to convert highly lipophilic molecules with long biological half-lives into more water-soluble metabolites that may be more readily excreted in urine

what are the various ways that kidneys excrete drugs?

• glomerular filtration for small polar drugs with low plasma protein affinity
• active tubular secretion for organic acids and metabolites actively secreted by endogenous transport system; org bases are secreted via a different transport mechanism; both are bi-directional
• re-absorption of nonpolar compounds at the pH of urine that easily dissolve in lipid membranes

• most drugs are hydrophobic & bind to plasma proteins which are not easily excreted
• few drugs are ionized at physiologic pH and so require mods to make them hydrophilic
• hydrophobicity often faciliates reabsorption in the tubules, and not excretion, thereby prolonging their activity in the body

in vivo:

• disappearance from plasma [does not distinguish between concentration changes due to metabolism or to tissue redistribution]
• measurement of metabolites [metabolite/drug ratio in urine, saliva, or plasma; high ratios in high metabolizers; more accurate than above method; must take into account *all* various forms of metabolites and intermediates]

in vitro:

• liver biopsy[although very accurate, unacceptable unless liver impairement suspected]

what are the potential pathways of biotransformation, and which is the more common pathway?

1. bioactivation- metabolites more active; may be desired (to produce a particular pharmacological effect) or undesired (causing organ toxicity or carcinogenesis)
2. inactivation- metabolites are inert or less active; the more common pathway

functionalization!!

make metabolite more polar by adding or unmasking a functional group that will make substance more easily excreted

FGs: hydroxyl, amino, sulfhydryl residues

in most cases, metabolites need more before being excreted and undergo phase II reactions

conjugation!!!

add an endogenous substrate: GLUCURONIC ACID, ACETIC ACID, SULFURIC ACID, or AMINO ACID  to the functional group added by phase I reactions.

goal to make metabolites even more polar to aid in excretion by kidneys via transport systems

usually phase I precedes phase II; in rare cases the reverse can occur if parent drug already has a FG that may acted upon by phase II enzymes

what is the most important component of the hepatic drug metabolism?

mixed function oxidase

• location: ER

• non specific system that works on variety of chemical substrates
• examples:
  • alchohol dehydrogenases
  • aromatases (estrogen biosynthesis/degradation)
  • aldehyde dehydrogenases
  • esterases (hydrolyze esters)
  • epoxide hydrolases (break epoxide bonds, common in carcinogenic intermediates)
  • cytochrome P450s

what are the principal phase I enzymes and what do they metabolize?

Cytochrome P450s

metabolize:

• xenobiotics (drugs/medications; environmental pollulants, direct carcinogens)
• endogenous substances (fatty acids, PGs, leukotrienes, steroids)

what are some important characteristics of CYP450s?

• heterogenous group of heme proteins in ER MICROSOMES in liver (predominantly in liver; can also be found in kidneys, lungs, brain, intestine, skin, testes, placenta, adrenals)
• large multi-gene family: each CYP450 is product of a different GENE
• there are multiple CYP isoforms, so capable of metabolizing broad range of compounds
• each isoform has broad substrate specificity

what are some of the important requirements of CYP450 reaction?

1. NADPH
2. O2 (molecular oxygen)
3. NADPH-CYP450 reductase
4. FAD FMN (flavin containing co-factors)
5. cytochrome b5 (electron donor)

what are some of the general reactions catalyzed by CYP450?

• aliphatic oxidation
• aromatic oxidation
• demethylation
• deamination
• sulfoxide formation
• oxidation
• hydroxylation

HYDROXYLATION REACTIONS, even if end product is not hydroxylated, many of the above reaction types have hydroxylated intermediates

what is the role of cytochrome b5 or FMN/FAD (flavin containing cofactors)?

these cofactors contribute or accept electrons at particular times in the reaction

essentially they serve as electron vectors that shuttle e- where they need to be

Fe (from CYP450 porphyrin containing group) is oxidized and reduced throughout reaction

CYP450 reductase reduces the flavoproteins so that reaction is sustainable for consequent rounds

• glucuronidation
• conjugation with glutathione
• other types of conjugation:
  • sulfate
  • acetylation
  • methylation
  • amino acid conjugation

what are some important details of glucuronidation reaction?

most common route of drug metabolism bc of the plentiful nature of glucose in body

GLUCURONYL TRANSFERASE (multiple isoenzymes) catalyzes the interaction of UDPGA (uridine diphosphate glucuronic acid) + acceptor drug

enzyme located in ER (integral protein like CYP450)

high concentrations of glucuronyltransferase in the liver, and found in other tissues like kidney, GI, skin

what enzyme mediates the reaction of glutathione conjugation?

GLUTATHIONE-S-TRANSFERASE

located in the CYTOSOL of the liver, kidney, gut and other tissues

it is used as a free radical scavenger to remove potentially toxic electrophilic compounds

many drugs and xenobiotics metabolized by phase I rxns to strong electrophiles are converted to NON TOXIC conjugates by glutathione-s-transferase

what are some special characteristics of phase II conjugations?

• conjugates are usually polar molecules and easily excreted
• high energy intermediates formed
• endogenous substrates used for conjugations originate in DIET
• nutrition plays a critical role in regulation of drug conjugations

what is sulfate conjugation used for and what special cofactor is needed?

• major pathway for ethanol, alcohol, amide, and steroids
• cystolic sulfotransferase needs ATP-activated 3'phosphoadenosine-5'-phosphosulfate (PAPS) as a high energy intermediate cofactor

what does acetylation require for use by N-acetyltransferase?

acetyl-CoA as a cofactor

takes place mainly in the liver's Kuppfer cells

amino acid conjugation is a special form of Nacetylation, conjugationg erogenous carboxylic acids with amino acid (usually: glycine, glutamine, ornithine, arginine, or taurine)

what does the methylation enzyme, methyltransferase, require for activity?

S-adenosylmethione (SAM) as a co-factor with the methyltransferase

Kupffer cells of the liver's sinusoids

acetylation requires acetyl-CoA as a cofactor

in large intestine beta-glucuonidase cleaves the GA moiety and enables free drug to be re-absorbed and recirculate through hepatic system

end result: slowed elimation of drug through intestines

what are two examples of bioactivation of prodrugs?

• L-DOPA --> dopamine
• protonsil red --> sulfanilamide

what is one reason (of many) for the inter-individual variation in drug metabolism that relates to CYP450s?

there are likely more than 150 genes for CYP450 enzymes, and only 30-40 genes are likely expressed in any given individual

accounts in part for why some individuals are more susceptible to developing adverse reactions to medications than others

how can acetaminophen become toxic in some indivdiuals, leading to necrosis of the liver?

through sulfate conjugation, hydroxylation, and detox with glutathione, aspirin metabolites are usually excreted in urine w/o problem

in large doses, glutathione conjugation can become a limiting step when substrate overwhelms the conjugation machinery

build up in hydroxyparacetamol activates electrophilic attack on nucleophilic macromolecules-->covalent binding--> HEPATOCELLULAR NECROSIS

phase II acetylation occurs first

phase I hydrolysis then occurs

one metabolite, acetylhydrazine, is highly reactive and may directly cause hepatotoxicity upon acetylation of some of its proteins

this response is idiosyncretic

what are some examples of CYP450s and phase II enzymes activating carcinogens?

• p450: bioactivation of benzo[a]pyrene, a DNA adduct
• p450 & phase II combined bioactivation of  DNA adducts: AAF (acetylaminofluorene), nitrosamine (meat preservant), and aflatoxin (in legumes, like peanuts)

• sedatives/hypnotics (barbitol-classic one)
• steroids (testosterone)
• vitamins (Vit C)

physiological jaundice of the new born due to a failure to excrete bile pigment because of low levels of glucuronyltransferase (glucuronidation)

low capacity of both CYP450 enzymes and glucuronidation.  Increased toxicity to the antibiotic chloramphenicol.