Xenobiotics: Case Study

• The patient in the case study exhibited symptoms such as disorientation, chest and abdominal pain, shaking, and vomiting, along with yellow discoloration of the eyes.
• The woman took Lorcet (hydrocodone/acetaminophen), Soma (carisoprodol), and Cymbalta (duloxetine HCl) two days before presenting.
• Blood tests showed elevated alkaline phosphatase (302), alanine aminotransferase (ALT) (351), aspartate aminotransferase (AST) (1045), bilirubin (3.33 mg/dL), and prothrombin time (19.8 seconds).
• The patient also displayed reduced plasma bicarbonate, a reduced glomerular filtration rate, elevated serum creatinine and blood urea nitrogen, reduced blood glucose, and a plasma acetaminophen concentration of 75 mcg/mL with a positive hepatitis C virus (HCV) titer.

Xenobiotics

• Humans are exposed daily to foreign compounds called xenobiotics through the lungs, skin, or ingestion, whether unintentionally or deliberately.
• Xenobiotics can be innocuous or provoke biological responses, often dependent on their conversion into an active metabolite.

Purpose of Drug Biotransformation

• Mammalian drug biotransformation systems evolved to detoxify and eliminate plant, bacterial bioproducts, toxins and environmental xenobiotics and drugs.
• Lipophilic organic molecules tend to remain un-ionized and are reabsorbed at physiologic pH; they are also strongly bound to plasma proteins and may not be filtered easily at the glomerulus.
• Metabolism serves as an alternative to renal excretion for terminating or altering a drug's biological activity.
• Lipophilic xenobiotics are transformed into polar, readily excreted products.

Significance of Metabolism

• Lipid-soluble drugs would have extremely long half-lives without drug metabolism.

Metabolic Product Activity

• Metabolic products are often less active than the parent drug and may even be inactive.
• Some biotransformation products gain enhanced activity or toxic properties.
• The synthesis of endogenous substrates like steroid hormones and bile acids involves enzymes associated with xenobiotic metabolism.
• Drug-metabolizing enzymes can be exploited to design inactive prodrugs that become active in the body.

Role of Biotransformation in Drug Disposition

• Most transformations occur between drug absorption into circulation and renal elimination.
• Some transformations occur in the intestinal lumen or wall.
• All biotransformation reactions fall into two categories: phase I and phase II.

Phase I Reactions

• Phase I reactions convert the parent drug into a more polar metabolite by introducing or unmasking functional groups (-OH, -NH2, -SH).
• Phase I metabolites are often inactive.
• Activity is only modified or enhanced in some instances

Phase II Reactions

• Phase I metabolites that are adequately polar might be excreted readily.
• Phase I products often undergo a second reaction where an endogenous substrate (glucuronic acid, sulfuric acid, acetic acid, or amino acid) combines with the new functional group to form a polar conjugate.
• Phase II reactions are known as Conjugation or synthetic reactions, and a great number of drugs will undergo these sequential reactions.
• In some cases, a drug has a functional group which may form a coordinate directly.
• The hydrazide moiety of isoniazid forms an N-acetyl conjugate in a phase II reaction, then is hydrolyzed to isonicotinic acid in a phase I reaction.

P450-Mediated Bioactivation of Isoniazid

• Isoniazid (INH) can undergo P450-mediated bioactivation to an isonicotinic acyl radical which will then attack hepatic proteins that include the P450s that help metabolize it.
• In some people, this attack can result in idiosyncratic immunotoxicity as the INH-adducted proteins are proteolytically degraded into isonicotinic acylated peptides that act as autoantigens engendering pathogenic immunoreactive autoantibodies.
• INH-P450 autoantibodies can trigger liver cell injury by interacting with the small fraction of intracellular P450s integrated in the cell membrane.

Location of Biotransformation

• Although all tissues have some capacity to metabolize drugs, the liver is the main organ.
• Other active tissues include the gastrointestinal tract, lungs, skin, kidneys, and brain.
• After oral administration, many drugs are absorbed intact from the small intestine, transported to the liver, and undergo the first-pass effect.
• Some drugs undergo more metabolism in the intestine.
• Intestinal metabolism can contribute to the overall first-pass effect.
• Compromised liver function patients may rely more on intestinal metabolism for drug elimination and clinically relevant drug-drug interactions.
• First-pass effects can limit bioavailability, requiring alternative administration routes.
• The lower gut contains intestinal microorganisms that conduct biotransformation reactions.
• Gastric acid digests drugs- penicillin.
• Digestive enzymes such as polypeptides digest insulin.
• Enzymes in the wall of the intestine will impact sympathomimetic catecholamines.

Drug Biotransformation in Vivo

• In vivo drug biotransformation is catalyzed by specific cellular enzymes versus simple chemical reactions.
• The enzymes are located in the endoplasmic reticulum, mitochondria, cytoplasm, lysosomes, or even the nuclear envelope or plasma membrane.

Microsomal Mixed Function Oxidase System and Phase I Reactions

• Many drug-metabolizing enzymes are in the endoplasmic reticulum of the liver and other tissues.
• Isolated membranes re-form into vesicles called microsomes.
• Microsomes retain morphologic (rough/smooth surfaces) and functional characteristics.
• Rough microsomes are dedicated to protein synthesis.
• Smooth microsomes have enzymes for oxidative drug metabolism.
• Smooth microsomes contain mixed function oxidases (MFOs), or monooxygenases and the activity of MFOs requires both a reducing agent, such as NADPH and molecular oxygen.
• During reactions, a molecule of oxygen is consumed (reduced) and split, with one oxygen atom appearing in the product, and the other in the form of water.
• Two microsomal enzymes play a key function in the oxidation-reduction process.

Enzymes in Oxidation-Reduction

• NADPH-cytochrome P450 oxidoreductase (CPR, or POR) contains flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
• Cytochrome P450, a hemoprotein, serves as the terminal oxidase.
• The body contains multiple forms of hemoprotein, and this multiplicity is increased by repeated exposure to exogenous chemicals.
• The hemoprotein Cytochrome P450 (P450 or CYP) gets its name from spectral properties and gives the name spectral properties from when in its reduced (ferrous) form, given it binds carbon monoxide.

P450 Reductase

• A major aspect of P450's relative abundance, compared with the reductase, contributes to making P450 heme reduction a rate-limiting step in hepatic drug oxidations.
• P450 reductase and molecular oxygen are required for microsomal drug oxidations
• Oxidized P450 combines with a drug substrate to form a binary complex.
• NADPH donates an electron to P450 reductase, reducing the oxidized P450-drug complex.
• A second electron is introduced from NADPH, reducing molecular oxygen.
• The oxidized P450 reductase also gives rise to an "activated oxygen"-P450-substrate molecule.
• This complex will transfer oxygen to the molecule to form order.

Activated Oxygen

• Activated oxygen allows the oxidation of a number of materials.
• Substrate specificity is quite low of the enzyme complex.
• The only common structural feature is high lipid solubility of the wide variety of structurally unrelated drugs and chemicals that serve as substrates in this system.
• Compared with other enzymes such as phase II, P450s are remarkably sluggish.

Phase I Reactions

• Examples of phase I reactions include oxidations or cytochrome P450-dependent oxidations.
• Cytochrome P450-dependent oxidations include aromatic hydroxylations and aliphatic hydroxylations, as well as epoxidation, oxidative dealkylation, an N-oxidation, as well an S-oxidation, Deamination, and Desulfuration.
• An example of phase I reactions are also cytochrome P450-independent oxidations.
• Further examples in phase I are amine oxidases and dehydrogenations.

Reductions in Phase I

• Examples of some reductions are Azo reductions and Nitro reductions.
• Phase I can be Carbonyl reductions.

Hydrolyses in Phase I

• Hydrolyses is also an aspect of phase I
• Phase I involves esters and amides.

Human Liver P450 Enzymes

• Human Gene arrays combined with immunoblotting analyses of microsomal preparations, as well as the use of relatively selective functional markers and selective P450 inhibitors, have identified humans have a liver P450s.
• Human P450s include CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, CYP3A4,
• Human liver P450s appear to be the most important forms, accounting for approximately 15%, 4%, 1%, 20%, 5%, 10%, and 30%, respectively, of the total human liver P450 content.
• Together, they are responsible for catalyzing the bulk of the hepatic drug and xenobiotic metabolism, and some P450 substrates can be potent competitive inhibitors and/or mechanism-based inactivators.

Enzyme Induction

• P450 substrate drugs, on repeated administration, induce P450 expression by enhancing the rate of its synthesis or reducing its rate of degradation.
• P450s are affected by environmental chemicals, and air pollutants.
• Increased P450 synthesis requires enhanced transcription and translation, along with increased synthesis of heme, its prosthetic cofactor.
• P450 enzymes can be stabilized by a substrate versus its degradation.

Enzyme Inhibition

• Substrates drugs will inhibit cytochrome P450 enzyme activity
• The drugs cimetidine and ketoconazole bind tightly to the P450 heme iron effectively reducing the metabolism of any coadministered drugs to follow.
• Other medications like antibiotics will also attack CYP3A, to metabolites that complex the cytochrome, rendering it catalytically inactive.
• Compound proadifen (SKF-525-A is used for research.

Suicide Inhibitors

• Suicide Inhibitors include steroids like ethinyl estradiol.
• A suicide inhibitor like chloramphenicol attacks CYP2B1
• Other things to inactivate CYP2B1 are carbon disulfide and grapefruit furanocoumarins and ritonavir.

Phase II Reactions

• Phase I result in coupling reactions with other the endogenous substances in the body to produce drug conjugates
• Conjugates are polar and excreted.
• Formation involves high-energy intermediates and transfer enzymes.
• Such enzymes may be located in microsomes or in the cytoplasm.
• Uridine 5'-diphosphate (UDP)-glucuronosyl transferases (UGTs) are dominant and will catalyze the coupling of a substance like UPDGA.

Human Transferases

• Nineteen are human
• Encode UGT proteins involved in metabolizing drugs and xenobiotics.

Human Sulfotransferases

• Catalyze sulfation of substrates using PAPS as sulfate donor.

Cytosolic and Microsomal Glutathione

• Cytosolic and microsomal glutathione (GSH) transferases (GSTs) are also involved in metabolizing drugs and xenobiotics, also leukotrienes and prostaglandins.

Cytosolic N-Acetyltransferases (NATs)

• Chemicals with a aromatic amine are substrates of NATs that utilize acetyl-coA

Metabolism of Drugs to Toxic Products

• Metabolism can lead to reactions
• Exposure to materials will turn metabolites too
• The lack of toxicity is Expanding rapidly so, an example is acetaminophen APAP, is normally Safe but has a low effect.

P450 Dependent GSH

• If APAP intake far exceeds therapeutic doses, glucuronidation, sulfation pathways are saturated, the P450-dependent pathway becomes significantly important with long term exposure.

Clinical Relevance of Drug Metabolism

• The dose and frequency of administration must be just right to achieve therapeutic numbers.