Theme 3: Factors affecting drug metabolism (L13-17) Flashcards
1
Q
How were cytochrome P450s discovered?
A
Using spectrophotometry at 450nm with e- (Fe2+) and CO2 bubbled through
2
Q
How does administration of certain drugs increase metabolism of other drugs?
A
Over time more CYP450s metabolites are produced
3
Q
How can inducers modulate the action of drugs?
A
Drugs are addd at the same time and time taken for the symptom to show is measured in comparison with other drugs
4
Q
What is a cistron?
A
the smallest unit of genetic material which coded for a single polypeptide for the transmission of genetic information
5
Q
What happens when enzymes are induced?
A
Expression of the genes increases as they are in one cistron
6
Q
What is the stability like of the metabolising enzyme mRNA?
A
Low, not long lifetime
7
Q
How does protein stability impact metabolising protein expression?
A
Proteases can degrade protein as they have a short lifetime
8
Q
When are the majority of CYP450s regulated?
A
At transcriptional level
9
Q
How is CYP1A1 inducible?
A
By polyaromatic hydrocarbons
Basal mRNA and protein is low
Northern blot used to measure mRNA
BetaNF used to CYP1A1 so mRNA expression increased
10
Q
What are B-13 cells?
A
They are A pancreatic cell line transformed into hepatocytes for CYP450 expression and metabolism activity of different drugs
11
Q
How does TCDD induce CYP1A1 to promote reporter gene expression?
A
Using a response element (XRE1)
12
Q
What is the principle of electro mobility shift assay?
A
To bind target oligonucleotides to protein resulting in change in oligonucleotide mobility during electrophoresis
13
Q
How is NFkappaB TXN induced?
A
Using TNFalpha
14
Q
What is the function of gliotoxin in NFkappaB TXN induction?
A
It breaks NFkappaB down meaning TXN cannot be induced
15
Q
What happens in PXR knockout mice in response to CYP3A?
A
The inducer of CYP3A is abolished which is confirmed in western blotting
16
Q
What are other approaches of measuring CYP450 inducer levels?
A
Knockout mice
Knockin mice (hPXR and keep mouse PXR)
Knockout and knockin (out PXR in hPXR)
17
Q
How does TCDD induce CYP1A1?
A
AhR and Hsp90 dissociate which allows TCDD complex to form and Arnt complex forms which binds to an enhancer/promoter allowing increase in transcription forming CYP1A1
18
Q
Which ligand-dependent intracellular receptors does CYP2B depend on?
A
CAR
19
Q
Which ligand-dependent intracellular receptors does CYP3A depend on?
A
PXR
20
Q
Which ligand-dependent intracellular receptors does CYP4A depend on?
A
PPARalpha
21
Q
Which ligand-dependent intracellular receptors does CYP7A depend on?
A
LXR, FXR
22
Q
What is the superfamily of receptors for CYP2B, 3A, 4A and 7A?
A
Nuclear receptor
23
Q
What is the mechanisms of action for nuclear receptors?
A
They heterodimerise as a ligand binds which increases TXN
24
Q
Which nuclear receptors bind steroids?
A
Glucocorticoid
Mineralocorticoid
Androgen
Oestrogen
25
What nuclear receptors do other ligands bind?
Retinoid X receptor
Retinoic acid receptor
Thyroid hormone receptor
Vitamin D receptor
26
What nuclear receptors do bile acids bind?
Pregnane X receptor
Constitutive activated receptor
27
What half sites do nuclear receptors bind?
AGGCTA
steroid hormone receptors
Bind as homodimers/ heterodimers
Imperfect palindromic sequences (RNA polymerase)
3BP spacing
28
what are the different classes of genes of inducers?
CYP1A
CYP2B
CYP3A
CYP4A
CYP7A
29
what are the different receptor classes of inducers?
AhR
CAR
PXR
PPARalpha
LXR, FXR
30
what are the AHR receptor substrates?
poly aromatic hydrocarbons
Tryptophan derived products (origin)
31
What are the CAR receptor substrates?
Phenobarbitone drug
Bile acids
32
What are the PXR receptor substrates?
many drugs (xenobiotics)
Bile acids
33
what are the PPAR alpha receptor substrates?
Fibrate drugs (small class)
Fatty acids
34
what are the LXR, FXR receptor substrates?
Cholesterol
Bile acids
35
what is the effect of rifampicin on the combined pill?
It induces expression of liver enzyme resulting in more extensive clearance of the pill effects which reduces their effect on inhibiting ovulation
36
What is Rifampicin?
It is an antibiotic used to treat tuberculosis and leprosy
37
What is the effect of barbiturates on clearance?
Barbiturates and induce CYP’s that metabolise them which increases clearance so long-term use results into intolerance to clearance
38
how does theophylline impact metabolism?
it causes enhanced metabolism in smokers as well as people who eat barbecued food which can cause a rapid clearance of certain asthma treatments causing cardiac effects
39
what happens when drug metabolism is inhibited?
It often results into toxic levels of parent drug or a metabolite
40
how does the the CYP 450 reductase cause oxidation?
there is a small electron transport chain which uses NADPH2 to oxidise the iron centre of the CYP 450
41
what effect do both NADH and NADPH have on CYP metabolism?
When used together they give an increased reaction rate which involves cytochrome B5
42
What state is Fe3+ in when when a drug hasn’t bound?
hexavalent
43
what is the structure of the central complex in cytochrome P450?
It is a hexavalent shape with nitrogen surrounding Fe3+ with a H2O and cysteine residue
44
What is the peak of a cytochrome P450 when the water is unbound?
390nm
45
when does the peak at 450nm occur?
Only when carbon monoxide and electrons are present
46
what is the state when water is unbound to the Fe3+?
Pentavalent
47
what happens when a substrate interacts with the protein site in a CYP450?
It dictates specificity
Suggest he binding is less influential re-specificity
48
which kind of substrates bind to the iron group in CYP450?
normally nitrogen containing drugs or chemicals
Most of broad spectrum inhibitors
49
what do active CYPs require for activity?
Haem/O2
50
Where do non-specific CYP inhibitors bind?
The haem prosthetic group
51
Give one example of a non-specific CYP inhibitor
SKF525a
52
give examples of CYP isoform-specific inhibitors
CYP1A2 furafylline (bronchodilator)
CYP3C9 sulfaphenazole (sulfonamide antibacterial)
CYP2D6 quinidine (antiarrhythmic Na+ channel blocker)
CYP3A4 troleandomycin (macrolide antibiotic, inhibits bacterial protein synthesis)
53
give an example of a probe activity for CYP1A2
Methyl resorufin O-deethylase
54
Which inhibitors are important for P450 metabolism?
Reversible inhibitor:
Competitive
Mixed
Irreversible
55
what happens to the Km and Vmax when a reversible competitive inhibitor is added?
The Km increases
The Vmax stays the same
56
what happens to Km and Vmax when a reversible mixed inhibitors added?
Km increases
V-max decreases
57
what happens to Km and Vmax when an irreversible inhibitor is added?
Km and Vmax stay the same
58
What is an example of a reversible competitive inhibitor?
quinidine (CYP2D6)
59
How does competitive inhibition by another substrate work?
Drugs are administered together to compete for the active site
60
what is an example of competitive inhibition by another substrate?
Omeprazole and diazepam CYP2C19
61
what is mixed competitive /uncompetitive inhibition?
The full substrate is an added therefore by interacting with the prosthetic group further metabolism is inhibited
62
How do mixed inhibitors bind to CYP 450s?
They covalently bind as they often have a nitrogen group which binds to the Fe in the haem prosthetic group
63
how do metyrapone and ketoconazole interact?
They are mixed inhibitors therefore they compete to bind
64
what is the irreversible inhibitor also known as?
A suicide substrate as the compound is lost
65
How do irreversible inhibitors work?
They used a substrate which is metabolised to a reactive inhibitor, but is not an inhibitor itself
66
what happens to CYP concentration when the suicide substrate concentration is increased?
The CYP concentration decreases
67
what is an example of a suicide substrate?
Troleandomycin - macrolide antibiotic (CYP3A4)
68
What are examples of how CYP?3A4 can be inhibited?
Azole antifungal agent (ketoconazole)
Macrolide antibiotic (erythromycin)
Grapefruit juice (cardiac arrhythmia)
69
What is in grapefruit juice that causes inhibition in CYP enzymes?
Furanocoumarins
70
what is a clinical example of the therapeutic inhibition with disulphiram?
It inhibits the enzyme aldehyde dehydrogenase
Which causes a buildup of acetaldehyde if an individual drinks alcohol
it is used as an aversion therapy as it causes flushing, nausea and vomiting
71
is inhibition or induction more important in CYP450s?
Inhibition is more important as a mechanism as there are many more problems that can occur
72
What are the different problems that occur from inhibition of CYP 450 enzymes?
Two drugs can be metabolised by the same enzyme
The therapeutic range of one or both drugs is narrow E.G. Warfarin
One drug binds more strongly to the metabolising enzyme than the other
73
How can a drug metabolism be investigated?
finding if it is a typical mono oxygenation reaction
NADPH dependence
Production of metabolite inhibited by CO
Other general CYP inhibitors
Specific isoform inhibitors
Inhibitory antibodies
74
How can inhibitory antibodies be used to find CYP mediating metabolism?
they combined to the active site as they are specific so they may only recognise an enzyme substrate complex
75
what is a factor of metabolising enzymes in fetus and neonates?
there are low levels of these metabolising enzymes
76
what effect does bilirubin have on fetus and neonates?
lack of glucose leads to buildup of bilirubin, which causes brain damage
77
what happens when chlorophenol is administered to neonates?
they are unable to clear it as effectively which stops the respiratory tract meaning the babies go grey (grey baby syndrome)
78
what are the side-effects for when babies cannot metabolise specific drugs?
development of abdominal distension, vomiting, cyanosis, cardiovascular collapse, irregular respiration and death
79
how does metabolism of caffeine range in age groups?
Different enzymes are used to metabolism caffeine so in foetus, neonates and infants the most predominant hydroxylation is 8-hydroxylation however in adults 3–demethylation is used
80
which type of testing no longer uses animal clinical trials?
Testing with cosmetics
81
what is coumarin and where is it found?
it is a naturally occurring compound
It is found in a wide variety of plants, MOs and some animal species
High levels occur in essential oils, particularly cinnamon bark oil, cassia leaf oil and lavender oil
It is also found in fruits, green tea and other foods
Derivatives have been identified in plants, in both the free state and as glucosides
82
Which cosmetic products is coumarin used in?
Perfumes
Soap and detergent
Toothpaste
Tobacco products
Alcoholic beverages
83
why was coumarin banned in the US?
when used as a food flavour it was discontinued as a result of finding toxic effects in rats and dogs fed coumarin the diet
It was found that the hepatotoxicity led to tumour formation in the liver
84
why was coumarin found to be so toxic?
It was found that it would bind to DNA and protein
85
what is the toxic metabolite formed from coumarin?
Coumarin 3,4-epoxied
86
what is coumarin in mostly metabolised to in humans?
7-hydroxycoumarin
87
which CYP 450 allowed the metabolism of coumarin to 3,4-epoxide?
CYP1A/CYP2E
88
Why is coumarin not authorised in the EU?
as CYP2A6 exhibit polymorphisms therefore they may have little to no function of this enzyme allowing carcinogenicity and toxicity of coumarin metabolism
89
how does species variation impact phase II metabolism?
when phenol is conjugated the proportion that uses glucuronyl transferases and sulphotransferases differs from species to species
90
What are the differences between species and strains of species?
they can show differences in metabolism, which is usually due to genetic deficiency
91
how can AhR differ in mouse strains?
there can be different levels of activation by binding to TCDD which stimulates CYP1A1 at different times
92
which mouse strains have higher affinity for ligand binding in cytosol?
C57BI6
C57BI6 X DBA
F1 X C57BI6
93
which mouse strain has lower affinity for Ligand binding in cytosol?
DBA
50% F1 X DBA
94
How can strains of rodents show differences in metabolism due to genetic deficiency?
female DA rats: lacks rat equivalent of CYP2D6 and is unable to hydroxylate debrisoquine
gunn rat: unable to synthesise certain phenol glucuronides
95
Which hormones affect P-450 levels in rats?
Growth hormone
Oestrogen
Progesterone
Testosterone
Insulin
Thyroid hormone
Glucocorticoids
96
which enzymes do rats have differences in expression patterns?
CYP2A, CYP2B, CYP2C and CYP3A
97
what are the consequences of different expression patterns in metabolising enzymes in rodents?
Each isoform has a different specificity so there are different metabolism patterns between sexes (hormones and growth hormones)
98
how does growth hormone impact the expression levels of metabolising enzymes?
there are specific effects on CYP2C
Levels increase at puberty and upper regular expression of CYP2C7, 2C11, 2C12 AND 2C22
Female rats have continuous high levels inducing CYP2C7 and 2C12
Male rats have intermittent levels inducing CYP2C11 AND 2C22
99
What is a hypophysectomy?
it is the surgical removal of the pituitary gland
100
which metabolising enzymes do glucocorticoids induce in rats?
CYP3A family, natural forms may inhibit drug metabolism
101
Which diseases of the liver affect drug metabolism?
Alcoholic liver disease
Cirrhosis
Porphyria
102
In acute intoxication, what are the disadvantages when being converted to acetic acid?
there is a reduction in NAD as it is used up so supply becomes limited meaning it cannot be used for the citric acid cycle
103
What happens when there is a limited supply of NAD for the citric acid cycle?
there is a high NADH/NAD+ ratio which can inhibit the synthesis of UDP glucuronic acid
104
Which metabolising enzymes are interrupted by acute intoxication?
CYP2E1 - which also metabolites other drugs e.g. paracetamol
105
How does drug metabolism impact chronic alcoholics?
they have induced CYP2E1 which result in more rapid clearance e.e anaesthetics
105
what is porphyria?
It is an impaired synthesis of heme and an accumulation of toxic precursor
106
what effect does porphyria have on drug metabolising enzymes?
The levels may be lower due to limited supply of haem
Drugs e.g. barbiturates which induce CYP450s trigger increase synthesis of heat precursor triggering a clinical attack
107
What effect does inflammation have on metabolising enzymes?
inflammation has a general repressive effect on CYP450s expression and on drug metabolism
108
What is pharmacogenetics?
The study of variations in drug response between individuals that have hereditary bias
109
What are the different ways of having a genetic polymorphism?
Substitution (SNP), insertion or deletion
110
How often do polymorphisms approximately occur?
1 in 1000 bases of DNA
111
What are the properties of functional polymorphisms?
They have an effect on biological activity
Due to AA substitution, splice site change or effect in TXN factor binding
112
How can xenobiotic metabolism polymorphisms be analysed?
Phenotypically - measure enzyme activity using a probe and measuring metabolites
Genotypically - looking directly for the presence of mutation in DNA - need to know gene responsible (screening for)
113
Why is it important knowing the polymorphisms of xenobiotic metabolism?
Kinetics and dynamics
Safety - toxicology
Efficacy - response
Economic consideration (cost:benefit)
Therapeutic effect
114
What are the properties of the CYP2D6 enzyme?
Located on 22q13.2
9 exons
Metabolism of ~1/4 of all drugs
highly polymorphic
>100 alleles reported
115
What does metabolic ratio show?
It shows the different types of metabolisers in a population (slow, normal and ultra rapid)
116
What does it mean when someone is an ultra rapid metaboliser?
They have a duplicated gene of a normal allele
117
What does it mean when someone is an intermediate metaboliser?
They have 2 partially defective alleles or a defective and a null allele
118
What does it mean when someone is a poor metaboliser?
They have 2 null alleles
119
What are the common isoforms of CYP2D6 that are loss of activity?
CYP2D6*3, CYP2D6*4 and CYP2D6*5
120
What happens in CYP2D6*3 isoform?
There is a frameshift in the gene therefore it is non-functioning
121
What happens in the CYP2D6*4 isoform?
Alternative splicing site therefore no function
122
What happens in the CYP2D6*5 isoform?
Entire gene is deleted
123
How can people be ultra rapid metabolisers?
They can have multiple copies of the CYP2D6 gene adjacent to wt CYP2D6 which mean faster drug metabolsim
124
What is the most common isotope in europeans?
CYP2D6*4
125
What are the consequences of being poor or ultra rapid metabolisers?
Poor - failure to metabolise means greater risk of toxicity, cannot activate prodrugs
Ultra - poor response to antidepressants or may suffer toxicity to prodrugs
126
How can metabolising enzymes different activities be measured?
Using an activity score from 0-2.25
0 = poor
2.25 - ultra rapid
127
What are the contraindications of codeine use in children?
They can have severe respiratory depression if they are ultra rapid metabolisers
128
What is the problem when administering codeine for breast feeding mothers?
There can be increased morphine in the breast milk if they are ultra rapid metabolisers
129
What effect can the type of metabolisers have on codeine administration?
It can change the dosage required for the patient
UM - avoid codeine usage
PM - avoid codeine usage (diminished analgesia)
130
How can the different metaboliser status impact TCA antidepressants?
UM - avoid as less active compound in plasma so use of different antidepressant
PM - avoid as potential side effects may arise as higher plasma concentration so use different antidepressant
131
What are the properties of the NAT2 phase II enzyme?
Arylamine N-acetyltransferase 2
Chromosome 8p22
isoniazid acetylation
132
What is the status of individuals with normal NAT2 activity?
Fast acetylators
133
Which population lacks NAT2 activity?
Europeans - slow acetylators
134
What has happened to the gene in NAT2 slow acetylators?
They have 2 mutated copies of the gene
135
How can NAT2 activity be phenotyped?
Using caffeine or genotyping
136
Where are the mutations in NAT2 located?
T341C, G590A and G857A
137
What happens to people with mutated NAT2 and metabolism of isoniazid?
They have increased risk of hepatotoxicity
Fast show poor response with intermittent dosing
138
What is the consequence of slow acetylation of hydralazine?
It can cause drug induced systemic erythematosus
139
What is hydralazine?
It is an antihypertensive drug using vasodilation
mainly metabolised using NAT2
140
Where are the different NAT2 isoforms more prevalent?
NAT2*5 - 50% Europeans
NAT2*7 - East Asians
141
What is disadvantageous about biotransformation?
It is a major cause of toxicity
-toxic metabolites from parent drug
-metabolites can be metabolised to other toxic metabolites
142
What can inadequate assessment of metabolism lead to?
Adverse reactions in trials
Drug attrition rates (failure to reach clinical)
Drug recall even after approval
143
What can drug-drug interactions be impacted by?
Inhibition or induction of enzymes
144
What are the different ways of reaction phenotyping?
Finding major metabolites (toxicity and activity)
Enzymes involved in metabolism of parent drug and metabolites (proportion of metabolism, polymorphisms in enzymes, impact metabolism otherwise)
145
What are the different methods of phenotyping?
Hepatocytes - hard to come across
Microsomes
S9 fraction (cytosol and microsomes)
Recombinant enzyme systems
Animal models
Hepatic cell lines
In silico (AI for ideas on metabolism studies)
146
What are the most important studies for phenotyping?
Induction and inhibition studies
147
How can hepatocytes be used for phenotyping?
Assessment of compound passing through membrane
Cryopreservation required
Strategies for co-culturing
148
What are the advantages of hepatocyte models?
They are the gold standard, physiologically relevant
In-vivo like functions
149
Which cells are specifically assessed in hepatocytes?
Parenchymal
150
What are the disadvantages of hepatocyte models?
Limited sources
Cryo can result in loss of enzyme function
Culturing over long periods can result in loss of functionality
151
What are the advantages of microsome models?
Most widely used and basic
Pooled vs individual
Genotyped
Useful for CYPs and UGTs (Uridine 5'-Diphospho-Glucuronosyltransferases)
Easily derived from any organ
Cryopreserved - no loss in enzyme function
152
Why do activity levels vary in banks of human liver microsomes?
Genetic variation
Lifestyle differences (smoking)
Different personal lifetime exposure to inducers
153
How can P450s be measured in the microsomes?
Enzyme assays and immunoblotting
154
How can activity levels be measured with new compounds?
High-Performance Liquid Chromatography
155
What are the advantages of S9 fraction models?
Cytosolic and microsomal fractions
Almost same as hepatocytes for phase I and II enzymes
More representative
Soluble co-factors may be diluted
156
What are the most suitable systems for CYP450s MOs?
Microsome, S9 and hepatocytes
157
What are the most suitable systems for monoamine oxidase?
Hepatocytes
158
What are the most suitable systems for Flavin-containing MO?
Microsomes, S9 and hepatocytes
159
What are the most suitable systems for Alcohol and aldehydeDH?
S9 and hepatocytes
160
What are the most suitable systems for esterases?
Microsomes, S9 and hepatocytes
161
What are the most suitable systems for UDP-glucuronyl transferase?
Microsomes, S9 and hepatocytes
162
What are the most suitable systems for Phenol sulfotransferases?
S9 and hepatocytes
163
What are the most suitable systems for N-acetyl transferase?
Microsomes, S9 and hepatocytes
164
What are the most suitable systems for glutathione-s-transferases?
S9 and hepatocytes
165
What are the most suitable systems for membrane bound GST?
Microsomes, S9 and hepatocytes
166
What are the different heterologous recombinant systems?
E.coli, yeast, insect cells and mammalian cells
167
What are the different mammalian cells used for in vivo studies?
COS cells (monkey)
HepG2 cells
Human lymphoblastoid cells
V79 cells (Chinese hamster)
168
What is required for P450 expression?
Full length cDNA for isoform of interest (NADPH-P450 reductase, cytochrome b5)
169
What is transient expression?
Disappears over time
cDNA in nucleus no genomic integration
Gene not reproduced
Short period of expression
SHORT TERM
170
What is stable expression?
Maintained throughout cell lineage
Genomic integration of cDNA
Gene inherited through mitosis
Daughter cells express product
LONG TERM
171
What are the properties of using E.coli as an expression system?
After optimisation:
Amount of active enzyme is variable
Remove part of N-terminus making P450 soluble
172
What is required to use E.coli as an expression system?
Required optimisation:
cDNA N-terminal sequence modification
Change residue immediately after initial Met
Inc AT content 5' end
Add His residue at 3' end
173
What are the properties of using yeast as an expression system?
Endogenous P450 oxidoreductase
Some strains express human oxidoreductase
More postranslational modifications
Membrane organelles
Isolate P450-containing microsomes
Cheap and scalable
174
What are the issues of using E.coli as an expression system?
No internal membrane system
P450s have nothing to anchor to
Express>purify>reconstitute>investigate
175
What are the issues of using yeast as an expression system?
Already express own P450s
Difficult to isolate/investigate P450 of interest
176
What are the properties of using insect cells as an expression system?
High level, transient expression
More complex PT modification than bac/yeast
Cotransfect P450 and oxidoreductase
Baculovirus vectors are demanding
Higher cost and longer duration
Supersomes commercially available
177
Which non-P450s can get expressed into which systems?
E.coli: sulfotransferases, GSTs and other families
Insect cells: UGT and FMO (baculovirus)
178
Why are mammalian cells useful for P450 expression?
Toxicity studies: xenobiotics on cell survival, assessment of mutagenicity
More relevant and related to intracellular environment
Proper protein folding and PT modification and localisation
Similar control pathways
179
What are the properties of transient expression in COS cells?
Monkey kidney cells (P450 oxidoreductase)
P450 expression through cloning with promoter and transfecting cells (SV40 origin replication)
180
Which studies does transient expression limit?
In toxicology studies
181
How are P450 expressed in human lymphoblastoid cells?
G0 to G1 stimulation = immobilisation
Trasfected with cDNA
Treat with compound
Isolate metabolites
Investigate toxicity and mutagenicity
182
How can chemical inhibition be used for P450 enzymes?
Isoform specific inhibitors added to mix of human liver microsomes
Examples
Furafylline (CYP1A2)
Sulfaphenazole (CYP2C9)
Quinidine (CYP2D6)
Troleandomycin (CYP3A4)
183
How can antibody inhibition be used for P450 enzymes?
Polyclonal or monoclonal (better) antibodies can affect activity of isoforms
Preincubate antibody with human liver microsomes before adding substrate
184
What are the best ways of finding the specific P450 isoform?
Using recombinant P450 in purified form or microsomes
Inhibition studies using chemical or antibodies
185
How many donors are required for approval?
At least 10
186
How is CYP inhibition potential assessed?
Probe substrate/marker reaction experiments
187
What is high-throughput screening used for in CYP screening?
Gives initial idea of CYPs involved for further studies and ideas of methods
Less labour intensive
188
How can CYP induction potential be assessed?
qRT-PCR (measure mRNA produced) and reporter gene assays (used as marker)
189
What are the animal models used for drug metabolism studies?
Rodents
190
What do animal studies show in drug metabolism?
Benefits of whole body and toxicity
Species dependent differences in metabolism enzymes and transporters
Ethical concerns
191
What are humanised mice?
Specific CYPs are knocked in after knocking out mouse genes
Transplant human hepatocytes into immunodeficient mice after destruction of mouse liver
192
What are the different sources of human hepatocytes?
Primary human hepatocytes
Tumour cell lines (hepatoma)
Adult stem cells
hESC (embryonic stem cell)
iPSC (pluripotent stem cells) - less ethical concern
193
What is the most common hepatoma cell line studied?
HepG2
194
What are the disadvantages of using liver tumour cells?
They have altered metabolism - greater energy demand
195
What are the differences in metabolic activity in tumours and hepatocytes?
Significantly lower metabolic activity
Not recommended for studies where appropriate drug metabolism is important
Stable or transient expression of CYP/drug metabolising enzymes
196
What can in silico methods be used for?
Predict regioselectivity
Predict metabolites
Predict interactions of drugs with metabolising enzymes
Predict toxicological effects of metabolites
197
Why are in silico methods used?
Cost effective, high throughput
Only supplement and support biological assays
