1. I. Why is Biotransformation Necessary?

2. Lipophilicity is an obstacle to drug excretion.
I. Why is Biotransformation Necessary?
Introduction:
Lipophilic drugs pass through biological membranes; which contributes to the drug to reaching its site of action.
Lipophilicity is an obstacle to drug excretion.
Note: renal excretion of unchanged drug contributes only slightly to elimination, since the unchanged, lipophilic drug is easily reabsorbed through renal tubular membranes.

3. I. Why is Biotransformation Necessary?
Biotransformation of drugs to more hydrophilic molecules is required for elimination from the body
Biotransformation reactions produce more polar, hydrophilic, biologically inactive molecules -- that are more readily excreted.
Sometimes metabolites retain biological activity and may be toxic.
Drug biotransformation mechanisms are described as either phase I or phase II reaction types.

4. II. Drug Biotransformation: Phase I and Phase II
Phase I characteristics:
Parent drug is altered by introducing or exposing a functional group (-OH,-NH2,-SH)
Drugs transformed by phase I reactions usually lose pharmacological activity
Occasionally, Inactive, prodrugs are converted by phase I reactions to biologically-active metabolites

5. II. Drug Biotransformation: Phase I and Phase II
Phase I characteristics:
Phase I reaction products may:
*be directly excreted in the urine
*react with endogenous compounds to form water soluble conjugates.
(Phase II)

6. II. Drug Biotransformation: Phase I and Phase II
Phase II characteristics:
Parent drug participates in conjugation reactions that:
form covalent linkage between a parent compound functional group and:
glucuronate (UDP glucuronosyltransferase)
sulfate (sulfotransferase)
glutathione (glutathione-S-transferase)
amino acids (eg. Acyl-CoA glycinetransferase)
acetate (N-acetyltransferase)

7. II. Drug Biotransformation: Phase I and Phase II
Phase II reactions often use phase I metabolites to catalyze the addition of other groups, e.g. acetate, glucuronate, sulfate or glycine to the polar groups present on the intermediate. 
Following phase II reactions, the resultant metabolite is typically more readily excreted.
II. Drug Biotransformation: Phase I and Phase II

8. II. Drug Biotransformation: Phase I and Phase II
Important enzymes for phase II reactions include glutathione-S-transferases,
UDP-glucuronosyl transferases,
sulfotransferases,
N-acetyltransferases,
methyltransferases and
acyltransferases.
II. Drug Biotransformation: Phase I and Phase II

9. II. Drug Biotransformation: Phase I and Phase II Metabolism
Phase II characteristics:
Conjugates are:
highly polar
generally inactive*
rapidly excreted in the urine
*at least one exception to the rule: morphine glucuronide metabolite-- more potent analgesic then parent compound

10. II. Drug Biotransformation: Phase I and Phase II Metabolism
Phase II characteristics:
High molecular weight conjugates:
*excreted in the bile
*conjugate bond may be cleaved by intestinal flora
*parent drug released back to the systemic circulation
*this process, "enterohepatic recirculation":
delayed parent drug elimination
prolongation of drug effect
(eg. Morphine, tetracyclines)

11. II. Drug Biotransformation: Phase I and Phase II Metabolism
Principal Organs for Biotransformation:
Principal Organ: Liver
Other metabolizing organs:
gastrointestinal tract
lungs
skin
kidney

12. II. Drug Biotransformation: Phase I and Phase II Metabolism
Metabolism is responsible for “First-Pass” effects:
Example I
Oral administration of drug
Absorbed intact (small intestine)
Transported first to the liver (portal system) and
Extensive metabolism -- first-pass effect

13. II. Drug Biotransformation: Phase I and Phase II Metabolism
Principal Organs for Biotransformation (cont):
Sequence II
oral administration of drug:
absorbed intact (small intestine)
extensive intestinal metabolism -- contributing to overall first-pass effect

14. II. Drug Biotransformation: Phase I and Phase II Metabolism
Issues in bioavailability: reduced bioavailability
First pass effect: bioavailability of orally administered drugs -- so limited -- alternative routes of administration must be used
Intestinal flora may metabolize drugs
unstable in gastric acid-- penicillin
metabolized by digestive enzymes -- insulin
metabolized by intestinal wall enzymes-- sympathomimetic catecholamines

15. II. Drug Biotransformation: Phase I and Phase II Metabolism

16. Phase I Reactions

17. Phase I Reactions

18. Phase I Reactions

19. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions
The reaction:
one molecular oxygen (O2) is consumed per substrate molecule
one oxygen atom -- appears in the product; the other in the form of water
Oxidation-Reduction Process:
Two important microsomal enzymes:
*NADPH cytochrome P450 reductase
*CYP450

20. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions
The reaction:
Oxidation-Reduction Process:
Two important microsomal enzymes:
*flavoprotein--NADPH cytochrome P450 reductase
*Cytochrome P450:
multiple forms
named cytochrome P450 because:
the reduced (ferrous) form, binds carbon monoxide: -- the resulting complex exhibits of absorption maximum at 450 nm.

21. Phase I Reactions
Mechanism of P450

22. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps):
NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION
1. The binding of a substrate to a P450 causes a lowering of the redox potential by approximately 100mV, which makes the transfer of an electron favourable from its redox partner, NADPH-CYP450 Reductase.
START HERE

23. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps):
NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION
2. The first reduction -The next stage in the cycle is the reduction of the Fe3+ ion by an electron transferred from the NAD(P)H CYP450 reductase via an electron transfer chain.

24. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps):
NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION
3. Oxygen binding An O2 molecule binds rapidly to the ion Fe2+ forming Fe2+-O2

25. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps):
NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION
4. A second reduction is required by the stoichiometry of the reaction. This has been determined to be the rate-determining step of the reaction

26. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps):
NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION
5. O2 cleavage: The O2 reacts with two protons from the surrounding solvent, breaking the O-O bond, forming water and leaving an Fe3+-O complex.

27. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps):
NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION
6. Product formation The Fe-ligated O atom is transferred to the substrate forming, in this case, a hydroxylated form of the substrate.

28. Drug Biotransformation: Phase I and Phase II Metabolism
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions. The reaction (7 steps):
NOTE in the Figure Below the CONVERSION OF RH to ROH representing DRUG OXIDATION
7. The product is released from the active site of the enzyme which returns to its initial state

29. In general, conjugates are polar molecules
Phase 2 Reactions
Phase II
In general, conjugates are polar molecules
that are readily excreted

30. Phase II Metabolism: Some Phase II Reactions
Type of Conjugation
Endogenous Reactant
Transferase Location
Type of Substrates
Examples
Glucuronidation
UDP-glucuronic acid
UDP-glucuronosyl transferase (microsomes)
Phenols, alcohols, carboxylic acids, hydroxylamines, sulfonamides
Nitrophenol, morphine, acetaminophen, diazepam, meprobamate, digoxin
Acetylation
Acetyl-CoA
N-acetyltransferase (cytosol)
amines
Isoniazid, sulfonamides, clonazapam
Glutathione Conjugation
glutathione
GSH-S-transferase
(cytosol, microsomes)
Epoxides, arene oxides, nitro groups, hydroxylamines
Ethacrynic acid, bromobenzene
Glycine conjugation
glycine
Acyl-CoA glycinetransferase (mito)
Acyl-CoA derivatives of carboxylic acids
Salicylic acid, benzoic acid, nicotinic acid, cinnamic acid

31. Phase II Metabolism: Some Phase II Reactions
Type of Conjugation
Endogenous Reactant
Transferase Location
Type of Substrates
Examples
Sulfate Conjugation
Phosphoadenosyl phosphosulfate (PAPS)
Sulfotransferase (cytosol)
Phenols, alcohols, aromatic amines
Estrone, aniline, phenol, acetaminophen, methyldopa
Methylation
S-adenosyl methionine
Transmethylases (cytosol)
Catecholamines, phenols, amines
Dopamine, epinephrine, pyradine, histamine, thiouracil
Water Conjugation
water
Epoxide hydrolase, (microsomes)
Arene oxides, cis-disubstituted and mono- substituted oxiranes
Benzopyrene 7,8- epoxide; carbamazepine epoxide

32. Phase II Metabolism: Some Phase II Reactions
Overview: Phase II reactions:
(Non-microsomal enzymes) microsomal
Reaction types:
conjugation glucuronidation
hydrolysis
oxidation
reduction
Location (non-microsomal enzymes):
primarily hepatic (liver); also plasma & gastrointestinal tract
Non-microsomal enzymes catalyze all conjugation reactions except glucuronidation

33. Phase II Metabolism: Some Phase II Reactions
Overview: Phase II reactions:
Non-microsomal enzymes
Nonspecific esterases in liver, plasma, gastrointestinal tract hydrolyze drugs containing ester linkages,
e.g.: succinylcholine (Anectine)

34. Phase II Metabolism: Some Phase II Reactions
Overview: Phase II reactions:
Conjugation reactions:
Usually "detoxification reaction”
Conjugates:
  *more polar
  *easily excreted
*typically inactive

35. Drug Biotransformation
Toxicity:
Drugs can occassionally be metabolized to toxic products.
Acetaminophen hepatotoxicity -- normally safe in therapeutic doses
Therapeutic doses:
glucuronidation + sulfation to conjugates
(95% of excreted metabolites);
5% due to alternative cytochrome P450 depending glutathione (GSH) conjugation pathway
Drug Biotransformation

36. Drug Biotransformation
Acetaminophen Overdose
UDP-
glucuronosyltransferase
Ac-Glucuronide Ac
Ac-Sulfate
therapeutic
CYP450
(2E1)
toxic
Glutathione Precursor
N-acetylcysteine
Ac-TOXIC Metabolite
Glutathione-S-Transferase
GSH
Ac-SG
(mercapturate)
excreted and harmless
Cell Macromolecules
AC-protein
Hepatic Cell Death

37. Phase II Metabolism: Some Phase II Reactions
Acetaminophen
Toxicity:
At high doses:
*Glucuronidation and sulfation pathways become saturated
*Cytochrome P450 dependent pathway: now more important
with depletion of hepatic glutathione, hepatotoxic, reactive, electrophilic metabolites are formed
Antidotes: N-acetylcysteine
protects patients from fulminant hepatotoxicity and death following acetaminophen overdose.

38. Basis for individual to individual variation in drug responses
Response Variation Due to Pharmacokinetic Differences
*Bioavailability
*Renal function
*Liver function
*Cardiac function
*Patient Age 
Response Variation Due to Pharmacodynamic Differences
*Enzyme activity
*Genetic differences
Response Variation Due to Drug Interactions

39. Basis for individual to individual variation in drug responses
Genetic Factors: in Biotransformation of Drugs
Genetic influences: Variation in drug metabolism rates or in receptor sensitivity
Metabolism:
Patients can be categorized as either rapid or slow acetylators; a classification which refers to the patients ability to relatively rapidly or slowly catalyze acetylation reactions. 
Biotransformation of some drugs are affected by acetylation rates, examples include isoniazid (INH); here Phase II can proceed Phase I.

40. Influence of Age on Drug Responses
Variation in drug responses --usually due to:
diminished cardiac output
reduces hepatic perfusion (decreases delivery of drug to the liver for metabolism)
can prolong the duration of action of drugs

41. Influence of Age on Drug Responses
increased body fat
increases Vd (another contributing factor is decreased plasma protein binding)
promotes accumulation of highly lipid-soluble agents such as:
diazepam (Valium)
thiopental (Pentothal)

42. Drug-Drug Interactions
Definition:
Drug interaction -- when one drug affects the pharmacological response of a second drug given at the same time.
Drug interactions may be due to:
pharmacodynamic effects
  pharmacokinetic effects

43. Drug-Drug Interactions
Consequences of drug interactions:
  increased drug effects; decreased drug effects
Drug1= CYP450 1A2 inducer
Drug2= CYP450 1A2 substrate
Effect: increased clearance of Drug2 than would otherwise be expected.
Drug1= CYP450 1A2 inhibitor
Effect: decreased clearance of Drug2 than would otherwise be expected.

44. Drug-Drug Interactions
Consequences of drug interactions:
 Adverse effects -- toxic reactions
  *one drug may interact with another to impede absorption
  *one drug may compete with another for the same plasma protein-binding sites
   *one drug may change the renal excretion rate of the other.
*one drug may affect metabolism of another by either enzyme induction or enzyme inhibition

45. III. Cytochrome P450 Metabolizing Enzymes
Mixed Function Oxidase System (cytochrome 450 System)--Phase I Reactions
Microsomes have been used to study mixed function oxidases
Drug metabolizing enzymes:
located in lipophilic, hepatic endoplasmic reticulum membranes
smooth endoplasmic reticulum:
contains enzymes responsible for drug metabolism

46. III. Cytochrome P450
Cytochrome P450 isoform naming conventions:
Review -- drug biotransformation usually involves two phases, phase I & phase II.  
Phase I reactions are classified typically as oxidations, reductions, or hydrolysis of the parent drug. 
Following phase I reactions, the metabolites are typically more polar (hydrophilic) which increases the likelihood of their excretion by the kidney.  Phase I metabolic products may be further metabolized

47. III. Cytochrome P450
Most phase I reactions are catalyzed by the cytochrome P450 system (CYP). 
This superfamily consists of heme-containing isoenzymes which are mainly localized in hepatocytes, specifically within the membranes of the smooth endoplasmic reticulum. 
The primary extrahepatic site containing CYP isoforms would be enterocytes of the small intestine. 

48. III. Cytochrome P450
Genetic Factors (cont):
The gene family name is specified by an Arabic numeral, e.g. CYP3.
> 40% of sequence homology characterize CYP isoforms within a family.
CYP families are subdivided into subfamilies designated by an upper case letter, it e.g. CYP3A .
Gene numbers of individual enzymes are noted by a second Arabic numeral following the subfamily letter, e.g. CYP3A4.

49. III. Cytochrome P450
Genetic Factors (cont):
CYP isoforms not only metabolize many endogenous substances including prostaglandins, lipids, fatty acids, and steroid hormones but also metabolize (detoxify) exogenous substances including drugs
Major CYP isoforms responsible for drug metabolism include:CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP1A2, CYP2E1 in in certain cases CYP2A6 and CYP2D6

50. IIB. Clearance
Dietary considerations:
Grapefruit juice contains chemicals that are potent inhibitors of CYP3A4 localized in the intestinal wall mucosa
Cruciferous vegetables such as brussels sprouts, cabbage, cauliflower and hydrocarbons present in charcoal-broiled meats can induce CYP1A2.
Calcium present in dairy products can chelate drugs including commonly used tetracyclines and fluoroquinolone antibiotics.

51. IIB. Clearance
Age:
Neonates have reduced hepatic metabolism and renal excretion due to relative organ immaturity. 
On the other hand, elderly patients exhibit differences in absorption, hepatic metabolism, renal clearance and volume of distribution.

52. IIB. Clearance
Genetic Factors:
Genetic polymorphism affecting CYP2D6, CYP2C19, CYP2A6, CYP2C9, and N- acetyltransferase result in significant inter- individual differences in drug-metabolizing abilities (the drug of course must be a substrate for one of the above cytochrome P450 isoforms)

53. IIB. Clearance
Genetic Factors:
Certain genetic polymorphisms are associated with ethic groups. 
For instance, 5%-10% of Caucasians are poor metabolizers of CYP2D6 substrates. 
By contrast, the frequency of CYP2D6 poor metabolizers in Asian populations is about 1%-2%. 
On the other hand, the incidence of poor metabolizers of CYP2C19 drugs is about 20% in Asian populations, but only about 4% in Caucasian populations.

54. III. Cytochrome P450 Metabolizing Enzymes
Some drugs stimulate
Expression of CYP450.

55. Drug Biotransformation: Phase I and Phase II Metabolism
Cytochrome P450 Enzyme Induction:
Following repeated administration, some drugs induce cytochrome P450 (increase amount of P450 enzymes) usually by:
*increase enzyme synthesis rate
*reduced enzyme degradation rate

56. III. Cytochrome P450 Metabolizing Enzymes
DRUG
Some drugs can inhibit P450s
CYP450 Inhibition

57. Drug Biotransformation: Phase I and Phase II Metabolism
Cytochrome P450 enzyme inhibition:
Certain drugs, by binding to the cytochrome component, act to competitively inhibit metabolism. Examples:
  Cimetidine (CYP3A4; CYP2D6)
(anti-ulcer --H2 receptor blocker) and
Ketoconazole (Nizoral) (antifungal)
Mechanism of Action: competitive inhibition

58. Drug Biotransformation: Phase I and Phase II Metabolism
Cytochrome P450 enzyme inhibition:
Chloramphenicol (antibiotic):
metabolized by cytochrome P450 2B1 to an alkylating metabolite that inactivates that cytochrome P450 isoform.

60. III. Cytochrome P450 Metabolizing Enzymes
Carbamazepine (anticonvulsant) is 76% bound to plasma proteins. Carbamazepine is primarily metabolized in the liver.
Cytochrome P450 3A4 was identified as the major isoform responsible for the formation of carbamazepine-10,11-epoxide.

61. III. Cytochrome P450 Metabolizing Enzymes
Since carbamazepine induces its own metabolism, the half-life is also variable.
Following a single extended-release dose of carbamazepine, the average half-life range from hours and hours on repeated dosing.
The apparent oral clearance following a single dose was 25 ± 5 mL/min and following multiple dosing was 80 ± 30 mL/min.

62. 5%-10% of Caucasians are poor metabolizers of CYP2D6 substrates. 

64. the incidence of poor metabolizers of CYP2C19 drugs is about 20% in Asian populations, but only about 4% in Caucasian populations.