygen species. The key point for triggering fibrosis is undoubtedly the accumulation of malondialdehyde, which leads to the formation of inflammatory infiltrates, fibrosis and activation of stellate cells. Stellate cells trigger the induction of cytokines such as tumor necrotic factor and transforming growth factors. The depletion of the antioxidant system leads to the launch of Fas-legand, the mitochondrial reactive oxygen species, hepatocyte necrosis occurs, and further fibrous tissue develops, which is the basis for the development of cirrhosis.

This slide shows a diagram, you see an excess of lipids that accumulate in the hepatocyte. Mitochondrial dysfunction and dysfunction of cytochrome P450 leads to activation of lipid peroxidation, triggering of Kupffer cells, inflammatory cytokines, activation of stellate cells and apoptosis, which further leads to the development of hepatocyte necrosis.

The metabolic syndrome is very important because non-alcoholic fatty liver disease is part of the metabolic syndrome. And not only on the hepatocyte, in which there is an increase in the level of low-density and very low-density lipoproteins, triglycerides (this is very important), but it also affects the endothelial cell. Endothelial dysfunction occurs and a moment is also triggered, which is associated with lipid peroxidation, the accumulation of substances that affect atherosclerosis, sudden death, heart attacks.

Undoubtedly, the increase in the level of free fatty acids is associated with adipocytes. And a decrease in esterified cholesterol in particular also leads to various stresses on the nuclear receptor. And the so-called activated peroxisome proliferation receptor is especially important at present, it is to it that all the eyes of scientists who work with obesity, diabetes, and non-alcoholic fatty liver disease are directed.

A monocyte (macrophage), in some cases, an increase in the level of inflammatory responders (tumor necrotic factor, interleukins-6, membrane toll-like receptors, free fatty acids) also triggers moments that are associated precisely with the pathological effects of fatty acids.

The criteria for assessing insulin resistance have been known to everyone since 1985. It is determined by the HOMA index - Homeostasis Model Assessment, and the more modern QUICKI index - Quantitative Insulin Sensitivity. Here are the concentration of insulin, serum glucose, as well as norms.

We would like to point out that not all patients with non-alcoholic fatty liver disease need to undergo a liver biopsy. We currently have moments that enable us to determine the level of fatty infiltration of the liver. And in particular, this is a fibrotest.

In the algorithm for diagnosing non-alcoholic fatty liver disease, we pay attention not only to specific signs, but also to the activity of the enzymes of alanine and aspartic transaminase, gamma-glutamyl transpeptidase, alkaline phosphatase, we pay attention to alcohol intake, which was discussed by previous colleagues. And I would like to pay attention, of course, to the risk factors: metabolic syndrome, insulin resistance, diabetes mellitus. Treatment is prescribed to correct this situation, if necessary, a liver biopsy. Undoubtedly, absolute indications for biopsy are required. And if the body mass index exceeds 35 and 40, then measures are already being taken that are associated with surgical treatment.

I would like to draw your attention to a number of drugs (non-steroidal - anti-inflammatory glucocorticosis, and steroid drugs, tetracycline antibiotics), a number of nutritional factors (starvation, rapid weight loss, surgical interventions, metabolic genetic factors, in particular, hereditary hemochromatosis, various poisons) and other comorbidities. This is very important for differential diagnosis.

In the stage of steatosis, the treatment of obesity, insulin resistance, and dyslipidemia is important. In the stage of steatohepatitis, the most important point is the elimination of oxidative stress, inflammation and fibrosis.

Excessive induction of cytochrome P450 2E has a detrimental effect on hepatocytes due to the release of free radicals. Essential phospholipids act not only as antioxidants, but also serve as a very important point for reducing the activity of cytochrome 2E1, as shown in the works of M. Aleinik. The results of some studies suggest that the introduction of essential phospholipids can reduce the induction of cytochrome P450 2E (works by Vladimir Trofimovich Ivashkin, which were presented with Marina Viktorovna Maevskaya in Russian sources in 2004).

Stellate cells are involved in the formation of the end stage of non-alcoholic fatty liver disease. And in laboratory experiments, it has been demonstrated that the complete prevention of stellate cell activation with the use of CYP2E1 inhibitors prevents the development of cirrhosis.

I would like to draw your attention to the fact that not only the Russian author M. Aleinik, but also the Japanese author Akiyama in the journal "Hepatology" in 2009, based on the model of alcoholic liver damage, also pays attention to cytochrome P450 2E, acetyl-CoA oxidase and nicotinamide adenine dinucleotide oxidases, that essential phospholipids exhibit anti-inflammatory, anti-apoptotic and anti-fibrotic activity in this pathology.

This is a theoretical version of the assumption of the use of inhibitors of cytochromes P450, and in particular the drug "Essentiale", which is a reference and is the most important moment for the inhibition of cytochromes P450 2E and, accordingly, P450 4A11. This prevents lipid oxidation, glycolysis and reduces fatty acid synthesis.

In the treatment of non-alcoholic fatty liver disease, drugs are presented: insulin sensitizers, antioxidants, hepatoprotectors, antimicrobials.

But I would like to pay attention to membrane phospholipids. They are the main lipid components of cell membranes. Damage to phospholipid membranes leads to a syndrome of cytolysis, and an excess of reactive oxygen species leads to damage to phospholipid membranes based on microsomal γ-oxidation and peroximal β-oxidation. Accordingly, damage to phospholipid membranes is cell death, which leads to the launch of fibrosis and activation of stellate cells.

Damage to the structure of the liver is damage to the membranes. In the variant of essential phospholipids, it is a material that restores cell membranes instead of lipids. Restoration of the structure of the liver makes it possible to restore the function of the liver.

Our patients suffer not only from alcoholic fatty liver disease, alcoholic hepatitis, but also other liver diseases, this is an undeniable fact. I would like to draw your attention to the fact that according to E. Kunz (2008 monograph), essential phospholipids have an antifibrotic effect, an effect that stabilizes bile and the hepatocyte membrane.

This is a publication that was released in 2008 based on pharmacological and clinical data. Essential phospholipid therapy seems to be the preferred choice for significantly reducing the manifestation and elimination of fatty liver disease of various etiologies, developed due to alcohol consumption, obesity, and even if the cause cannot be identified.

I would like to point out that there are several studies on Essentiale. These studies are well known to everyone. But I would like to say that with diabetes "Essentiale" makes it possible for patients with non-alcoholic liver disease to normalize the level of glucose, glycated hemoglobin, serum cholesterol.

Finally, I would like to say that liver damage characterized by the accumulation of fat in the absence of alcohol abuse is known as non-alcoholic fatty liver disease. Risk factors are obesity, type 2 diabetes. In the pathogenesis of non-alcoholic fatty liver disease, excessive activity of cytochromes P450 2E1 is of particular importance. Clinical variants of the course of the disease: pain in the right hypochondrium, asthenovegetative and dyspeptic disorders, hepatomegaly. And our diagnostic algorithm is based on the consistent exclusion of alcoholic and iatrogenic, as well as viral liver lesions.

Cytochrome P450(CYP450) is a large group of enzymes responsible for the metabolism of foreign organic compounds and drugs. Enzymes of the cytochrome P450 family carry out oxidative biotransformation of drugs and a number of other endogenous bioorganic substances and, thus, perform a detoxification function. Cytochromes are involved in the metabolism of many classes of drugs, such as proton pump inhibitors, antihistamines, retroviral protease inhibitors, benzodiazepines, calcium channel blockers, and others.

Cytochrome P450 is a protein complex with a covalently bound heme (metal protein) that provides oxygen addition. Heme, in turn, is a complex of protoporphyrin IX and a divalent iron atom. The number 450 indicates that the reduced CO-bound heme has a maximum absorption of light at a wavelength of 450 nm.

Cytochromes P-450 are involved not only in the metabolism of drugs, but also in the conversion of hemoglobin to bilirubin, the synthesis of steroids, etc. All cytochrome P-450 isoforms are grouped into the CYP1, CYP2, CYP3 families. Within the families, the subfamilies A, B, C, D, E are distinguished. Within the subfamilies, the isoforms are indicated by a serial number. For example, CYP2C19 is the name of the 19th cytochrome of the subfamily "C", family "2". In total, there are about 250 different types of cytochrome P-450, of which approximately 50 are in the human body, and only six of them (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4) are related to drug metabolism.

The activity of cytochromes P-450 is influenced by many factors - smoking, alcohol, age, genetics, nutrition, diseases. These factors are responsible for the formation of the individual characteristics of the work of P-450 enzymes and determine the effects of drug interactions in a particular patient.

Importance of cytochromes P450 for gastroenterology

The recently significantly increased interest of gastroenterologists in cytochrome P450 isoforms CYP2C19 and CYP3A4 is due to their role in the metabolism of benzimidazole derivatives, which include all drugs from the ATC group A02BC "Proton pump inhibitors" (omeprazole, pantorazole, lansoprazole, rabeprazole and esomeprazole) . It is clinically significant that the CYP2C19 gene is polymorphic, and the magnitude of the therapeutic effect of various PPIs largely depends on the state of this gene in a patient.

Among PPIs, lansoprazole exhibits the greatest inhibitory effect on CYP2C19, and to a lesser extent omeprazole and esomeprazole. The effect of rabeprazole is even lower, however, its thioester, which is formed during non-enzymatic metabolism, has a significant inhibitory effect on CYP2C19 activity. Pantoprazole has the least effect on CYP2C19. Pantoprazole has the greatest inhibitory effect on CYP3A4 in vitro, followed (as the effect decreases) by omeprazole, esomeprazole and rabeprazole and lansoprazole. For patients receiving multiple drugs, pantoprazole is the preferred PPI (Bordin D.S.).

Metabolism of five proton pump inhibitors.
Darker arrows indicate more significant metabolic pathways.
Figure taken from Marelli S., Pace F .

With the active participation of CYP3A4, domperidone, cisapride and a large number of other drugs are metabolized.

A number of gastroenterological drugs inhibit cytochrome CYP3A4, thereby affecting the pharmacokinetics of co-administered drugs.

The drug interaction problem

In modern clinical practice, the combined use of drugs is widespread, which is associated with the presence of several diseases in a patient or insufficient effectiveness of monotherapy. With combination therapy, drug interactions are possible. More than one medication is taken by approximately 56% of patients under 65 years of age and 73% of patients over 65 years of age. Taking two drugs leads to their interaction in 6% of patients. Prescribing 5 (or 10) drugs increases the frequency of interactions to 50 (or 100)%.

Potentially dangerous drug combinations are a serious clinical problem. There is evidence that between 17 and 23% of drug combinations prescribed by physicians are potentially dangerous. In the US alone, 48,000 patients die each year due to unintended drug interactions. The FDA has removed several drugs (including the prokinetic cisapride) from registration due to their potentially dangerous interactions with other drugs, including fatal ones.

The main mechanisms of drug interactions are associated with changes in their pharmacokinetics or pharmacodynamics. The most significant, according to modern concepts, are changes in pharmacokinetics during drug metabolism with the participation of cytochromes P-450.

An example of a dangerous interaction is the recently discovered interaction between PPIs and clopidogrel, which is widely used in the treatment of patients with coronary heart disease. To reduce the risk of gastrointestinal complications, patients receiving acetylsalicylic acid in combination with clopidogrel are prescribed PPIs. Since the bioactivation of clopidogrel occurs with the participation of CYP2C19, the use of PPIs metabolized by this cytochrome may reduce the activation and antiplatelet effect of clopidogrel. In May 2009, at a conference of the Society for Cardiovascular Angiography and Interventions (SCAI), data were presented indicating that the simultaneous use of clopidogrel and PPI significantly increases the risk of myocardial infarction, stroke, unstable angina, the need for repeated coronary interventions and coronary death (Bordin D .WITH.).

Cytochrome CYP2C19

The cytochrome P450 isoform CYP2C19 (S-mephenytoin hydroxylase) catalyzes the reactions of 5-hydroxylation of the pyridine ring and 5 "-demethylation in the benzimidazole ring. In the human body, CYP2C19 is located in hepatocytes.

All types of mutations in the CYP2C19 gene can be divided into three groups:

1. Without mutations (homozygotes), they are also fast PPI metabolizers.
2. Having a mutation in one allele (heterozygotes), an intermediate type of metabolism.
3. Having mutations in both alleles, they are also slow PPI metabolizers.

The prevalence of CYP2C19 genotypes, type of metabolism and the effect of PPIs in the treatment of acid-related diseases are given in the table:

Genotype CYP2C19	Prevalence (Tkach S. M. et al., 2006)			type of metabolism	Half-life of PPI, T½, hour (Lapina T.L.)	Acid-inhibiting effect of PPIs
		caucasian race	Mongoloid race
No mutations (homozygotes)	90% Caucasian population	50,6 %	34,0 %	Quick	1	Short
Mutation in 1st alley (heterozygotes)	10% Caucasian population	40,5 %	47,6 %	Intermediate	-	Average
Mutation in both alleys	20-30% Asian population	3,3 %	18,4 %	Slow	2–10	High

Slow metabolizers differ from fast and intermediate metabolizers by a twofold higher concentration of PPIs in blood plasma and half-life. The polymorphism of the gene encoding the 2C19 isoform determines the different rate of PPI metabolism in patients. In connection with the above, the selection of IPP is recommended to be carried out under the control daily pH-metry(Havkin A.I., Zhikhareva N.S., Drozdovskaya N.V.).

• CYP2C19 actively metabolizes the following drugs: tricyclic antidepressants (amitriptyline, clomipramine, imipramine), antidepressant - selective serotonin reuptake inhibitor citalopram, antidepressant - MAO inhibitor moclobemide, anticonvulsants and antiepeliptics (diazepam, primidone, phenytoin, phenobarbital, nordazepam), proton pump inhibitors (omeprazole, pantorazole, lansoprazole, rabeprazole, and esomeprazole), the antimalarial proguanil, the NSAIDs diclofenac, and indomethacin, as well as: warfarin, gliclazide, clopidogrel, propranolol, cyclophosphamide, nelfinavir, progesterone, teniposide, tetrahydrocannabinol, carisoprodol, voriconazole, and others
• strong CYP2C19 inhibitors: moclobemide, fluvoxamine, chloramphenicol (levomycetin)
• non-specific inhibitors of CYP2C19: PPI omeprazole and lansoprazole, H2 blocker cimetidine, NSAIDs indomethacin, as well as fluoxetine, felbamate, ketoconazole, modafinil, oxcarbazepine, probenecid, ticlopidine, topiramate
• CYP2C19 inducers: rifampicin, artemisinin, carbamazepine, norethisterone, prednisone, St. John's wort.

Effect of different CYP2C19 genotypes on the efficiency of Helicobacter pylori eradication

In patients with the genotype of "fast" metabolizers, a rapid metabolism of proton pump inhibitors is noted, therefore, the antisecretory effect of taking the latter is less pronounced in them than in individuals with the phenotypes of "intermediate" and "slow" metabolizers. Difference in antisecretory effect may determine lower eradication rate Helicobacter pylori at "fast" metabolizers. Thus, there is a higher efficiency of eradication therapy in patients with genotypes of "slow" (88.9%) and "intermediate" (82.7%) metabolizers compared to "fast" metabolizers (see figure).

Effect of different CYP2C19 genotypes on the efficiency of Helicobacter pylori eradication.
BM - "fast" metabolizers, PM - "intermediate" metabolizers, MM - "slow" metabolizers (Maev I.V. et al.)

Due to the fact that molecular genetic studies are inaccessible to a practicing physician, it is possible to suspect "fast" metabolizers based on the persistence of abdominal pain on the 3rd–4th day from the start of PPI administration, and also taking into account the slow endoscopic dynamics during epithelialization of erosions and scarring. ulcers in a patient. In turn, the insufficiency of the antisecretory effect of PPI therapy can be verified by daily intragastric pH-metry (Maev I.V. et al.).

Cytochrome CYP3A4

The CYP3A4 enzyme catalyzes the sulfoxidation reaction leading to the formation of a sulfo group. CYP3A4 is one of the most important cytochromes for pharmaceuticals, since it biotransforms, at least partially, about 60% of oxidized drugs. Although CYP3A4 activity varies widely, it is not subject to genetic polymorphism. The location of CYP3A4 on the apical membranes of enterocytes of the small intestine and hepatocytes facilitates its metabolism of drugs prior to entry into the systemic circulation, which is known as the “first pass effect”.

A genetic defect in CYP3A4 may be the cause of the development of a secondary long QT syndrome when taking cisapride and, as a result, the development of cardiac arrhythmia (Khavkin A.I. et al.).

• CYP3A4 is the main enzyme in the metabolism of the following drugs: immunosuppressants (cyclosporine, sirolimus, tacrolimus), chemotherapy agents (anastrozole, cyclophosphamide, docetaxel, erlotinib, tyrphostin, etoposide, ifosfamide, paclitaxel, tamoxifen, teniposide, vinblastine, vindesine, gefitinib) , antifungal agents (clotrimazole, ketoconazole, itraconazole),

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