Cytochrome P450steroid hormone biosynthesis from cholesterolmetabolism of Xenobiotics-compounds which are not normally found in the bodyDrugsCompounds in food produced by cooking (polyaromatic hydrocarbons, also in tobacco smoke) or microorganisms typically organic molecules which are poorly soluble in water
*Reactions *Enzymes involvedPhase I - CYP450 , FMO , hydroxylases (alcdehydrogenase , xanthineoxidase , MAO)Phase II – conjugation - ….
The figure shows increasingly microscopic levels of detail, sequentially expanding the areas within the black boxes. CYPs embedded-phospholipidbilayerof the endoplasmic reticulum (ER). located on the cytosolic surface of the ER. NADPH-cytochrome P450 oxidoreductase, transfers electrons to the CYPA single NADPH-CYP oxidoreductase species transfers electrons to all CYP isoforms in the ER. Each CYP contains a molecule of iron-protoporphyrin IX that functions to bind and activate O2. Substituents on the porphyrin ring are methyl (M), propionyl (P), and vinyl (V) groups.
Existence of inter-individual differences in DNA sequences coding for one specific gene, giving rise to different functional and (or) morphological traitsGene – made of DNA containing exons & introns which have coding n non coding sequenceChromosome– contains multiple copies of genes or many genesChromatin – condensed chromosomesAllele – (e.g. Aa)one member of a pair or a series of different forms of a gene.(members of a gene that produce different traits in gene charesteristics)Could be coding or non coding sequence. Genotype for the gene is the set of alleles that the individual posses.Diploid organisms (2 chromosomes) 2 alleles make up the genotype.A phenotype is any observable characteristic or trait of an organism: such as its morphology, development, biochemical or physiological properties ...The genotype is the characteristic that cannot be observed. The genotype is the genetic constitution of a cell, an organism, or an individual
Patients with alcohol dependence are at increased risk of acetaminophen toxicity!
This analgesic antipyretic drug is quite safe in therapeutic doses (1.2 g/d for an adult). It normally undergoes glucuronidation and sulfation to the corresponding conjugates, which together comprise 95% of the total excreted metabolites. The alternative P450-dependent GSH conjugation pathway accounts for the remaining 5%. When acetaminophen intake far exceeds therapeutic doses, the glucuronidation and sulfation pathways are saturated, and the P450-dependent pathway becomes increasingly important.
Astemizole (torsades de pointes )Terfenadine (active metabolite fexofenadineprolonged QT interval , torsades de pointes ,polymorphic ventricular tachycardia due to blockage of the delayed rectifer K ch in the heart) Cisapride (ventricular arrhythmias) Felodipine (vascular selective Ca ch block dihydropyridine , long t1/2) Cyclosporine (immunosuppressant - nephrotoxicitySaquinavir (PI)TriazolamTheophylline (cvscnsrs)
Interactions become a problem when multiple drugs are simultaneously administered, for example in elderly patients, who on a daily basis may take prescribed antiinflammatory drugs, cholesterol-lowering drugs, blood pressure medications, a gastric acid suppressant, an anticoagulant, and a number of over-the-counter medications.
Cytochrome P450<br />The P450 enzymes have unique spectral properties, and the reduced forms combine with carbon monoxide to form a “PINK” compound (hence 'P') with absorption peaks near 450nm (range 447-452nm).<br />Dr. Pavitra Raj Dewda<br />1<br />
Dr. Pavitra Raj Dewda<br />2<br />Contents<br />
1.BIOTRANSFORMATION<br />What is it?<br />What are the Components?<br />What is the Process?<br />Dr. Pavitra Raj Dewda<br />3<br />xenobiotics metabolism<br />substances absorbed across the lungs or skin or, more commonly, ingested either unintentionally as compounds present in food and drink or deliberately as drugs for therapeutic or "recreational" purposes.<br />Organs of Biotransformation<br />LIVER (hepatocytes)<br />kidney (proximal tubule)<br />lungs (type II cells)<br />testes (Sertoli cells)<br />skin (epithelial cells)<br />intestines (microbial flora)<br />
Clinical CorrelateMetabolites can alter the pharmacological action of drug qualitatively<br />TerfenadineFexofenadine<br />FluoxetineNorfluoxetine<br />Dr. Pavitra Raj Dewda<br />6<br />METABOLITE<br />CYP3A4<br />Blocks histamine H 1 receptors only & not cardiac K+ channels<br />Non sedating antihistaminic <br />Can Block K+ Channel Cause Arrhythmias<br />METABOLITE -demethylated<br />T1/2 – 50 hrs<br />T1/2 – 10 days , COMPETES for hepatic oxidases with TCA ect..days after administration of the parent drug has been stopped.<br />CYP2D6<br />
Dr. Pavitra Raj Dewda<br />8<br />In its reduced (Fe2+) form, it binds carbon monoxide to give a complex that absorbs light maximally at 450 nm<br />Proteins with at least 40% amino acid sequence homology.<br /> In humans up to 21 families have been described.<br /><ul><li>In humans - 21 families, 20 subfamilies and 57 genes
Of these CYP 1, 2 and 3 constitute 70% of total hepatic CYPs which responsible for 94% of drugs metabolism
Some show genetic polymorphism while others can be induced or inhibited</li></ul>>55 amino acid sequence homology & 57 such isotype<br />40-55% amino acid sequence homology & about 20 subfamilies are imp in man<br />
What are CYPs ?<br />Superfamily of haemoprotein enzymes found on the membrane of SER <br />The heme is a cofactor which contains iron-protoporphyrin IXthat functions to bind & activate Oxygen and transfer electrons.<br />CYPs are also known as <br />Mixed function oxidases<br />Poly substrate Mono-oxygenases<br />Location <br />liver , intestine , lungs , kidneys , brain etc.<br />Dr. Pavitra Raj Dewda<br />9<br />
Microsomal drug oxidations <br />Reaction<br />step 1<br />Oxidized (Fe3+) P450 combines with a drug substrate to form a binary complex. <br />step 2<br />NADPH donates an electron to the flavoprotein P450 reductase, which in turn reduces the oxidized P450-drug complex . <br />step 3<br />A second electron is introduced from NADPH via the same P450 reductase, which serves to reduce molecular oxygen and to form an "activated oxygen"-P450-substrate complex”<br />step 4 <br />This complex in turn transfers activated oxygen to the drug substrate to form the oxidized product<br />Sometimes the oxidised product is a Epoxide (highly reactive electrophiles that can bind to cellular nucleophiles found in protein, RNA, and DNA, resulting in cell toxicity and transformation)<br />EpoxideHydrolaseshydrolyse such epoxides<br />microsomalepoxidehydrolase (mEH)<br />soluble epoxidehydrolase (sEH)<br />Dr. Pavitra Raj Dewda<br />12<br />
3.Genetic Polymorphism<br />What is it?<br />Phenotypic (allelic) variants <br />Examples <br />New approach to personalized Rx<br />PRINCIPLE<br />Testing for Polymorphisms (genotyping) could predict therapeutic failures or ADRs therefore used to optimize drug choice, avoid serious ADRs and decrease medical costs!<br />Dr. Pavitra Raj Dewda<br />14<br />
Genetic Polymorphism of CYP450 <br />Polymorphism - www.imm.ki.se/CYPalleles/<br />Is Genetic variations in a population when both gene or allelic variants exist with a frequency of at least one percent<br />CYP enzymes in families 1-3 are polymorphic<br />CYP2D6, CYP2C19 and CYP2C9 –main polymorphic forms responsible for almost 40% of P450 metabolism<br />Homozygous mutations in the CYP1B1 gene are associated with primary congenital glaucoma<br />Dr. Pavitra Raj Dewda<br />15<br />
P450 Phenotypes based on the allelic variation & clinical importance<br />Dr. Pavitra Raj Dewda<br />16<br />
From impossible to possible<br />Dr. Pavitra Raj Dewda<br />18<br />
The AmpliChip (Roche Molecular Systems, Inc.) <br /><ul><li>Tests the DNA from a patient's white blood cells collected in a standard anticoagulated blood sample for 29 polymorphisms and mutations for the CYP2D6 gene and 2 polymorphisms for the CYP2C19
According to FDA labeling, "Information about CYP2D6 genotype may be used as an aid to clinicians in determining therapeutic strategy and treatment doses for therapeutics that are metabolized by the CYP2D6 product"</li></ul>Dr. Pavitra Raj Dewda<br />19<br />
CYP450 Enzyme Induction<br />What is enzyme induction?<br />What are the consequences of enzyme induction?<br />What is the mechanism of enzyme induction?<br />Examples<br />Dr. Pavitra Raj Dewda<br />20<br />
What is enzyme induction?<br />Increase in the CYP450 metabolizing capacity<br />Requires repeated drug administration<br />Onset & Duration of induction<br />Depends on ‘kinetics ’ & ‘half life’ of the drug (ranges from 1-6 days)<br />Peak induction4-14 days <br />On withdrawing inducer the CYP returns to their original level in 1-3 weeks<br />Mechanism<br />Enhancing the rate of its synthesis* <br />Increasing transcription<br />Reducing its rate of degradation /substrate stabilization<br />Increasing translation<br />Dr. Pavitra Raj Dewda<br />21<br />
What is the mechanism of enzyme induction?<br />Is incompletely understood but is similar to a steroid that bind to nuclear receptors (induction/regulatory receptors). <br />Drugs bind to the ligand binding domain of a soluble protein, termed the aromatic hydrocarbon (Ah) receptor which is cytosolic in nature. <br />This binding leads to conformational change in receptor<br />Transportation of this complex to the nucleus by an Ah-receptor nuclear translocater <br />In the DNA binds Ah-receptor response elements thereby promoting transcription of the gene<br />Dr. Pavitra Raj Dewda<br />22<br />Non-transcriptional mechanisms <br />Protein / Substrate stabilization or increased protein translation i.e. decreased degradation.<br /> e.g. CYP2E 1 by ethanol and isoniazide<br /> CYP3A by clotrimazole<br />
High dosing requirements of drugs like ‘Theophylline’ in smokers</li></ul>Carcinogen<br />
Enzyme Inhibition<br />What is enzyme inhibition?<br />What are the types?<br />Specific examples<br />Dr. Pavitra Raj Dewda<br />28<br />
What is enzyme inhibition?<br />Certain substrates can block P450 enzyme isoforms (specifically) that metabolize other drugs/substrates <br />A drug may inhibit one P450 isoenzyme while being metabolized its self by another.<br />E.g. Quinidine metabolized by CYP3A4 and inhibits CYP2D6<br />Also some times a product of drug’s oxidation may inhibit its own metabolism “Suicide Inhibitor”<br />E.g. Chloramphenicol , Spironolactone etc… <br />Unlike induction (1-3 weeks for onset) , enzyme inhibition usually begins with the first dose of the inhibitor and has a fast time course.<br />Dr. Pavitra Raj Dewda<br />29<br />
The determinant of potency of an inhibitor is the strength of the bond between the prosthetic heme iron and the lone electron pair of inhibitors<br />30<br />H2-receptor antagonist has a imidazole ring which binds competitively binding to the H 2 receptor & also selectively to the heme prosthetic group of CYP3A4 and 2D6<br />imidazole-containing antifungal & CYP3A4 inhibitor<br />Ketoconazole (/Itraconazole>fluconazole )is a more potent inhibitor because of higher lipophilicity resulting in stronger hydrophobic interactions to CYP3A4 <br />Ketoconazoleinhibits metabolism of anti-HIV viral protease inhibitors<br />
Dr. Pavitra Raj Dewda<br />31<br />e.g. of Irreversible Inhibition due to formation of a MI<br />Inhibitors <br />Erythromycin, Clarithromycin (but not azithromycin)<br />Grape fruit juice (furanocoumarins)<br />Metabolism of drugs Inhibited (increasing Bioavalibility)<br />Astemizole<br />Terfenadine<br />Cisapride<br />Felodipine<br />Cyclosporine <br />Saquinavir<br />Theophylline<br />Triazolam<br />Illustrative Case <br />A 47-year-old man recently diagnosed with HIV infection visited his physician with flushing, dizziness and swelling of the feet and ankles. He had been taking sustained-release nifedipine for treatment of hypertension for about three years. Approximately two weeks earlier, his physician had prescribed a combination of lamivudine, zidovudine and the protease inhibitor ritonavir.<br />Ritonavir (inhibitor CYP3A)increases the Bioavalibility of Nifedipine<br />Erythromycin - oxidised by CYP3A4<br />Nitroso Metabolite + Haem portion of the CYP enzyme<br />Stable MI complex is inactive<br />CYP3A4<br />(torsades de pointes / prolonged QT interval)<br />(ventricular arrhythmias ; <br />the new drug ITOPRIDE is metaboilsed by FMO3)<br />nausea, diarrhea, increase in heart rate, arrhythmias, and CNS excitation<br />
Suicide inhibitors—inactivators that attack the heme or the protein moiety—includes ;<br />Steroids (ethinyl estradiol, norethindrone, and spironolactone)<br />Fluroxene<br />Allobarbital<br />The analgesic sedatives allylisopropylacetylurea<br />Diethylpentenamide<br />Ethchlorvynol<br />Carbon disulfide<br />Chloramphenicol<br />Grapefruit furanocoumarins<br />Deprenyl<br />Phencyclidine<br />Ticlopidine and clopidogrel<br />Ritonavir<br />Propylthiouracil<br />Dr. Pavitra Raj Dewda<br />32<br />
e.g. of drugs inhibiting CYP2D6<br />In order of inhibiting potency<br />Paroxetine(is also metabolized)>Fluoxetine>Sertaline<br />Dr. Pavitra Raj Dewda<br />33<br />CYP2D6<br />(substrates)<br />If combined with Trazadone<br />Postural Hypotension & probably Pirpaism<br />If combined with TCAs<br />Cardio toxicity <br />Convulsions<br />Coma<br />
ROLE OF DRUG METABOLISM IN THE DRUG DEVELOPMENT PROCESS<br />Efficacy and Safety ; Both depend on drug metabolism<br />It is necessary to determine how and by which enzymes a potential new drug is metabolized<br />drug-drug interactions <br />genetic polymorphisms <br />Ideally, the best drug for a trial would be - <br />metabolized by several CYPs so that variability in expression levels of one CYP or drug-drug interactions would not significantly impact its metabolism and pharmacokinetics<br />Dr. Pavitra Raj Dewda<br />35<br />
Summary <br />Most important CYPs involved in drug metabolism are CYP3A4/5, CYP1A2, CYP2D6, CYP2C9, CYP2C19, CYP2E1<br />Knowledge about polymorphisms in CYPs (CYP2D6, CYP2C9, CYP2C19), enzyme induction & inhibition helps filtering out drugs for clinical trials and prevents unnecessary patient drug and drug interactions . <br />Dr. Pavitra Raj Dewda<br />36<br />
Dr. Pavitra Raj Dewda<br />38<br />Have a great day!<br />
Extra <br />Dr. Pavitra Raj Dewda<br />39<br />
How can we demonstrate a role for cytP450 in biotransformation of a XB?<br />cytP450 activity increases after exposure to XB <br />Enzymatic inhibitors of cytP450 (e.g. aminobenzotriazole) have an effect<br />Absorption spectrum of cytP450 is modified in presence of XB<br />Reaction can be reconstituted with components of the catalytically cycle (e.g. NADPH)<br />Dr. Pavitra Raj Dewda<br />40<br />
Hydroxylation of substrates by cytochrome P450-Deactivation (detoxication)<br />Dr. Pavitra Raj Dewda<br />41<br />
Role of cytochrome P450 and peroxidase in activation of benzene in bone marrow<br />Dr. Pavitra Raj Dewda<br />42<br />PHS – prostaglandin H synthase<br />
Halothane hepatitis and activation by oxidation and reduction<br />Dr. Pavitra Raj Dewda<br />43<br />