Cytochrome P450 Pharmacology

Sections

Biotransformation
P450 Drug-Drug Interactions
CYTOCHROME P450 ISOFORMS

• Here, we'll learn about drug biotransformation and we'll focus on key cytochrome P450 (P450) drug-drug interactions.

Biotransformation

Overview

• To begin, start a table.
• Denote that we divide drug biotransformation reactions into phase 1 and phase 2 reactions.

Phase 1

• Phase 1 reactions either reveal or introduce a functional group to make the lipophilic chemical more polar (ionized) and better able to be to be renally excreted.
• They are primarily executed by cytochrome P450 enzymes, which are anchored to smooth endoplasmic reticulum membranes.

P450 Reactions

• As we address in our P450 catalytic cycle tutorial, three types of P450 reactions predominate:
  • Hydroxylation – the insertion of oxygen into a carbon (or non-carbon) hydrogen bond.
  • Heteroatom oxidation – the addition of oxygen to a heteroatom (a non-carbon or hydrogen atom, such as nitrogen or sulfur).
  • Epoxidation – the addition of an oxygen across a carbon-carbon double bond (C=C) to form an epoxide (a cyclic ether).

Phase 2

• Phase 2 reactions couple an endogenous, polar moiety to the substrate (rather than reveal or introduce a functional group, like phase 1 reactions do) and are carried out by a wide variety of different transferases that can be located within specific organelles or freely within the cytoplasm (whereas smooth ER membrane-bound P450 enzymes carry out the majority of phase 1 reactions).
• Phase 2 reactions involve the conjugation of multiple endogenous reactants, including:
  • UDP glucuronic acid via glucuronidation (eg, lamotrigine)
  • Acetyl-CoA via acetylation (eg, Isoniazid (INH))
  • Glutathione via glutathione conjugation (eg, acetaminophen)
  • S-adenosylmethionine (SAM) via methylation (eg, dopamine and epinephrine)

BASIC STOICHIOMETRY

• To begin, let's address the basic stoichiometry of P450-mediated hydroxylation, the most common form of drug biotransformation; we address its biochemistry in detail elsewhere.
• P450 is a mixed function oxidase (aka monooxygenase), which means that it transfers one ("mono") oxygen to a substrate; they add a single oxygen into a bond. We highlight this definition because it is the over-arching mission of the catalytic cycle.

Reactants

Indicate that the reactants are:

• NADPH, which serves as a reducing agent; it is involved in electron transfer via P450 reductase.
• Hydrogen, which serves a key role in oxygen activation.
• Molecular oxygen; as just discussed, the reaction involves the insertion of an oxygen atom into a bond.
• And the substrate with a hydrogen bond (R-H).
• Consider that NAPDH is an important co-factor, so it is present in the basic stoichiometry; whereas the enzymes and prosthetic groups are not.

Summary

• Reactants: NADPH + H+ + O2 + R-H

Products

And show that the products are:

• NADP+, which is the result of NADPH oxidation
• H2O, which results from the introduction of protons.
• And R-OH, which is the result of hydroxylation of the substrate.

Summary

• Products: NADP+ + H2O + R-OH

• We see that an oxygen is inserted between the carbon-hydrogen bond to make this an hydroxylation and two hydrogens bind with the other oxygen to form water.

CATALYTIC CYCLE OVERVIEW

• Let's also generate a short-hand version of the major steps of the catalytic cycle, which we learn in detail elsewhere.

Reactants

First, the key reactants:

• Drug (which we draw as a pill) with hydrogen bond
• 2 protons
• Molecular oxygen

Summary

• Substrate-H + 2H+ + O2

Products

Next, the products:

• We show the drug with an hydroxyl.
• And water.

Summary

• Substrate-OH + H2O

Enzymes & Co-factors

• Show that NADPH (a co-factor in the stoichiometry) is oxidized to NADP+ via P450 reductase which transfers the electron from this oxidation to the P450 enzyme catalysis.
• We show that electron transfer occurs two times during the catalytic cycle.
• Note that one of these electron transfers can occur via cytochrome b5 (rather than NADPH reductase).
• Finally, show that the major catalytic enzyme is P450, which contains a central ferric iron (Fe3+) atom.

HISTOLOGY

Smooth Endoplasmic Reticulum

• So, now, let's take a look at where the reaction actually occurs in the cell: at the smooth endoplasmic reticulum membrane.
• First, draw a portion of a cell: the nucleus (with a nucleolus and chromatin) and include endoplasmic reticulum (ER). * Show that rough ER is lined with ribosomes (rough ER is essential for protein synthesis).
• Then, show smooth ER is void of ribosomes (it has a smooth surface) and is the location of P450, which is anchored to the membrane.
• Show a magnified view of the smooth ER membrane.
• Indicate the ER lumen and the cell cytoplasm.

Smooth ER Membrane

• Then, show that P450 is anchored to the smooth ER membrane, facing into the cell cytoplasm.
• Also show its neighboring P450 reductase, which provides electrons to P450.
• And cytochrome b5 (CYB5), which can provide the second electron in the catalytic cycle (instead of P450 reductase).

Phase 2 Transferases

• In contrast, as mentioned earlier, some phase 2 transferases are located within specific organelles (like P450 is) but others are freely located within the cytoplasm.

Microsomal Enzymes

• Note that P450s are commonly referred to as microsomal enzymes; this is because, in general, scientists do not study these enzymes in their in vivo environments but rather in broken-down in vitro in samples.
• During centrifugation, tissue samples are fractionated and then allowed to reform into microsomes (vesicles).
• The microsomes retain certain morphological and functional properties of their un-fractionated in vivo origins, however – smooth microsomes are derived from smooth ER and rough microsomes are derived from rough ER.
• And it is the smooth microsomes that contain the P450s whereas the rough microsomes contain the materials for protein synthesis.

DISTRIBUTION

Now, let's address where we find P450 on a macroscopic level.

Liver

• Draw a large liver and underlying gallbladder to indicate that the liver has the largest levels of P450 enzymes of any organs.

Additional P450 locations

However, P450 enzymes are found in numerous other organs, as well.

• Show that they are found in the adrenal glands and kidneys, lungs, as well as the pancreas and small intestine. The full distribution of these enzymes is beyond our scope, here, but even further examples include the testes and ovaries, skin, etc…
• Make special note of the CYP3A4 P450 isoform, which accounts for the metabolism of ~ 50% of the drugs in use, because the liver has the highest activity of this isoform; this fact can help us remember that the liver has the highest distribution of P450.

NOMENCLATURE

Before we address P450 drug-drug interactions, let's breakdown its nomenclature.

• Use CYP3A4.

Indicate that nomenclature for the P450 enzymes is as follows:

• CYP: Cytochrome
• 1st Number: Family
• 1st Letter: Subfamily
• 2nd Number: Individual enzyme
  • We'll learn about key aspects of a selection of isoforms later.

Cytochrome P450

What does CYP450 stand for?

• We can work our way through the term cytochrome P450, itself, as follows:
• "Cyto" refers to cell
• "Chrome" is shorthand for "chrome yellow", which is a specific bright yellow color; it's actually that yellow precipitate color we are used to seeing in organic chemistry lab.
• "P450" refers to the spectral wavelength that is maximally absorbed when spectrophotometry is performed on this group of enzymes (when they are bound to carbon monoxide): 450 nm, which is a blue color.
  • When blue light is absorbed, yellow light is perceived, so the absorption of wavelength at 450 nm generates that "chrome yellow" (organic chemistry precipitate) color we just discussed.
  • Consider, for instance, bilirubin, which is strongly yellow because it is a breakdown product of heme and cytochrome P450 enzymes are heme-proteins, which absorb blue light and give off a yellow appearance.

P450 Drug-Drug Interactions

Overview

• First, let's generate a list of P450 inducers and inhibitors because one of the key takeaways from this tutorial is that certain commonly prescribed drugs can greatly impact the blood level of other commonly prescribed drugs, and it's critical that we have a working knowledge of some of these major interactions for the care of our patients.

Inducers

• First, the inducers, which increase the metabolic activity of certain P450 isoforms and reduce certain drug levels:

Antiepileptic drugs (older generation)

• Carbamazepine
• Phenobarbital (and its prodrug, primidone)
• Phenytoin

Antimicrobials:

• Griseofulvin (an antifungal)
• Rifampin (a TB medication)

Diabetic medications

• Sulfonylureas (eg, glipizide)
• Troglitazone is a known inducer; note that there is less evidence that more modern "glitazones" (eg, rosiglitazone and pioglitazone) are also inducers.

Miscellaneous toxins and treatments

• Alcohol (in chronic ethanol use)
• Benzo[a]pyrene (which is a toxic byproduct of tobacco smoke, vehicle exhaust, as well as wood, coal, and petroleum burning).
  • Note that benzo[a]pyrene by itself is a weak carcinogen but via P450 metabolism, it is transformed over a series of steps to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, which is a much stronger carcinogen.
• St. John's wort (a dietary supplement)

Inducer mneomonic: CRAP TB GPS's

We can summarize many of the key inducers into an often-used mnemonic: CRAP TB GPS's

CRAP

• Carbamazepine
• Rifampin
• Alcohol (again, in chronic use)
• Phenytoin

TB

• Troglitazone
• Benzo[a]pyrene

GPS's

• Griseofulvin
• Phenobarbital (and other barbiturates)
• Sulfonylureas
• St. John's wort

INHBITORS

Now let's address the inhibitors, which reduce the rate of P450 metabolism and allow certain drug levels to rise:

Antiepileptic drug

• Valproic acid

Antimicrobials

• Chloramphenicol (a bacteriostatic antibiotic)
• Erythromycin (a macrolide)
• Isoniazid (a TB medication)
• -azole antifungals (eg, fluconazole, ketoconazole, etc…)
• Sulfonamides
  • They are used in toxoplasmosis management, and (more commonly) sulfamethoxazole (SMX) is co-formulated with trimethoprim (TMP) to as TMP/SMX (cotrimoxazole) in the treatment of routine urinary tract infections (UTIs).
  • This has very important implications in clinical practice because patients on warfarin can suffer from GI bleeds when warfarin metabolism is inhibited by sulfamethoxazole (as part of cotrimaxazole (TMP/SMX).
• Fluoroquinolones (eg, ciprofloxacin, another antibiotic commonly used to treat routine UTIs).

Cardiac rhythm medications

• Amiodarone
• Quinidine

H2 blocker

• Cimetidine

Antidepressants:

• SSRIs (selective serotonin reuptake inhibitors (eg, citalopram)
• Buproprion (a norepinephrine reuptake inhibitor)

Miscellaneous

• Grapefruit juice
• Alcohol in acute consumption
  • It competes with other substrates for binding to CYPE1 (rather than chronic alcohol use, which causes P450 induction).

Inhibitor mneomonic: SICKFACES.COM.GJ

We can summarize many of the key inhibitors into an often-used mnemonic: SICKFACES.COM.GJ

SICK

• Sodium valproate
• Isoniazid
• Cimetidine (H2 blocker)
• Ketoconazole (which represents the -azole antifungals)

FACES

• Fluoxetine (which represents the SSRIs)
• Alcohol (in acute consumption, alcohol competes with other substrates for binding to CYPE1).
• Chloramphenicol
• Erythromycin
• Sulfonamides

COM

• Ciprofloxacin (which represents the fluoroquinolones)
• Omeprazole (a proton pump inhibitor) – this is misleading because omeprazole is also an inducer of certain isoforms
• Metronidazole (another -azole)

GJ

• Grapefruit Juice

Mixed Inducer/Inhibitor Function

• Lastly, let's list a few medications of the numerous medications that have complicated mixed induction/inhibition properties:
• Antiretroviral agents (eg, HIV medications)
• Isoniazid (a TB medication)
• Proton pump inhibitors (PPIs) (eg, omeprazole, lansoprazole)

CYTOCHROME P450 ISOFORMS

• Finally, let's address a selection of the P450 isoforms.

P450 Structure

• First, to provide some structural context, let's show the three-dimensional cytochrome P450 heme protein structure, which comprises:

Central iron atom

• A central iron atom with six binding sites.

Protoporphyrin

• Four of these go to the nitrogen (N) of protoporphyrin (protoporphyrin IX), which is a planar structure with four pyrrole rings that are linked via methine bridges.
  • A pyrrole ring is a 5-membered ring that comprises consists of 1 nitrogen, 4 carbon atoms, and 5 hydrogens.

Cysteine and Oxygen

• The heme protein binds cysteine and is able to bind up oxygen, which is key to the cytochrome P450 catalytic cycle.

P450 ISOFORMS

Now, let's list some of the key isoforms and their substrates.

CYP1A2

• CYP1A2 metabolizes a variety of drugs and toxins, including acetaminophen and theophylline, and caffeine and benzo[a]pyrene. Remember benzo[a]pyrene is a carcinogen in tobacco smoke and other products.

CYP2C9

• CYP2C9 metabolizes multiple drugs, including, notably: warfarin, phenytoin, and NSAIDs. Star the role of CYP2C9 is warfarin metabolism because inhibitors can easily cause warfarin levels rise and cause hemorrhages and inducers can cause warfarin levels to fall and cause clot formation.

CYP2C19

• CYP2C19 can be remembered as metabolizing (amongst other drugs) the 3 P's: Plavix (which is the tradename drug for clopidogrel); proton pump inhibitors (PPI's); and psychoactive/CNS medications. Note that CYP2C19 testing is sometimes done by cardiologists prescribing clopidogrel.

CYP2D6

• CYP2D6 metabolizes up to 25% of commonly used drugs. Make a notation that it metabolizes codeine to morphine (which has 200 times the effect of codeine), this is especially important because poor CYP2D6 metabolizers fail to achieve the analgesic effect from codeine that normal metabolizers do (who are able to convert codeine to morphine).

CYP2E1

• CYP2E1 notably metabolizes ethanol (amongst other substances and pharmaceuticals).
  • Think: you have to be "21" to drink "E"thanol.

CYP3A4

• CYP3A4 metabolizes up to 50% of drugs, so we star it. Notably, suvorexant (an orexin receptor antagonist sleep aid) is a CYP3A4 substrate and so it is contraindicated in people taking CYP3A4 inhibitors, which impairs its metabolism.