Pharm - #5 & 6 Flashcards
_____ is chemical transformation of xenobiotic (i.e. drug) within living organism
Drug metabolism
- enzyme based - most drugs
- non-enzyme based - some drugs
What are the consequences of drug metabolism?
important point - drugs may be considered poisons:
high doses can be harmful (toxic effects)
therapeutic doses can also be harmful (side effects)
State some examples of inactivation & activation
- Inactivation!
- rid body of foreign chemicals
- lipid soluble to less lipid soluble
** facilitate excretion via kidney
(not reabsorbed; excreted in urine) - Activation
- ex: Ldopa to dopamine
- clopidrogel (inactive) to active metabolite
BUT can have LETHAL SYNTHESIS
- non - toxic to toxic metabolite
ex: parathion to paraoxon - Chemical modification of drug terminates activity
- lock & key
- Maintenance of Activity
ex: diazepam to oxazepan (active)
- same activity
First pass biotransformation occurs where?
Subcellular level:
Where are the enzymes responsible for phase I reactions?
Phase II reactions?
LIVER
- Subcellular level: endoplasmic reticulum, mitochondria, cytosol, lysosome, nuclear envelope, plasma membrane
Phase I = endoplasmic reticulum
Phase II enzymes: reactions are mainly in the cytoplasm
What are the functional groups that are added in Phase I reactions?
What happens in phase I?
Phase II functional groups? What happens here?
- Amine (NH)
- OH (hydroxyl)
- SH (sulfhydral)
phase I reactions: small chemical change makes drugs more HYDROPHILIC and also provides
functional group used to complete phase II reactions
phase II reactions: conjugation with small, endogenous substance on functional group added
during phase I reaction
** usually of lower pharmacologic activity
CONJUGATION reaction
GAS
G - glucuronic acid / glutathione
A - acetyl group
S: sulfate group
Phase I and Phase II reactions mostly make drugs move from ____ to ___ phillic
Lipophillic to hydrophillic
In phase I reactions (which are referred to as functionalization reactions), the parent drug is converted to more ____metabolite by introducing or unmasking a functional
group on the molecule.
What are the 4 main Phase I Reactions?
POLAR
-polar molecules are more water soluble and less likely to be reabsorbed by the glomeruli in the
kidney and are excreted
- Microsomal Oxidations
a) hydroxylation
b) among others - Non-microsomal Oxidations
a) alcohol oxidations (hydrolysis) - Hydrolysis Reactions (require water)
a) esterases
b) amidases
c) peptidases
Microsomal Oxidations: Phase I rxn
Where are cytochrome P450 enzymes mostly found?
What is microsomal fraction?
Assigning P450 enzyme to a drug _____
- in ER of liver & intestine
- mixed-function oxidase system - isolated by tissue homogenization and differential centrifugation
- pellet-vesicles formed from ER membrane (microsomes) used for in vitro studies - Promiscuity!
What is an example of a microsomal drug oxidation?
Cytrochrome P450 cycle
- hydroxylation of drug metabolite (OH group introduced
& molecular oxygen is added
NADPH-cytochrome P450 reductase - transfers reducing equivalents from
reducing cofactor NADPH to hemoprotein- net effect = one oxygen atom incorporated into drug substrate and one into water
- balanced equation: RH + O2 + NADPH + H+ ROH + H2O + NADP+
Cytochrome P450 isozymes catalyze xenobiotic metabolic reactions such as _____.
Microsomal enzymes are
responsible for oxidative drug metabolism and are known as _______.
These enzymes are also known as ______ because one atom of oxygen is incorporated into drug substrate while the other atom of oxygen is used in the formation of water.
hydroxylation
mixed functional oxidases (MFOs)
monooxygenases
The cytochrome P450 cycle:
- The drug (RH) binds with cytochrome P-450 - hemoprotein enzyme in its oxidized _____ form
- The resulting drug-cytochrome complex is then reduced by the flavoprotein ______ to its ferrous form (Fe2+).
- ______ serves as primary electron donor.
- Molecular _____ reacts with the reduced cytochrome P450-drug complex to generate a ternary complex.
- A second electron transfer occurs probably via the same _____
- The oxygen-oxygen bond is broken with the uptake of _____ protons
- Water is released and _____ is generated (Fe – O)3+.
- The Fe-ligated O atom is transferred to the substrate forming a hydroxylated form of the substrate____.
The product is released from the active site of the enzyme that returns to its original ____ form.
- ferric form (Fe3+).
- NADPH-cytochrome P-450 reductase
- NADPH
- oxygen
- NADPH-cytochrome P-450 reductase.
- two protons.
- activated oxygen
- (ROH)
- oxidized
What performs the reduction in the P450 reaction?
What is incorporated and what is added to form water?
- NADPH reductase
2. Oxygen is incorporated into drug substrate and one oxygen added to form water
What is an example of a non-microsomal oxidation reaction? (not mediated by P450)
What are 2 important enzymes? Where are they found?
What is the most important liver enzyme to metabolize alcohol?
Alcohol Oxidation
- alcohol dehydrogenase (in the cytosol)
- Acetaldehyde dehydrogenase (mitochondria)
make ethanol to acetaldehyde into acetate
- ALCOHOL DEHYDROGENASE!!
- some ethanol is metabolized by catalase & CYP2E1
- relevant when p450 status changes
What are 3 hydrolysis reactions in Phase I?
What is the talk home message?
How does the metabolite of these reactions affect sulfonamide? (sulfa drug)
Clinical application for using procaine and procaine amide concomitantly with a sulfa drug for bacterial infection?(2)
- Esterases that make PABA from local anesthetic like Procain
- Amidases from porcine amide make PABA
**PABA is structurally similar to sulfonamides (antimicrobial)
**PABA will compete with sulfonamide for drug site of action and lead to reduced therapeutic effectiveness
a) CLINICALSIGNIFICANCE: Do not use procaine for local anesthesia when treating an infection with a sulfa drug (sulfonamide).
b) CLINICAL APPLICATION: Substitute another antiarrhythmic agent instead of procainamide in patients receiving sulfa drug therapy.
What happens in Phase II reactions? Result?
What are 4 examples?
Why do Phase I reactions precede Phase II reactions?
CONJUGATIONS: functional group (Phase I Rx) combines with endogenous substrate
RESULT = highly polar conjugate - easily excreted in urine
- Glucuronic acid conjugation
- Acetylation
- Sulfation
- Glutathione conjugation
- oxidation reactions often precede conjugation (phaseII) because
the generated hydroxyl group during phase I reaction is easily conjugated with several compounds
What enzyme is important in glucuronic acid conjugation?
free \_\_\_\_\_ does not couple to drugs; it has to be activated to uridine diphosphate glucuronic acid (UDP-GA)
UDP - glucosyltransferase
- found in ER
- P450 enzymes found in the ER as well so Phase I and II reactions occur in close proximity!
- glucoronic acid
UGT is a ____ enzyme.
It is proximal to phase I metabolites, also formed in ____
- microsomal
2. ER
What factors affect drug metabolism?
- Drug age interactions
- differential expression of enzymes - Drug - drug incompatibilities
a) competitive inhibition (paba & sulfanomide)
b) INDUCTION - pharmocokinetic tolerance - Drug - endogenous substrate interactions
a) competition with endogenous substrate - Drug disease interactions
a) liver disease = impact MICROSOMAL OXIDASES
b) cardiac disease - impact on HEPATIC BLOOD FLOW - Drug egentic interactions
- gene mutations
a) poor metabolism = toxicity
b) enhanced metabolism = reduced therapeutic effect
ex: mutliple copies of p450 enzymes
What is gene induction?
What is the result?
GENE INDUCTION
upon repeated administration chemically dissimilar drugs can “induce” cytochrome P450
mechanism - transcriptional or posttranscriptional
-enzyme inducer may stimulate
metabolism of SIMULTANEOUSLY administered drug
RESULT: decrease in the therapeutic effectiveness of drug
NOTE: pharmacodynamic tolerance involves down-regulation of drug targets (e.g. receptors, ion channels, enzymes)
What is induction?
What does it typically involve?
What enhances? What reduces?
What are 3 factors that may INDUCE CYp 450s?
Clinical significance?
upon repeated administration chemically dissimilar drugs can “induce” cytochrome P450
mechanism - transcriptional or posttranscriptional
1, Involved CYp450s
- Enhances rate of synthesis
- Reduce rate of degradation
- environmental pollutants (benzopyrene, PCBS, dioxin)
- tobacco smoke
- Charcoal broiled meat
Clinical Significance: Altered Dosing
RESULT - increased metabolism - dose adjustment to obtain desired effect
- sometimes genetically determined
What is inhibition?
What is reversible: competitive or non-competitive inhibition?
Some drugs can inhibit drug metabolizing enzymes
Two drugs metabolized by same enzymes (competition between the two drugs)
Competitive = reversible
non-competitive = irreversible
PERMANENT –> covalent modification
The following describes active or inactive drugs?
- very lipid soluble
- less polar
- weak electrolyte
- less ionized
- more able to penetrate membrane
ACTIVE
What is the final outcome ofdrug metabolism?
Active to inactive drug
FINAL OUTCOME: Properties of drug metabolite serve to PREVENT the renal REABSORPTION of the drug metabolite, thereby allowing the body to eliminate the foreign compound
What is a SNP?
Where can they occur?
Single Nucleotide Polymorphism (SNP)
- a polymorphism due to a change in a single nucleotide - human genome = 3 109 nucleotides
average frequency of 1 per 1000 base pairs = interindividual difference 3 106 base pair - useful for genome wide association studies
COding or non- coding region:
SNP in coding region may change amino acid = nonsynonymous coding SNP (cSNP)
- SNP in non-coding region may be in promoters, enhancers, splice sites, or other sites that control gene transcription or mRNA stability
Pharmacogenomics can be divided into what 4 general areas?
- Avoiding adverse drug reactions
- Identifying causes of disease
- Designing clinical trials
- Treating specific diseases
What is the general rule for number of individuals plotted against maintentenace dose?
General rule: number of individuals plotted against maintenance
dose for a given drug will show a bell-shaped distribution
low maintenance dose = poor metabolizers
large maintenance dose = ULTRARAPID metabolizers
Alot of genetic disorders are due to ___ enzymes
These defective enzymes are like found in what 2 areas?
Protein folding enzymes
- metabolism
- drug receptor
drug metabolism enzymes are polymorphic - variability in responses: efficacy and
ADRs
What happens to efficacy and toxicity in a homozygous mutant?
toxicity increases due to the increase in efficacy
Most Phase I enzymes that digest toxins are which?
Which are the most polymorphic?
Which phase II drugs are most important? Which are highly polymorphic?
- CYP3A4 = not polymorphic
- CYP 2C9 & CYP 2D6 are polymorphic!
- UGT - highly polymorphic
& metabolize up to 50% of drugs
What are some drug classes metabolized by CYp2d6?
- tricylclic anti-depressants
- anti-arrhythmics
- b-blockers
- neuroleptics
Which p450 enzyme does the following:
- metabolizes 20 - 25% of all drugs
- highly polymorphic gene - inactive in 2-10% of population
- deficiency can have diverse consequences: dependent upon drug
detected by modern pharmacogenetic methods
(microarrays and PCR)
P450 CYP2D6
-– first discovered to affect the metabolism of debrisoquine and sparteine
Roche Diagnostics - DNA sequence microarray - pharmacogenomic profiling of
CYP2D6
Which p450 enzyme is described by the following:
Phase I enzyme - metabolizes ≈ 15% of all drugs
Incidence: estimated 40% of population are heterozygotes
Homozygote mutant ≈ 1%
CYP2C9
What is the drug associated ADR for CYP2C9?
What is another variability factor
Warfarin (anti-coagulant)
- ppl. with mutations can have excessive bleeding & hemorrhage
= fatal
- accounts for 10% population variability
- other variability factors – site of drug action
– vitamin K epoxide reductase (VKORC1) – key warfarin target – clotting factor
– 25% of warfarin variability due to VKORC1
Which p450 enzyme is described by the following:
Phase I enzyme - metabolizes ≈ 5% of all drugs
Incidence: ~ 24% of white population has one allele
~ 33% of African American population has one copy
~ 54% of Asian population has one copy
Homozygote mutant - 1%
CYP2C19
What is the drug associated ADR for CYP2C19?
Clopidogrel (PLAVIX) anti-coagulant given prophylactically
for thrombosis- prodrug coverted to active metabolite
Patients with two CYP2C19*2 alleles are unresponsive to this drug
** prescribe warfarin or other related drug
Which p450 enzyme is described by the following:
Phase I enzyme - metabolizes ≈ 50% of all drugs
Inactivating polymorphic variants: NONE
CYP3A4!!!
What is the contraindication for CYP3A4?
- grapefruit juice has
- plyphenolic compounds
- flavonoids
- fluranocoumarins
these bind to CYP3A4 and
inhibit CYP3A4
Where is N-acetyltransferase (NAT2) found?
What is poor with a slow acetylator phenotype?
How is this identified?
What is the danger?
LIVER
- phase II enzyme
- Inactivating polymorphisms
- inefficient conjugation
- RFLP
- danger = INCREASED TOXICITY due to SLOW METABOLISM
(because need a higher concentration to reach efficacy!)
What are the drug associated ADRS of NAT2? (3)
- isoniazid - peripheral neuropathy (anti-biotic; tuberculosis)
- hydralazine - lupus erythematosus (antihypertensive)
- sulfonamides - hypersensitivity reactions (antimicrobial)
What is pharmacodynamic variability?
What is an example? (2)
Pharmacodynamic variability : drug variability due to due polymorphisms/mutations
at the site of drug action
- Warfarin
- site of action for warfarin is at VKORC1 gene
- has polymorphisms in CYPC29 or in VKORC1
- the mutations render warfarin INACTIVE
Poor responders that have an inactive VKORC1 gene need a lower or higher dose?
HIGHER dose
- poor responders have an inactive VKORC1
excellent responders need a lower dose!
How can causes of disease be identified?
- Sequencing of human genome
- DEEP sequencing, cloning, bioinformatics - Markers identified
- SNP map
- mapping disease loci easier once family identified
- genome wide association studies
- most genes encoding proteins involved in molecular pathways have been identified
What are some therapeutics based on RECOMBINANT proteins?
What is a pharmacogenomic alternative ?
- Hromones
- hormone replacement!
(BEST EXAMPLE OF PHARMACOGENETICS BENEFIT) - Pharmacogenomics alternatice = GENE CLONING
- genes encoding hormones identified
- relatively small proteins
- produced and purified from bacteria
What is an example of Gene Cloning used clinically?
What is the only caveat?
Erythropoietin (epogen) - - used to treat chronic kidney failure on dialysis
- stimulates production of red blood cells
- contraindicated in patients with uncontrolled hypertension
Delivery
What is the result of pharacogenetic screening?
Increasing statistical significance, saving years and money to find safer & more efficacious medicines
What are the phases of clinical trials?
Phase I
- human subjects
- healthy volunteers – small sample size - usually healthy volunteers
- safety profile and dosage levels
- pharmacokinetic analysis
Phase IIA
- drug tested for desired clinical effect-safety concerns are examined
- relatively small sample size (100 patients)
costly: millions of dollars**
o Phase IIB
- further efficacy and dose ranging are examined *** larger sample size
o Phase III
- very large sample size – hundreds - thousands of individuals
cost: tens of millions of dollars
- very large sample size – hundreds - thousands of individuals
- could take years before the drug is marketed as a medicine to treat patients
o Phase IV
-
** Post marketing follow-up – adverse events –
advantage: potentially large sample size
Use __________ data to determine who would be good responders
– smaller clinical trials, less time to complete, reduce cost
– get medicines to patients faster
– reduce number of failed trials - focused clinical trials
- greater chance of obtaining
statistically significant results with reduced side-effects
pharmacogenetic
What is a drug that is a good example of pharmacogenomic clinical trials?
What is it an inhibitor of? (2)
What is it used to treat?
LAPATINIB
- EGFR and HER2 receptor tyrosine kinases
- used to treat specific cancers
Phase I ; 16 of 107 ppl had diarrhea/skin rash
What is lapatinib metabolized by?
DNA microarray of the genes encoding these enzymes revealed
association of diarrhea and rash with ______genotype
Take home message?
– metabolized by CYP3A4 and CYP3A5
- some is metabolized by CYP2C19***
- polymorphic CYPRA4 is not!!!
-SINGLE DRUG CAN BE
METABOLIZED BY MANY GENES
- CYP2C19*2
- Perform trial using LOWER tolerable dose
What can pharmacogenomic identification help with?
What about targeted therapy?
- Diagnosis
2. Less side effects
Give an example of targeted therapy.
What is the drug of choice to treat this amplification?
What is a serious side effect?
Treatment of Breast Cancer
- Fish analysis to identify HER2- Neu amplification!
- Trastuzumab (herceptin)
- anti - Her2/Neu humanized mAb antibody
- reduces tumor sizes, slows disease progression & cancer recurrence
- drug would not work in BRCA mutation - CHF
What can PCR be used for?
What about FISH?
- ideal for detecting leukemias caused by chromosomal rearrangements & viral infections
- fluorescent in situ hybridization - identify gene location on chromosome - useful for detecting amplifications, translocations
What are 2 examples of a targeted use of FISH for therapy?
- Her2
2. BCR-ABl
How can Chronic Myelegenous Leukemia be identified? Treated?
- FISH
- Imatinib (gleevec)
- inhibits Bcr-Abl kinase
- constitutivly active kinase associated with CML
**reduces proliferation and activates apoptosis in the cancerous white blood cells
associated with CML
– one chromosome 9 longer than normal and one chromosome 22 shorter than normal
- translocation leads to the fusion of two genes, bcr1 and abl (abl encodes is a non-receptor
tyrosine kinase) - phosphorylates several cytoplasmic substrates, activating signal transduction cascades that control growth and differentiation
