Cyp450 Pavitraraj

Cytochrome P450

Cytochrome P450The 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).Dr. Pavitra Raj Dewda1

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Dr. Pavitra Raj Dewda2Contents

1.BIOTRANSFORMATIONWhat is it?What are the Components?What is the Process?

Dr. Pavitra Raj Dewda3

xenobiotics metabolismsubstances 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.Organs of BiotransformationLIVER (hepatocytes)kidney (proximal tubule)lungs (type II cells)testes (Sertoli cells)skin (epithelial cells)intestines (microbial flora)

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

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Location SER / CytosolSite liver ,kid lungs/ hepatic plasmaEnzymes Reaction catalysednote4


*Reactions *Enzymes involvedPhase I - CYP450 , FMO , hydroxylases (alc dehydrogenase , xanthine oxidase , MAO)Phase II conjugation - .5

Clinical Correlate Metabolites can alter the pharmacological action of drug qualitatively

Terfenadine Fexofenadine

FluoxetineNorfluoxetineDr. Pavitra Raj Dewda6

Non sedating antihistaminic Can Block K+ Channel Cause ArrhythmiasBlocks histamine H 1 receptors only & not cardiac K+ channelsT1/2 50 hrsT1/2 10 days , COMPETES for hepatic oxidases with TCA ect..days after administration of the parent drug has been stopped.METABOLITEMETABOLITE -demethylatedCYP3A4CYP2D6

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2.Cytochrome P450 isoenzymes (CYPs)What are they?NomenclatureCYP reaction


Dr. Pavitra Raj Dewda8Proteins with at least 40% amino acid sequence homology. In humans up to 21 families have been described.In its reduced (Fe2+) form, it binds carbon monoxide to give a complex that absorbs light maximally at 450 nm40-55% amino acid sequence homology & about 20 subfamilies are imp in man>55 amino acid sequence homology & 57 such isotype

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 metabolismXenobiotic metabolising - 12 CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5)Some show genetic polymorphism while others can be induced or inhibited

What are CYPs ?Superfamily of haemoprotein enzymes found on the membrane of SER The heme is a cofactor which contains iron-protoporphyrin IX that functions to bind & activate Oxygen and transfer electrons.CYPs are also known as Mixed function oxidasesPoly substrate Mono-oxygenasesLocation liver , intestine , lungs , kidneys , brain etc.


CYP Oxidation ReactionDr. Pavitra Raj Dewda10

Dr. Pavitra Raj Dewda11NADPH +H++ O2+RH

NADP++H2O+R-OH

The figure shows increasingly microscopic levels of detail, sequentially expanding the areas within the black boxes. CYPs embedded-phospholipid bilayer of 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. 11

Microsomal drug oxidations Reactionstep 1Oxidized (Fe3+) P450 combines with a drug substrate to form a binary complex. step 2NADPH donates an electron to the flavoprotein P450 reductase, which in turn reduces the oxidized P450-drug complex . step 3A 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 complexstep 4 This complex in turn transfers activated oxygen to the drug substrate to form the oxidized productSometimes 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) Epoxide Hydrolases hydrolyse such epoxides microsomal epoxide hydrolase (mEH)soluble epoxide hydrolase (sEH)Dr. Pavitra Raj Dewda12

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Carbamazepine v/s newer antiepilepticsCarbamazepine (prodrug)CYP3A Carbamazepine -10,11-epoxide (active)mEH

Dihydrodiol (inactive)

Newer Gabapentine & Levetiracetam are metabolized only by CYPsDr. Pavitra Raj Dewda13CYP3A4ValnoctamideValproic acid

DrowsinessHeadachesMigraines Motor coordination impairmentUpset stomach

3.Genetic PolymorphismWhat is it?Phenotypic (allelic) variants Examples New approach to personalized Rx

PRINCIPLETesting for Polymorphisms (genotyping) could predict therapeutic failures or ADRs therefore used to optimize drug choice, avoid serious ADRs and decrease medical costs!Dr. Pavitra Raj Dewda14

Genetic Polymorphism of CYP450Polymorphism - www.imm.ki.se/CYPalleles/Is Genetic variations in a population when both gene or allelic variants exist with a frequency of at least one percentCYP enzymes in families 1-3 are polymorphicCYP2D6, CYP2C19 and CYP2C9 main polymorphic forms responsible for almost 40% of P450 metabolismHomozygous mutations in the CYP1B1 gene are associated with primary congenital glaucomaDr. Pavitra Raj Dewda15

Existence of inter-individual differences in DNA sequences coding for one specific gene, giving rise to different functional and (or) morphological traits

Gene 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 15

P450 Phenotypes based on the allelic variation & clinical importanceDr. Pavitra Raj Dewda16


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From impossible to possibleDr. Pavitra Raj Dewda18

The AmpliChip (Roche Molecular Systems, Inc.)

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"Dr. Pavitra Raj Dewda19

CYP450 Enzyme InductionWhat is enzyme induction?What are the consequences of enzyme induction?What is the mechanism of enzyme induction?ExamplesDr. Pavitra Raj Dewda20

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What is enzyme induction?Increase in the CYP450 metabolizing capacityRequires repeated drug administrationOnset & Duration of inductionDepends on kinetics & half life of the drug (ranges from 1-6 days)Peak induction 4-14 days On withdrawing inducer the CYP returns to their original level in 1-3 weeksMechanismEnhancing the rate of its synthesis* Increasing transcriptionReducing its rate of degradation /substrate stabilizationIncreasing translation


What is the mechanism of enzyme induction?Is incompletely understood but is similar to a steroid that bind to nuclear receptors (induction/regulatory receptors).

Drugs bind to the ligand binding domain of a soluble protein, termed the aromatic hydrocarbon (Ah) receptor which is cytosolic in nature.

This binding leads to conformational change in receptor

Transportation of this complex to the nucleus by an Ah-receptor nuclear translocater

In the DNA binds Ah-receptor response elements thereby promoting transcription of the geneDr. Pavitra Raj Dewda22

Nuclear Receptors That Induce Drug MetabolismREGULATORY RECEPTORLIGANDSCAR (constitutively active receptor)*CYP3A, CYP2B6, CYP2C9Phenobarbitallong t1/2 ; induction delayed for 14-22 daysPregnane X receptor (PXR)*CYP3ADexamethasone[Short t1/2 ; induction time -4 days]Antibiotics (rifampicin and troleandomycin) Ca2+ channel blockers (nifedipine)Statins (mevastatin)Antidiabetic drugs (troglitazone) HIV protease inhibitors (ritonavir) Anticancer drugs (paclitaxel)St. John's wortAh-receptor CYP1APolycyclic aromatic hydrocarbons eg, benzo[a]pyrene, dioxinAryl hydrocarbon receptor (AHR) CYP1A1/2 activates procarcinogenOmeprazoleFarnesoid X receptor (FXR)Bile acidsVitamin D receptorVitamin DPeroxisome proliferator activated receptor (PPAR)FibratesRetinoic acid receptor (RAR)all-trans-Retinoic acidRetinoid X receptor (RXR)9-cis-Retinoic acid*type 2 nuclear receptors also PPAR

Non-transcriptional mechanisms Protein / Substrate stabilization or increased protein translation i.e. decreased degradation. e.g. CYP2E 1 by ethanol and isoniazide CYP3A by clotrimazole

CYP3A4- inducers macrolides , glucocorticoids , rifampin (ocp failure) , st johns wort (cycloporin)CYP2E1 inducer chr alc , INH (paracetamol)CYP1A2 inducer non pharmacological agents charcoal boriled meat , smoke , PAHC22

Pharmacological inductionOral contraceptives & steroids

Rifampicin

Inactivated & Excreted

Result Contraceptive Failure Dr. Pavitra Raj Dewda23

Induction

CYP3A4Other CYP3A inducersGlucocorticoidsAnticonvulsants

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Pharmacological induction/Herbal DrugCyclosporin

St. Johns Wort

Metabolite

Result Impaired Immune responseRejectionDr. Pavitra Raj Dewda24

Induction

CYP3A4

Pharmacological inductionParacetamol

Ethanol(chronic)INH

Toxic Metabolite(N-acetylbenzoiminoquinone)

Result Hepatotoxicity Dr. Pavitra Raj Dewda25

Induction

CYP2E1

Patients with alcohol dependence are at increased risk of acetaminophen toxicity!25

Pharmacological induction - paracetamol contDr. Pavitra Raj Dewda26

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.26

Cigarette smoke Benzpyrene CYP1A2 7-8epoxidation

Dr. Pavitra Raj Dewda27activatesCarcinogen+induces synthesis +Polycyclic hydrocarbons- Epoxide formationCharcoal barbecued foodPlasticizers (polychlorinated biphenyls)

Non Pharmacological (CYP1A2) inducersHigh dosing requirements of drugs like Theophylline in smokers

Enzyme InhibitionWhat is enzyme inhibition?What are the types?Specific examplesDr. Pavitra Raj Dewda28

What is enzyme inhibition?Certain substrates can block P450 enzyme isoforms (specifically) that metabolize other drugs/substrates A drug may inhibit one P450 isoenzyme while being metabolized its self by another.E.g. Quinidine metabolized by CYP3A4 and inhibits CYP2D6Also some times a product of drugs oxidation may inhibit its own metabolism Suicide InhibitorE.g. Chloramphenicol , Spironolactone etc

Unlike induction (1-3 weeks for onset) , enzyme inhibition usually begins with the first dose of the inhibitor and has a fast time course.Dr. Pavitra Raj Dewda29

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

30H2-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

imidazole-containing antifungal & CYP3A4 inhibitorKetoconazole (/Itraconazole> fluconazole )is a more potent inhibitor because of higher lipophilicity resulting in stronger hydrophobic interactions to CYP3A4

Ketoconazole inhibits metabolism of anti-HIV viral protease inhibitors

REVERSIBLE INHIBITION30

nausea, diarrhea, increase in heart rate, arrhythmias, and CNS excitationDr. Pavitra Raj Dewda31Inhibitors Erythromycin, Clarithromycin (but not azithromycin) Grape fruit juice (furanocoumarins)

Metabolism of drugs Inhibited (increasing Bioavalibility)Astemizole Terfenadine CisaprideFelodipine Cyclosporine Saquinavir TheophyllineTriazolam

CYP3A4(torsades de pointes / prolonged QT interval)(ventricular arrhythmias ; the new drug ITOPRIDE is metaboilsed by FMO3)e.g. of Irreversible Inhibition due to formation of a MIErythromycin - oxidised by CYP3A4

Nitroso Metabolite + Haem portion of the CYP enzyme

Stable MI complex is inactive

Illustrative Case 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.

Ritonavir (inhibitor CYP3A)increases the Bioavalibility of Nifedipine

Astemizole (torsades de pointes )Terfenadine (active metabolite fexofenadine prolonged 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 (cvs cns rs)31

Suicide inhibitorsinactivators that attack the heme or the protein moietyincludes ; Steroids (ethinyl estradiol, norethindrone, and spironolactone)Fluroxene Allobarbital The analgesic sedatives allylisopropylacetylureaDiethylpentenamide Ethchlorvynol Carbon disulfideChloramphenicolGrapefruit furanocoumarinsDeprenylPhencyclidine Ticlopidine and clopidogrelRitonavir Propylthiouracil Dr. Pavitra Raj Dewda32

e.g. of drugs inhibiting CYP2D6In order of inhibiting potencyParoxetine(is also metabolized)>Fluoxetine>SertalineDr. Pavitra Raj Dewda33If combined with Trazadone

Postural Hypotension & probably Pirpaism

If combined with TCAs

Cardio toxicity ConvulsionsComa(substrates)

CYP2D6


ROLE OF DRUG METABOLISM IN THE DRUG DEVELOPMENT PROCESS Efficacy and Safety ; Both depend on drug metabolismIt is necessary to determine how and by which enzymes a potential new drug is metabolizeddrug-drug interactions genetic polymorphisms Ideally, the best drug for a trial would be - metabolized by several CYPs so that variability in expression levels of one CYP or drug-drug interactions would not significantly impact its metabolism and pharmacokineticsDr. Pavitra Raj Dewda35

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.

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Summary Most important CYPs involved in drug metabolism are CYP3A4/5, CYP1A2, CYP2D6, CYP2C9, CYP2C19, CYP2E1Knowledge 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 . Dr. Pavitra Raj Dewda36


Chapter 15Text 14


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Extra


How can we demonstrate a role for cytP450 in biotransformation of a XB?cytP450 activity increases after exposure to XB Enzymatic inhibitors of cytP450 (e.g. aminobenzotriazole) have an effectAbsorption spectrum of cytP450 is modified in presence of XBReaction can be reconstituted with components of the catalytically cycle (e.g. NADPH)Dr. Pavitra Raj Dewda40

Hydroxylation of substrates by cytochrome P450-Deactivation (detoxication)Dr. Pavitra Raj Dewda41

Role of cytochrome P450 and peroxidase in activation of benzene in bone marrowDr. Pavitra Raj Dewda42

PHS prostaglandin H synthase

Halothane hepatitis and activation by oxidation and reductionDr. Pavitra Raj Dewda43

Reactions catalyzed by cytochrome P450Dr. Pavitra Raj Dewda44

Dr. Pavitra Raj Dewda451A22A62B62C82C92C182C192D62E13A4,5,7amitriptylinecaffeineclomipramineclozapinecyclobenzaprineestradiolfluvoxaminehaloperidolimipramine N-DeMemexilletinenaproxen olanzapineondansetron phenacetinacetaminophenpropranololriluzoleropivacainetacrineTheophyllinetamoxiphenetizanidineverapamil(R)warfarinzileutonzolmitriptanCoumarintobacco nitrosaminesnicotineArtemisinbupropioncyclophosphamideefavirenzifosfamideMethadonePropofolSelegilineSertalinpaclitaxeltorsemideamodiaquinecerivastatinrepaglinideibuprofenmeloxicamS-naproxenpiroxicamsuprofenOral Hypoglycemic Agents:tolbutamideglipizideAngiotensin II Blockers:losartanirbesartanSulfonylureas:glyburide/glibenclamideglimepiridetolbutamideamitriptylinecelecoxibfluoxetinefluvastatinglyburidenateglinidetamoxifentorsemideS-warfarin1TolbutamideProton Pump Inhibitors:lansoprazoleomeprazolepantoprazolerabeprazoleAnti-epileptics:diazepamphenytoin(O)S-mephenytoinphenobarbitoneamitriptylinecarisoprodolcitalopramchloramphenicolclomipraminecyclophosphamidehexobarbitalimipramine indomethacinR-mephobarbitalmoclobemidenelfinavirnilutamideprimidoneprogesteroneproguanilpropranolol teniposideR-warfarinBeta Blockers:amitriptyline clomipramine desipramineimipramine paroxetinehaloperidolalprenololamphetaminearipiprazoleatomoxetinebufuralolchlorpheniraminechlorpromazinecodeine fluvoxaminelidocaine metoclopramidemethoxyamphetamineoxycodonesparteinetamoxifentramadolvenlafaxineAnesthetics:enfluranehalothaneisofluranemethoxyfluranesevofluraneerythromycin telithromycin Benzodiazepines: cyclosporine tacrolimus (FK506) HIV Antivirals Antihistamines: astemizole chlorpheniramine terfenadine Calcium Channel Blockers HMG CoA Reductase Inhibitors: atorvastatin cerivastatin lovastatin simvastatin estradiol hydrocortisone progesterone testosterone cafergot caffeine cilostazol cocaine codeine dapsone dexamethasone dextromethorphan fentanyl finasteride gleevec haloperidol lidocaine methadone nateglinide quinine risperidone tamoxifen taxol vincristine

CYP450 ENZYME & SUBSTRATES

Some inducers of cytochrome P450 isozymes


Some inhibitors of cytochrome P450 isozymes



1A2

chromosome 15

2B6

chromosome 62C8

chromosome 102C19

chromosome 102C9

chromosome 10

2D6

chromosome 222E1

chromosome 103A4,5,7

chromosome 7

broccoli

brussel sproutschar-grilled meat

insulin

methylcholanthrene

modafinil

nafcillin

beta-naphthoflavone1

omeprazole

tobaccophenobarbital

rifampinrifampincarbamazepine

norethindrone

NOT pentobarbital

prednisone

rifampinrifampin

secobarbitaldexamethasone

rifampinthanol

isoniazidHIV Antivirals:

efavirenz

nevirapine

barbiturates

carbamazepine

efavirenz

glucocorticoids

modafinil

nevirapine

oxcarbazepine

phenobarbital

phenytoin

pioglitazone

rifabutin

rifampin

St. John's wort

troglitazone

1A2

2B62C82C192C92D62E13A4,5,7

fluvoxamine

ciprofloxacin

cimetidine

amiodarone

fluoroquinolones

furafylline

interferon

methoxsalen

mibefradilthiotepa

ticlopidinegemfibrozil

trimethoprim

glitazones

montelukast

quercetinlansoprazole

omeprazole2

pantoprazole

rabeprazole

chloramphenicol

cimetidine

felbamate

fluoxetine

fluvoxamine

indomethacin

ketoconazole

modafinil

oxcarbazepine

probenicid

ticlopidine

topiramatefluconazole

amiodarone

fenofibrate

fluvastatin

fluvoxamine

isoniazid

lovastatin

phenylbutazone

probenicid

sertraline

sulfamethoxazole

sulfaphenazole

teniposide

voriconazole

zafirlukastbupropion

fluoxetine

paroxetine

quinidine

duloxetine

terbinafine

amiodarone

cimetidine

sertraline

celecoxib

chlorpheniramine

chlorpromazine

citalopram

clemastine

clomipramine

cocaine

diphenhydramine

doxepin

doxorubicin

escitalopram

halofantrine

hydroxyzine

levomepromazine

methadone

metoclopramide

mibefradil

midodrine

moclobemide

perphenazine

ranitidine

red-haloperidol

ritonavir

ticlopidine

tripelennaminediethyl-dithiocarbamate

HIV Antivirals:

indinavir

nelfinavir

ritonavir

clarithromycin

itraconazole

ketoconazole

nefazodone

saquinavir

telithromycin

aprepitant

erythromycin

fluconazole

grapefruit juice

verapamil

diltiazem

cimetidine

amiodarone

chloramphenicol

delaviridine

diethyl-dithiocarbamate

fluvoxamine

gestodene

imatinib

mibefradil

mifepristone

norfloxacin

norfluoxetine

star fruit

voriconazoleram

Cyp450 Pavitraraj

Health & Medicine

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