Demystifying Cytochrome P450 for clinicians Alan P. Agins, Ph.D. President PRN Associates, Ltd Continuing Medical Education Tucson, AZ

Objectives At the conclusion of this program, the participant will be able to: Define the physiological and pharmacological roles of cytochrome P450 Describe the enzymatic process and clinical impact of cytochrome P450-dependent drug metabolism. List the major isoforms or subtypes of the cytochrome P450 enzymes that metabolize drugs Recognize the clinical ramifications of enzyme induction, inhibition and genetic polymorphism.

Cytochrome P450 Overview Major drug metabolizing enzyme system in the body Actually comprised of multiple proteins Active site or core of the enzyme system is a heme protein a.k.a. “mixed function monooxygenase” Reactions require molecular oxygen Liver and gut wall have the greatest concentration of P450 almost all tissues in body have some P450 (Lungs, Kidney, Skin, Brain)

Cytochrome P450: Raison d’etre Fat Soluble Water Soluble Kidneys Elimination In order for drugs to be absorbed into the body, that is, for them to get across the epithelial cell membranes lining the stomach and intestine, they have to be somewhat fat soluble. In contrast, in order for us to get rid of foreign chemicals (remember that drugs are actually considered foreign chemicals) the most rapid and efficient way is through the kidney in the urine. But urine is in reality water and so somewhere in the body it is necessary to convert those fat soluble chemicals into more water soluble chemicals . . . . and that’s where the liver and drug metabolism come into play. The liver helps to convert fat soluble drugs into water soluble metabolites so that the kidney can more efficiently get rid of them. Liver Drug Metabolism

First Pass To Systemic Circulation Portal Tract From Gut CYP CYP CYP

Cytochrome P450 Mechanism Electron transport Metabolite OH Drug Fe+++ Fe+++ Fe++ Heme Oxygen 6

Fate of P450 Drug Metabolites OH Inactive Most drugs become inactive after P450 metabolism Examples: warfarin, amlodpine, atorvastatin, lorazepam

Fate of P450 Drug Metabolites Equally Active ”Active Metabolite” Examples: fluoxetine  norfluoxetine sildenafil  N-desmethylsildenafil alprazolam  4-hydroxyalprazolam loratadine  desloratadine (Clarinex) venlafaxine  O-desmethylvenlafaxine (Pristiq) risperidone  9-hydroxyrisperidone (Invega) Metabolite OH

Fate of P450 Drug Metabolites OH More Active Examples: losartan  E-3174 (10 – 40x) tamoxifen  endoxifen (1000x) oxycodone  oxymorphone (3 – 10x) hydrocodone  hydromorphone tramadol  M1 metabolite (1000x)

Fate of P450 Drug Metabolites OH Activation of “ProDrug” CYP450 necessary to convert drug to its active form Examples: codeine  morphine clopidogrel  active compound (ADP receptor blocker)

Fate of P450 Drug Metabolites OH Reactive (Toxic) Metabolite Examples: acetaminophen  hepatotoxic metabolite (N-acetyl-p-benzoquinone imine)

Cytochrome P450 Humans have 17 families of CYP450 genes 39 subfamilies Three families dedicated to drug metabolism CYPs 1, 2 and 3 Remaining 14 families involved in physiological / homeostatic functions biosynthesis or degradation of: cholesterol bile acids steroid hormones vitamin D3

How Cytochrome P450 got its name In hemoglobin, when the iron in the heme is reduced and binds to carbon monoxide (or oxygen) – the complex absorbs light in the visible spectrum such that it becomes blood RED (~ 650 nm). Ultraviolet infrared Wavelength (nm)

How Cytochrome P450 got its name When liver drug metabolizing enzyme preparations are subjected to the same conditions (ie., reduced heme iron plus carbon monoxide) – the complex absorbs light in the visible spectrum such that it becomes Blue-Violet in color. Ultraviolet infrared Wavelength (nm)

Cytochrome P450 Humans have 17 families of CYP450 genes 39 subfamilies Three families dedicated to drug metabolism CYPs 1, 2 and 3 Remaining 14 families involved in physiological / homeostatic functions biosynthesis or degradation of: cholesterol bile acids steroid hormones vitamin D3

Cytochrome P450 Isoforms

Some Drugs are metabolized by multiple CYP pathways Noroxycodone (inactive) Oxymorphone (active) Oxycodone CYP 2D6 CYP 3A4 Hydromorphone (active) Norhydromorphone (inactive) Hydrocodone Warfarin Anyhow, Keep in mind that many drugs are metabolized by more than one CYP450 enzyme. For most drugs there is one isoform that may be the major and most effcicient pathway but there may also be minor contibutions to the overall metabolism by other isoforms as well. Here’s an example. The opioid analgesic oxycodone is metabolized by CYP3A4 to noroxycodone (has weak analgesic), noroxymorphone, and alpha- and beta-noroxycodol. CYP2D6 mediated metabolism produces oxymorphone (has analgesic activity; low plasma concentrations), alpha- and beta-oxymorphol. HYdrocodone Hepatic; O-demethylation via primarily CYP2D6 to hydromorphone (major, active metabolite with ~10- to 33-fold higher or as much as a >100-fold higher binding affinity for the mu-opioid receptor than hydrocodone); N-demethylation via CYP3A4 to norhydrocodone (major metabolite); and ~40% of metabolism/clearance occurs via other non-CYP pathways, including 6-ketosteroid reduction to 6-alpha-hydrocol and 6-beta-hydrocol, and other elimination pathways (eg, fecal, biliary, intestinal, renal) (Hutchinson, 2004; Volpe, 2011; Zhou, 2009) CYP 1A2 CYP 3A4 CYP 2C9 Duloxetine CYP 1A2 CYP 3A4

Induction Inhibiton Pharmacogenetics Clinically Important Aspects of Cytochrome P450 Drug Metabolism Induction Elevated levels of Cytochrome P450 Inhibiton Blockade of hepatic drug clearance Pharmacogenetics Variability between patients

Induction resulting from administration of certain drugs reversible increase in enzyme concentration resulting from administration of certain drugs potential to increase rate of the “inducing” drug’s breakdown “Pharmacokinetic or Drug Disposition Tolerance” may increase the metabolism of other drugs taken concurrently

Induction Amount of CYP enzymes initiate “inducing” agent Time Discontinue “inducing” drug initiate “inducing” agent Time

Induction Systemic Circulation Portal vein CYP CYP CYP CYP CYP CYP Guniita © 123rf.com

Interactions potentially leading to sub-therapeutic drug levels CYP450 Induction Interactions potentially leading to sub-therapeutic drug levels Substrates Inducers CYP 3A4

Interactions potentially leading to sub-therapeutic drug levels CYP450 Induction Interactions potentially leading to sub-therapeutic drug levels Substrates Inducers CYP1A2

Interactions potentially leading to TOXICITY CYP450 Induction Interactions potentially leading to TOXICITY Substrates Inducers CYP2E1

Acetaminophen Toxicity CYP2E1 Alcohol Induces overdose Glucuronide Sulfate Glutathione

Inhibition of Drug Metabolism Due to two drugs competing for the same enzyme Drug with greater affinity typically wins! Some drugs get into active site and are slow to dissociate Drug that is not metabolized can build up to toxic levels

Cytochrome P450 Inhibition Competition for Enzyme by Substrates Drug 2 Drug Drug Drug Drug 1 Drug Drug 1 Drug 1 P450 P450 P450 Drug 1 P450

Interactions potentially leading to Toxic CYP450 Inhibition Substrates Inhibitors Interactions potentially leading to Toxic drug levels CYP3A4

Interactions potentially leading to Toxic CYP450 Inhibition Interactions potentially leading to Toxic drug levels Substrates Inhibitors CYP2D6

Interactions potentially leading to Sub-therapeutic CYP450 Inhibition Interactions potentially leading to Sub-therapeutic drug levels Substrates Inhibitors CYP2D6 Pro-drug Substrates (need activation by CYP2D6) codeine diminished analgesia tamoxifen decreased protection

Interactions potentially leading to Toxic CYP450 Inhibition Interactions potentially leading to Toxic drug levels Substrates Inhibitors CYP2C9

Interactions potentially leading to Toxic CYP450 Inhibition Interactions potentially leading to Toxic drug levels Substrates Inhibitors CYP2C19

Interactions potentially leading to Sub-therapeutic CYP450 Inhibition Interactions potentially leading to Sub-therapeutic drug levels CYP2C19

Interactions potentially leading to Toxic CYP450 Inhibition Interactions potentially leading to Toxic drug levels Substrates Inhibitors CYP1A2

Pharmacogenetics and Cytochrome P450 Major P450 Isoforms CYP3A4 CYP2D6 - Polymorphism CYP2C19 - Polymorphism CYP2C9 - Polymorphism CYP1A2 CYP2E1

CYP2D6 Poor Metabolizers (PM) Inheritance of two mutant CYP2D6 alleles No enzyme or very poor enzyme activity = impaired metabolism of CYP2D6 substrates Caucasians 8 – 14% African-Americans 2 – 3 % Hispanic 4 – 5 % Asian < 1% Higher plasma drug level due to decreased drug clearance; exaggerated clinical outcome and increased risk of dose-dependent side effects; may have to lower drug dose

Drugs Metabolized by CYP2D6 Potential Consequences in PMs   Drugs Metabolized by CYP2D6 Potential Consequences in PMs dextromethorphan Beta Blockers metoprolol carvedilol Opioids codeine hydrocodone oxycodone tramadol TCAs venlafaxine fluoxetine paroxetine haloperidol perphenazine risperidone atomoxetine tamoxifen

CYP2D6 Ultra-extensive Metabolizers (UEM) Inheritance of alleles with duplication or amplification of CYP2D6 genes Excessive amount of enzyme expressed, high metabolic capacity 15 - 30 % Northern Africans Eastern Africans Saudi Arabians Mediterranean 7 - 12% Northern European 2 - 5% Asian - Chinese ~1%

Drugs Metabolized by CYP2D6 Potential Consequences in UEMs   Drugs Metabolized by CYP2D6 Potential Consequences in UEMs TCAs venlafaxine fluoxetine paroxetine haloperidol perphenazine risperidone atomoxetine tamoxifen dextromethorphan Beta Blockers metoprolol carvedilol Opioids codeine hydrocodone oxycodone tramadol Possibly higher than normal drug dose required for efficacy; side effects if metabolites are toxic

Cytochrome P450 CYP2C9 Polymorphism Low activity in 3 – 16% of Caucasians 1 – 4% Asians, ~ 2% African-Americans May affect clearance of: Phenytoin, S-warfarin ARBs (losartan, valsartan) Sulfonylureas (glipizide, glyburide) NSAIDS (diclofenac, naproxen, ibuprofen, celecoxib) Misc (rosuvastatin, fluvastatin)

Summary of non-drug agents that may have clinically relevant impact of drug metabolism Diet, Lifestyle, Environment P450s can be induced or inhibited by alcohol caffeine constituents of tobacco charcoal-broiled foods cruciferous vegetables grapefruit juice air or water pollutants

Other factors that may affect Cytochrome P450 metabolism Age - Pediatric vs Adult vs Geriatric - Infants do not develop a mature enzyme system until more than 2 weeks after birth. Both quantitative & qualitative difference in pediatrics (neonates, infants, puberty) Elderly have age related decreases in liver mass, hepatic enzyme activity and hepatic blood flow.

Other factors that may affect Cytochrome P450 metabolism Gender Steroid induction of CYP3A may be responsible for some of the pharmacokinetic differences between men and women Also within an individual over his/her life cycle from the prepubertal years to puberty and on through menopause Liver Health Hepatic disease (ie., hepatitis, cirrhosis, etc.) will result in impaired metabolism of drugs by Cytochrome P450

Summary Cytochrome P450 plays a critical role in both physiological and pharmacological processes. It is important to recognize the clinical ramifications of induction, inhibition and genetics for drugs cleared through the CYP450 system. Cytochrome P450-related metabolism is one of the major sources of drug interactions Due to genetic polymorphism, it may be advantageous to include a family med history when taking the pts In addtion to drugs, P450 activity can be influenced by age, liver status, dietary and lifestyle habits

Alan Thanks for listening. You now know more about CYP450 than most clinicians (MDs, DOs, PAs, other NPs)! Thanks for listening. Alan