Lec 10, 11, 12 - Drug Biotransformation Flashcards
1
Q
explain the detoxification of doxorubicin including:
- what can it get converted into and by which enzyme
- which of the byproducts are cytotoxic? Why?
A
- quinone fg either converted to semiquinone by nadph cytochrome p450 reductatse (1e reduction rxn), or to hydroquinone by quinone reductase (2e reduction)
- semiquinone and radical oh, because they contain a radical atom
2
Q
what are reduction reactions? are they phase 1 or phase 2?
A
rxns where the product gets reduced (gets electrons)
phase 1
3
Q
which functional group in doxorubin do reductases act on?
A
quinone
4
Q
t/f: all enzymes will carry out the same effect on a drug
A
false
5
Q
is hydrolysis a phase 1 or 2 reaction?
A
1
6
Q
what are epoxides and how does hydrolysis combat toxicity?
A
chemically reactive electrophillic molecules, covalently bidn to proteins and lead to cytotoxicity
conjugates epoxide with water, gets rid of three ring reactive o and replaces with 2 oh groups
7
Q
what is the difference between microsomal and cytosolic forms of enzymes?
A
microsomal found in ER
cytosolic found in cytosol
8
Q
epoxide hydrolases convert epoxides into what? why is this byproduct less reactive?
A
- dihydrodiol (breaks open o ring and adds 2 hydroxyl)
- less reactive because less strain from 3 ring
9
Q
what is the main goal of phase 2 reactions?
A
- want to make the molecule more water soluble.
- some cellular pathway leads to the formation of endogenous substances that get tacked onto the drug via a transferase
10
Q
how is a glucuronide metabolite formed
A
UGT tacks glucouronic acid onto the drug
11
Q
generally speaking what is glucoronidation
A
transfer of glucauronic acid onto a drug
12
Q
what enzyme carries out glucoronidation? how many? where?
A
udp glucoronosyltransferase (UGT) - we have 22. happens inside er
13
Q
what are the substrates for UGT (ie: where does it attach to)? what drugs contain these groups?
A
hydroxyl, carboxyl, amine, sulfhydryl
acetominophen, morphine
14
Q
what is the cofactor for glucoronidation? which part of this cofactor actually gets attached to the drug to make the glucoronide metabolite?
A
UDPGA
just the gluc. acid gets attached
15
Q
explain how a glucouronide metabolite is formed, including the steps of UDPGA synthesis
A
utp joins with g1p
utp dephos by udp glucose pyrophos
udp glucose oxidised by nad and h20, makes udp nadh, h
udp gluc crosses into luman
drug joins into UDPGA, UDP separated
udp into cyto and gets phosphorylated intto UTP, process restarts
16
Q
explain the roles of the following enzymes:
1. UDP glucose pyrophosphorylase
2. UDP Glucose dehydrogenase
3. UDP Glucoronosyl transferase
A
- joins UTP and glucose 1 phos
- oxidises udp gluc
- transfers GA onto drug
17
Q
UGT action occurs on the lumenal or cytoplasmic side?
A
lumenal
18
Q
how is glucoronidation impacted in gilberts symptom?
A
inherited deficiency in the glucuronidation of endogenous bilirubin, resulting in its accumulation and jaundice, mild disorder, 2-5% of pop
19
Q
how is glucoronidation impacted in crigler-najjar syndrome?
A
UGT deficiency -> billirubin buld up -> impaired drug clearance and hyperbilli -> aplastic anemia from chloramphenicol -> grey baby syndrome
20
Q
how does chloramphenical lead to grey baby syndrome?
A
The gray baby syndrome is a type of circulatory collapse that can occur in premature and newborn infants and is associated with excessively high serum levels of chloramphenicol, chlo not broken down by glucoron. bc of genetic deficiency
21
Q
what is the end goal of glutathione conjugation?
A
attach cysteine (with sulfur) to drug for increased excretion
22
Q
t/f: the whole glutathione molecule gets added to the drug in glutathione conjugation
A
false, just the cysteine
23
Q
give a general overview of what glutathion conjugation is (the pathway) and how it leads to excretion?
A
drug joins onto tripeptide S with glutathion s transferase,
gammaglutamyltranspeptidase takes off and recycles the glutamine
cysteinyl glycinase takes off and recyvles the GLY
acetyltransferase attaches acetyl to cystein drug complex
now mercapturic acid, more water sol, excreted in urine
24
Q
where does glutathione conjugation occur?
A
cytosol, mitochondria, microsomal
25
what is the role of glutaythione s transferase in glut. conj?
attached the drug to tripeptide
26
glut. conj is carried out on which substrates? functional group examples include?
electrophillic centres (epoxides, arene oxides, nitro groups, hydroxylamines)
27
what is the cofactor in glut. conj? which part actually gets attached to the drug?
GSH (reduced glutathione) (glu, cys, gly), - just the cysteine
28
t/f; one enzyme is responsible for the entire glut. conj. pathway
false
29
what happens to the y-Glc-AA and Gly in glut. conj?
they get recycled
30
what is the end goal of sulfation?
attach sulfate to drug
31
what enzyme carries out sulfonation and how many do we have?
sulfotransferase (SULT), 14
32
where does sulfonation occur
cytosol
33
what substrates does sulfonation act on? drug examples?
phenols, alipathic alcohols
acetaminophen, steroid hormones
34
what is the cofactor for sulfonation? does the whole thing get attached to the drug? how is this cofactor formed?
1. PAPS (3 phosphoadenosine 5 phosphosulfate)
2. no, just the sulfate
3. sulfate joins with atp via atp sulfurylase, forms aps
aps phosphorylated, forming paps
sult removes pap, attaches sulfate to drug
35
t/f: only one phase 2 reaction occurs on each drug
false
36
what does acetominophen heptatoxicity demonstrate about phase two reactions?
many phase 2 rxns can happen on same drug, all wont have beneficial effects
37
explain the inactivation pathways of acetominophen, and what happens when these pathways get overwhelmed
either via glucoronidation or sulfonation. when overwhlemed, makes reactive intermediate napqi via p450s cyp2e1 and cyp3a4.
38
what is the backup detox pathway of acetominophen if glucoronidatoion and sulfation get occupied
GSH conjugation: addition of cysteine to napqi for excretion via glutathione s conjugation
39
how does NAC restore glutathione levels
its a cysteine precursor
40
what is NAPQI, how is it formed, and when would liver cell death occur
n-acetyl- p - benzoquinoneimine
formed when normal inactivation pathways of acetaminophen are full
liver cell death happens when napqi cant go through detox path, and it joins onto other macromols
41
what is the end goal of acetylation
add acetyl group onto drug
42
what enzyme carries out acetylation
n-acetyl-transferase (NAT)
43
how many NATs do we have?
2
44
where does acetylation occur
cytosol
45
what substrates does acetylation occur with? drug examples
aromatic amines and hydrazine
isoniazid, sulfamethoxazole
46
what cofactor is necessary for acetylation?how is this cofactor made? does the whole thing get attached to the drug?
acetyl coA
acetate joins onto coa
only acetyl gets attached, via NAT
47
what is the end goal of methylation?
attach a methyl group to the drug
48
what enzyme (general) carries out methylation? is there only one subtype?
methyltransferase
no
COMT, TPMT
49
where does methylation take place?
cytosol
50
what substrates does methylation use? why these substrates? drug examples?
phenol, catechol, alipathic and armoatic amine, n-heterocyclic, sulfhydryl
levodopa, 6mp
51
what cofactor is involved in methylation? does the whole thing get attached to the drug?
s-adenosylmethionine (SAM)
52
how is the methylation substrate formed?
methionine joins atp, makes sam and ppi, pi
53
what role does TPMT play?
transfers methyl from sam onto drug
54
does methylation detox or activate 6 mercap?
detox
55
what does 6 mercaptopurine get turned into, and by which rxn? what type of drug?
6 -methylmercaptopurine, by methylation via TMPT, cancer (leukemia) drug
56
what patient consideration is important when giving 6 mercap?
genetic polymorphs: do they have function tmpt to be able to detox cancer drug
57
what factors affect drug biotransformation
induction and inhibiton
diet
species
age
sex
hormones
disease states
genetics
58
t/f: individuals have the same capacity for drug met. throughout their lives
false
59
what does it mean to induct or inhibit an enzyme
induct: activate it
inhibit: block it
60
how can efficacy of drug be affected by induction/inhibtion
induction: may speed up drug met, negating or increasing effect dep on drug
inhibition: slows down drug met, causing slower elim, or less prodrug activation
61
how can toxicity of drug be affected by induction/inhibtion
induction: may lead to toxic byproducts
inhibition: not enough prodrug met, leads to tox
62
what does therpaeutic index have to do with impacts of induction/inhibition
narrow thera, need to understand how things that may induce or inhibit drug met may impact conc (very close between beneficial and toxic)
63
what does it mean for a p450 to be strongly inducible?
it can easily be activated
64
which is the most abundant p450? how is it induced?
cyp3a4/5. induced by pregnane x recep activation
65
this p450 metabolizes caffeine and is induced by cigarettes and charcoal broiled meats
cyp1a2
66
this p450 is induced by alcohol intake
cyp2e1
67
explain the relationship between aryl hydrocarbon receptors and p450s
activation of aryl hydrocarbon recepts can go on and induce p450s
68
what type of molecules have high affinity for aHr?
flat, cyclic, cl
69
TcdD, Indol[3,2,b]carbazole, 3methylcholanthrene, and benzo[a]pyrene are all examples of what
inducers of ahr
70
explain what it means to say that benzoapyrene induces its own biotransformation? what experimental evidence shows this?
the more benzoapyrene you have, the more benzoapyrene gets metabolized
the experiement where they injected mice microsomes with different levels of benzoa: dep on how much they had in their livers, the benzo was metabolised at different rates.
induction is reversible
71
why do smokers see a higher level of cyp1a1 activity?
cig smoke contains BP, activates ahr, activates cyp1a1
72
in which stage of enzyme modification does the most enzyme regulation occur?
gene transcription
73
at which stages can enzymes be regulated?
gene transcription, processing, mrna stab, translation, enzyme stabalization and degradation
74
cyp1a2 is regulated in which stage
processing
75
cyp2e1 is regulated in which stage
translation
76
which cyps are regulated in enzyme stabalization and degradation phase
2e1, 3a1, 3a2, 3a6
77
explain how an inducer leads to increased biotransformation (the whole pathway)
inducer goes into cell, attached to ahr along with chaperons, goes into nucleus via arnt, compex can activate phase 2 gst and ugt of p450s (1a1, 1a2, 1b1, 2s1), which increases biotrans
78
t/f: inducers only impact p450 action
false, also impact phase 2 ugt, gst
79
order these steps:
1. tcdd enters cell
2. p450s and phase 2 gst ugt get activated
3. chaperones hsp90 and aip join onto ahr tcdd complex
4. hsp unattaches, ARNt joins to AHR TCDD complex
5. AHR TCDD COMPLEX and chaperons enter nucleus
6. increased drug biotransformation
1, 3, 5, 4, 2, 6
80
what is the pregnane x receptor?
another receptor whos activation can induce p450s
81
how does st johns wort lead to increased metabolism of drugs? by which receptor?
joins onto pxr with chaperons, enters nucleus, induces cyp3a4 gene
82
t/f: increased metabolism of drugs is always bad
false
83
how does induction by phenobarbital help neonates with hyperbillirubinemia
induces UGT, which clears billirubin through glucoronidation
84
contrast therapeutic vs prophylactic
therapeutic is responsive, prophylactic is preventitative
85
how does induction by indole 3 carbinol provide reduced risk for breast cancer? why?
binds to ahr, induces cyp1a2, cyp1a1, turns estradiol into reduced form, non toxic and readily elim.
86
how does 13c reduce breast cancer risk?
reduces circulating levels of 17B estradiol
87
is the 2-oh or the 4-oh the more readily excreted estradiol
2-oh
88
treatment with ddt and subsequent less mammary tumours tells us what about the relationship between ddt and dmba?
ddt protects from dmba carcinogen
89
how does ddt provide dmba protection
enhances first pass clearance of dmba
90
t/f: inducers only induce one p450 species
false - they can induce more than one
91
t/f: inducers only induces p450s
false - can induce some phase 2
92
t/f: inducers never stimulate their own metabolism
false: can stim own (benzopyrene ex)
93
t/f: inducers can stimulate themselves and several other chemicals
true
94
t/f: inducers are active only in mammalian species
false; also in non mam
95
t/f: inducers only act in one given tissue at a time
false, act in many
96
t/f: inducers usually slow the rate of a certain p450s transcription
false, usually speed it up
97
explain the issues around the drug seldane in regards to inhibition
seldane metabolism was inhibited when taken with erythromycin and ketoconazole, therefore never got converted from terfenadine
98
how does terfenadine affect the heart
clocks k+ channels, leading to qt prolongation, and torsades de pointes
99
what is torsades de pointes
arrythymia
100
t/f: the metabolism of terfenadine leads to torsades de pointes
false - the blockage of metabolism does
101
what impact does erythromycin or ketconzale have on terfenadine
blocks its metabolism into fexofenadine, leading to torsades de pointes
102
why is allegra (fexofenadine) the preferred antihistamine
because it directly leads to antihistamine benefits, not reliant on metabolism that may be blocked
103
t/f: cyp3a4 is only involved in one metabolic pathway
false
104
why can cimetidine not be taken with wafarin, theophylline or phenytoin
drug drug interactions may occur because it may inhibit their metabolism
105
explain the metabolic bottleneck
too many drugs trying to go into one recep, recep cant handle them all, they dont all get appropriately metabolised
106
t/f: only drug interactions inhibit metabolism
false - diet, enviro, etc can as well
107
whats the relation of grapefruit and cyp3a4
grapefruit inhibits metabolism via cyp3a4
108
why is it important to know the specific p450 that metabolises a drug
so you can predict what factors might impact the metabolism of the drug
109
what is METEOR
a system to determine which p450 acts on which drug
110
what is species extrapolation
extrapolating how a drug may act in a human based on how it acts in an animal
111
what are concerns with p450 function exptrapolation from animal models?
the same genes in human vs animal dont always have the exact same function
112
contrast the two types of invitro tests to test p450 function
test in human tissues, or clone human ezyme into an animal and test (need to include p450 reductase)
113
what are the three steps to reaction phenotyping (xenotech approach)
find the p450s expressed in the cdna ,
correlation analysis with hlm from different ppl
inhibition of enzymes, see which one affects metabolism
114
what does the cdna bar plot show in reaction phenotyping?
the relative abundance of how each p450 metabolises the drug
115
what does the correlation in hlm plot show in reaction phenotyping
how much of the activated drug forms based on the activity of a certain enzyme (is there a correlation - this would be done for each enzyme that you get in the cdna plot)
116
what can you use as a predictor of enzymatic activity on an hlm scatterplot
how much a certain substrate gets acted on
117
what does the inhibition plot tell you in reaction phenotyping??
when you inhibit each of the enxymes, what happens to the prodrug metabolism? if the drug gets metabolised less when a certain p450 is inhibited, that means that that p450 is involved in its metabolism
118
explain what " can do != will do" means in reaction phenotyping, and exceptions to this rule
just because it does something in vitro, does not mean it always will invovo. unless: you use the right concentration of substrate, all pathways are considered, and each individual enzymes variation is considered.
119
why dont pharm companies want cyp3a4 to be the primary route of metabolism for a new drug?
cyp3a4 metabolizes so many drugs, therefore lots of competing pathways. highly variable, and many possibilities for it to be induced and or inhibited.
120
why dont pharm companies want cyp2d6 to be the primary route of metabolism for a new drug
lots of polymorphism -- variations in metabolism
121
whats a limitation of in vitro tests to predict drug metabolism
only show the potential enzymes that metabloize the drugs, not everything
122
what are the key issues when dealing with predicting drug met.
need to know all enzymes that are able to metabolise, all enzymes present, and all activity of enzymes
123
how can you figure out ratios of metabolic enzymes from probe drugs
See what metabolites are created, use that to figure out which enzymes are active
124
which p450 is heavily induced by alcohol intake, expressed in liver, kidney, lung, and some extrahepatic tissues, and biotransforms ethanol and halgenated alkanes?
cyp2e1
125
which p450 converts benzene and chloroform into heptotoxic metabolites
cyp2e1
126
which p450 was originally discovered as microsomal ethanol oxidsising system
cyp2e1
127
what is a polymorphic gene
one gene, many variations
128
which p450 has low abundance but biotransforms antiarrythmics, antidepressants, antipsychotics, bblockers, analgesics
cyp2d6
129
propafenone is a
antiaryhtmia drug
130
fluoxetine is a
antidepressant
131
chlropromazine is a
antipsychotic
132
propranolol is a
beta blocker
133
codeine is a
analgesic
134
which p450 is mainly expressed in the liver, polymorphic in humans, and catalyzes hydroxylation of s-mephenytoin
cyp2c19
135
s-mephenytoin is a
anticonvulsant
136
what is the main 2c enzyme in the liver
cyp2c9
137
which p450 is found in the liver, polymorphic, and catalyzes hydroxylation of tolbutamide, phenytoin, and warfarin
cyp2c9
138
tolbutamide is a
hypoglycemic
139
phenytoin is a
anticonvulsant
140
warfarin is a
anticoagulant
141
_____ is expressed in the liver, but not extrahepatically. its relative ____ is not in the liver, but is in other tissues
cyp1a2, cyp1a1
142
what induces cyp1a2
polycyclic aromatic hydrocarbons
143
what does cyp1a2 biotransform
caffiene, acetaminophen, aromatic amine carcinogens, aflatoxin
144
what is the most abundant p450 in the liver and small intestine
cyp3a4
145
which two enzymes catalyze 50% of known drugs
cyp3a4/3a5
146
erythromycin, codeine, lovastatin, diazepam, and taxol are all metabolized by:
cyp3a4
147
what is the main p450 in the human fetal liver
cyp3a7
148
t/f: only a limited range of xenobiotics induce cyp3a4
false
149
p450s were discovered by:
omura, sato
150
what does the 450 in p450 represent
wavelength og light that the heme group in the prtein absorbs
151
what is a microsome
circular vesicle formed from ER membrane after centrifugation
152
can microsomes be found in living cells
no
153
what did ronald estabrook show
that cytocrhom p450 metabolizes drugs and steroids
154
whats the main reaction carried out by cytochrome p450
monooxygenation (adding in an o and forming a water)
155
what type of protein are cytochrome p450s
heme
156
ferric vs ferrous
ferric = 3+
ferrous = 2+
157
what form must iron be in to bind oxygen
ferrous
158
which form of iron is reduced: ferric or ferrous
ferrous
159
which part of the p450 binds to the substrate
fe3+
160
overall p450 rxn
RH + o2 + nadph + h -> roh + h20 + nadp
161
order these steps:
1. p450 containing ferric iron binds the substrate
2. the ferrous p450 iron binds an oxygen
3. the o-o bond splits
4. the carbon radical and hydroxyl radical recombine. the hydroxylated metabolite is released
5. NADPH cytochrome p450 reductase takes an electron from nadph and reduces the p450 iron to a reduced state
6. the RH hydrogen is abstracted
7. a second electron either from p450 reductase or b5 reductatse gets added to the bound oxygen, reducing it into its activated form
8. one oxygen atom goes into water
9. ferric iron is regenerated
1, 5, 2, 7, 3, 8, 6, 4, 9
162
which side does POR act
cytosolic
163
what is the role of POR
reducing p450 so it can bind o2
164
which are the major p450 families for xenobiotic metabolism
1-3
165
what do p450 families 4-51 do
biosynthetic rxns, met of endo subs
166
how did p450 genes evolve
duplication of og genes, accumulation of mutations
167
how are p450s categorized
based on gene sequences
168
CYP1A and CYP1B are in the same ____ but not the same ______. this means their amino acid identity is roughly _________ the same
family, subfamily, >40%
169
the most dominant p450 is
cyp3a
170
is abundance correlated with importance
no
171
t/f: drug met is always protective
false
172
main consequences of drug met:
stable metabolites (inactivating, pro drug activating, activity maintaining)
chemically reactive metabolites (covalent binding -> toxicity)
173
example of drug inactivation
acetominophen to acet. sulfate via sulfonation or acet. gluc via glucoronidation
174
example of pro drug activation
cyclophosphamide to phosphoramide mustard (active metabolite to kill cancer cells)
175
example of stable metabolite formation
propafenone to 5 hydroxypropaenone (antiarrythmia)
176
example of chemically reactive metabolite
anticonvulsants (phenytoin, carbamazepine, phenobarbital), form arene oxide, covalently bind, forein body, autoimmune response
177
where does most drug biotrans take place
liver (but also happens almost everywhere
178
when can tissue specific toxicity occur
when there is expression of certain enzymes in a tissue (even if the tissue has low drug met, there are some hotspots)
179
how will first pass effect alter drug
drug gets activated or inactivated before reaching circ
180
how do we biotranform so many drugs
enzyme multiplicity, broad substrate selectivity
181
what do phase 1 rxns do
introduce new functional groups to the drug
182
what do phase 2 rxns do
make the drug water soluble for excretion
183
is it always phase 1 -> phase 2
no
184
example of drug going through both phase 1 and 2
codeine to morphine via p450, morphine to m3g via ugt
185
example of direct conjugation
morphine to m3g via ugt
186
t/f: morpheine is only made from codeine
false, also made from heroin via phase 1 deacetylation
187
phase 1 rxs
monooxygenation, oxidation, dehydrogenation, reduction, hydrolysis
188
phase 2 conjugating enzymes
udp glucoronosyltrans
glutatthion s trans
sulfotrans
n-acetyltrans
methyl trans
