Metabolism

Drugs are Altered by the Body for Excretion – Alteration Efficiency Determines Duration of Effect

Alteration of drugs (biotransformation, usually by the liver) is necessary for normal metabolism

most drugs are lipophilic and will be reabsorbed by the kidney unless converted to be hydrophilic Þ excretion

the metabolism can make drugs inactive, active, or toxic; many prodrugs require metabolism to become active

Animal experiments show that the duration of drug effect is inversely proportional to metabolic capacity (i.e., activity of inactivating enzymes)

Everyone’s metabolic enzymes are different

plasma half-life of a drug can differ by more than 10 times in different people
this impacts side effects – slower metabolism leads to higher concentration
there is a genetic basis for this – monozygotic twins have similar drug metabolism compared to dizygotic

Oral Bioavailability

Why Aren’t All Drugs Taken by Mouth? Because the liver is the major site of drug metabolism

When taken by mouth, not as much drug reaches the systemic circulation as when directly injected, for two reasons:

(1) First Pass Effect – some drugs (e.g., nitroglycerine) are inactivated by metabolism in the liver

100% of the blood from the gut passes through the portal vein to the liver, compared to 20% of systemic blood
if liver metabolism inactivates the drug, it will be much less effective when taken by mouth
nitroglycerine is usually administered sublingually or by patch to bypass liver and remain active

(2) Lack of Efficient Absorption by the Gut

Oral Bioavailability (Fo) is the proportion of orally administered drug that reaches the circulation

(Amount in bloodstream when taken by Mouth) / (Amount in bloodstream when taken by I.V.) = (0.0 – 1.0) / (1.0)

This is also equal to the proportion of the areas under the plasma concentration vs. time curves for oral and IV admin.

Drug Metabolism – A Two-Step Process

To make the drug water soluble, a polar handle is added to the drug or exposed; a water-soluble adduct is added to this

Phase I – Addition or exposure of a polar handle (-OH, -COOH, -NH₃) can take place in several ways:

oxidation using molecular O₂– e.g. P-450

hydrolysis to create an -OH from an ester (esterase) or amide (amidase) using H₂O

reduction not very common; reductases are rare (often from bacteria – antibiotics change drug handling)

Phase II – Conjugation to a water-soluble adduct (sulfate, nitrate, glucuronate) so that the drug will not be reabsorbed

glucuronotransferases (glucuronate), sulfotransferases (sulfonate), N-acetyl transferase, glutothionyl transferase, glycyl transferase

Example – codeine-O-CH₃ (inactive drug) Þ (phase I metabolism) Þ morphine (active drug) Þ (phase II metabolism) Þ morphine-O-glucuronide (inactive drug)

Cytochrome P-450 – a Phase I Oxidase

P-450 is a hemeprotein called P-450 because its absorbance peak is at 450 nm (most hemeproteins = 420 nm)

Unlike many hemeproteins (e.g., hemoglobin, catalase, peroxidase) the iron in cytochromes can exist in +2 or +3 state

this is because there is a cysteine thiolate to the heme group, allowing single electron donation

NADPH donates its electrons to flavin, and P-450 reductase hands them one at a time to P-450 (Fe⁺³ Þ Fe⁺²)

Cytochrome P-450 is thus a mixed function oxygenase that oxidizes a drug by reducing molecular O₂

R-H + O₂ + 2H⁺ + 2e^- (from NADPH) Þ R-OH + H₂O – this is catalyzed a monooxygenase (requires NADPH, O₂, drug in 1:1:1 ratio)

oxidation can end in many ways:

O-dealkylation (-O-CH₃ Þ O-H, as codeine Þ morphine)

deamination (-NH₂ Þ =O, as acetominophen)

sulfoxide formation (-S Þ -S=O)

side chain oxidation (-C-H Þ C-OH, pentobarbital)

aromatic hydroxylation (phenyl-H Þ phenyl-OH, as phenobarbitol)

N-dealkylation (N-CH₃ Þ N-H, methadone)

CO occupies the O₂ site and competitively inhibits the enzyme; but with drgard to drug substrate, leads to non-competitive inhibition

There are at least 500 isozymes of P-450 with distinct but overlapping substrate specificities

Rate of P-450 differs in different individuals–can distinguish different P-450 by selective substrates and selective inhibitors

Cycle of P-450 Function

iron atom (Fe) cycles between +2 and +3 oxidation states (Fe⁺² and Fe⁺³)

(1) normal resting state unbound with Fe⁺³

this binds the substrate (the drug)

(2) substrate binding causes P-450 reduction

one e^- from P-450 reductase–Fe⁺³ Þ Fe⁺²

(3) iron ion binds O₂ (if CO is present, it will bind instead)

(4) P-450 reduces O₂ to peroxide-like intermed.

Fe⁺³-OOH « Fe⁺⁴=O + OH^-

(5) the drug is oxidized with one oxygen atom

P-450 is reduced to resting state (Fe⁺⁴Þ Fe⁺³)

Functions of Hemoproteins

has both cyclic redox and single oxidation state functions
flavin deals out 1 e^- at a time to cyt P-450
NADPH gives 2 e^- to flavin
oscillates between +2 and +3 form

Drug Metabolism can Significantly Affect the Action of Many Drugs

Acetominophen can be toxic in large doses (many suicide attempts due to widespread availability)

phase II metabolism usually involves addition of sulfate or glucuronide to existing -OH

sometimes P-450 adds OH to nitrogen, producing an electrophilic species that must be reduced by glutathoine

if there is too much acetominophen nucleophilic macromolecules can be attacked, leading to cell toxicity – hepatic necrosis Þ coma/death in about 4 days

EtOH: Ý P-450 metbolism; is a competitive inhibitor

Procaine is originally local anesthetic that has antiarrythmic properties (esterases metabolize it too fast)

amidases are slower, so if ester bond is changed to amide (procainamide), it lasts longer
amide species was also metabolized – acetylated to NAPA, which is just as potent as procainamide
hence, total effect is proportional to sum of procaineamide and NAPA

AAF (2-acetylaminofluorine) is an insecticide that can undergo two different phase I pathways

one leads to safe elimination, the other leads to powerful carcinogen Þ depends on what part of AAF P-450 chooses to metbolize

Terfenidine (seldane) is an antihistamine that requires cyp-3A P-450 for its metabolism and elimination

simultaneous treatment with ketoconazole, an antifungal that inhibits cyp-3A, leads to toxic accumulation of terfenidine

Isoniazad (INH) is an antitubercular drug that non-competitively inhibits P-450 function by binding the O₂ site

simultaneous treatment with a drug that requires P-450 for its metabolism leads to toxicity in some individuals

e.g., diphenylhydantoin (DPH) is an antipsychotic that requires P-450 for its metabolism and elimination

Why do only a few individuals get DPH toxicity when treated with INH and DPH together?

INH itself is metabolized by N-acetylation in liver cytosol, causing it no longer to bind P-450.
some people have "slow" INH N-acetyltransferases – only these get DPH toxicity when treated with both drugs
there is a genetic basis for this – 50% of Caucasians and 90% of Eskimos have "slow" INH inactivation
INH binds to heme iron, which needs to bind O₂ (only occurs with slow metabolizers)

Intrinsic Clearance

According to Michaelis-Menton kinetics,

As long as [S] << K_m, the [S] in the denominator can be ignored, so that rate = (V_max/K_m) [S]

This means the rate is directly proportional to [S]

this constant of proportionality (V_max/K_m) is the clearance that would occur if all substrate were delivered to the enzyme

this is called the intrinsic clearance, or Cl_in, and has units volume/time – occurs in liver

Cl_in = (V_max) / (K_M)

Intrinsic clearance is reduced if:

(1) binding affinity ß

(2) enzyme effect ß

Factors Affecting Metabolism and Elimination – Can be Exogenous or Endogenous

Exogenous Factors:

(1) Non-Competitive Inhibition – metabolic enzyme is inhibited by binding to site other than active site

The inhibitor usually binds a domain of the metabolic enzyme that renders it inactive, like heme in P-450

V_max is reduced, so Cl_intr is decreased

Cl_in ß = (V_max ß) / (K_M)

isoniazid (INH) – binds P-450 until acetylated – electrons shared with acetyl group, so no longer binds heme

cimetidine (H2 receptor antagonist for reduction in gastric acidity) binds P-450, but rantidine does not

heme-binding electron-dense regions on cimetidine replaced on rantidine(nitrogen in ring Þ oxygen, CN Þ NO₂)

This is the most common exogenous factor affecting metabolism.

(2) Competitive Inhibition – metabolic enzyme is inhibited by binding to active site

The inhibitor could be another drug metabolized by the same enzyme (this is how theophylline inhibits erythromycin)

K_m is increased, so Cl_intr is decreased

Cl_in ß = (V_max) / (K_MÝ)

most drugs require a small concentration relative to Km

only a fraction of enzyme is used at any one time, so there is enough for both drugs to use without competition

some drugs require a large concentration relative to Km (e.g., theophylline, aspirin, phenytoin, and ethanol)

enzyme becomes saturated (1^st order Þ 0 order) –other drugs that use the same enzyme cannot be metabolized

mathematical basis for this:

as long as I is small compared to K_I, the term (I / K_I) will not be significant and the rate will be like Michaelis-Menton

(3) Induction (Cl_in Ý) – activity of metabolic enzyme is enhanced by one of two mechanisms:

(a) receptor-mediated endocytosis leading to increase in transcription of enzyme – make more enzyme

mechanism by which rifampin, phenobarbitol, phenytoin, and carbamazepine increase transcription of P-450

benzpyrine (found in cigarette smoke, charcoal, and agent orange) increases CYP1A family of P-450

(b) decrease in destruction of enzyme – destroy less enzyme

ethanol decreases P-450 destruction, leading to more active enzyme

This can occur between the drug and endogenous substances (auto-induction) or between two drugs (cross-induction)

auto-induction – drug induces enzyme activity directly
cross induction – second drug induces activity of the first drug, therefore indirectly

Endogenous Factors:

(1) Hepatic Blood Flow – most important factor for drugs that are very highly metabolized

metabolic capacity is measured by extraction fraction, E, which is fraction of drug that is cleared

this is dependent upon hepatic blood flow (Q), intrinsic clearance (C_in), and fraction unbound (f)

Hepatic clearance is defined as the product of hepatic blood flow (Q) and the extraction fraction (E)

For drugs with a very high metabolic capacity, (Cl_inx f) >> Q, so Q in the denominator can be ignored

as a result, Cl_H is dependent primarily upon hepatic blood flow for these drugs:

The steady state concentration (C_ss)is related to the Cl:

Thus, for these drugs, steady state concentration is inversely proportional to hepatic blood flow

since Cl_in is so high, hepatic blood flow is the limiting variable

Example: Lidocaine activity is not affected by any inducers or inhibitors because it is very rapidly metabolized (t_1/2 = 2 hours)

98% is cleared by the liver; only hepatic blood flow determines the steady state concentration

(2) Protein Binding – most important factor for drugs that are NOT very highly metabolized and have low Cl_in

Once again, hepatic clearance is defined as the product of hepatic blood flow (Q) and the extraction fraction (E)

in other words: dose/(clearance + fraction unbound)

For drugs with a low metabolic capacity, (Cl_in x f) << Q, so (Cl_in x f) in the denominator can be ignored

as a result, Cl_H is dependent primarily upon intrinsic clearance and fraction unbound

The steady state concentration (C_ss) is related to the Cl:

The fraction unbound (f) is very important for these drugs, and can significantly affect their plasma concentration.

Thus, steady state concentration is inversely proportional to fraction unbound and intrinsic clearance

since Cl_in is low, (Cl_in x f) is the limiting variable, and the value of f is significant
protein binding determines f

Example: Warfarin activity is proportional to the concentration of drug not bound to albumin

If another weak organic acid (such as valproic acid) that binds albumin is administered and the fraction of warfarin bound is reduced, the steady state concentration will decrease but the unbound concentration will not change.

However, during the initial period after administration of the displacing drug, there is an equilibration period during which the concentration of free drug is transiently higher than normal.

(3) Intrinsic Clearance – most important factor for oral drugs that are cleared primarily by the liver – Ý extraction, ß Fo

everything absorbed from the intestine goes to the liver before the systemic circulation (First Pass Effect)

oral bioavailability (Fo), or the fraction of ingested drug that reaches the circulation, is what is left over after extraction:

The steady state concentration (C_ss) related to the Cl and the Fo:

Therefore:

Thus, steady state concentration is inversely proportional to fraction unbound and intrinsic clearance

This is the same as #2 above, but for a different reason:

changes in hepatic blood flow are balanced by changes in processing efficiency (slower flow Þ more efficient)

ß hepatic blood flow Ý efficiency of clearance

Example: Propranolol activity is highly dependent on intrinsic clearance, and is highly susceptible to inhibition and induction

however, this is not a problem since propranolol has a very wide therapeutic range (can give 3 half-lives apart)