1
Q
What are the two processes that determine drug concentrations
A
- translocation of drug molecules
- chemical transformation
2
Q
how do drugs move around the body
A
- bulk flow (in the bloodstream)
- diffusional transfer (molecule by molecule over short distances)
3
Q
why does diffusional transfer differ with the chemical nature of the drug
A
- hydrophobic diffusion barrier
- aqueous diffusion
4
Q
what is a compartmentalised body
A
- body made of interconnected compartments separated by cell membranes
- the ability of drugs and other chemicals to move between these compartments depends on the selectivity of the membranes and the chemical properties of the drug
5
Q
what is the vascular endothelium
A
- acts as a filter (cut off MW 80-100k)
- the gaps between cells are filled with a protein matrix - tight packed - this acts as MW filter
6
Q
why is the endothelium discontinuous in liver and spleens
A
- large fenestrations allows drugs to exchange freely between blood and interstitium in the liver
7
Q
what is the structure of endothelial cell in liver
A
- endothelium
- basement membrane
- slit junctions
8
Q
structure of CNS and placenta
A
- astrocyte foot processes - lipophilic barriers
- tight junctions
- slit junctions
- basement membrane
9
Q
what is the importance of the structure of the barriers in CNS and placenta
A
- charged drugs cannot move through
- lipid soluble and carrier mediated transport can be used to move from plasma to CNS
10
Q
how can drugs be transferred across cell membranes
A
- diffusing direct through lipid
- diffusing through aqueous pores - most drugs are too big to move through
- combination with a transmembrane carrier protein
- pinocytosis (for macromolecules e.g. insulin
11
Q
how do drugs diffuse through lipid
A
- non-polar substances dissolve readily in non-polar solvents - cell membranes are lipid-rich environments
12
Q
how is permeability coefficient determined
A
- the number of molecules crossing the membrane per unit area of time (J)
- concentration difference across the membrane (delta C)
- J = P x delta C
13
Q
what are the physiochemical factors contributing to permeability
A
- partition coefficient
- diffusion coefficient
- diffusion coefficient is equal to 1/square root MW
14
Q
what is the relationship between lipid solubility and permeability
A
- close correlation between lipid solubility and permeability of cell membrane to different substances
- lipid solubility an important determinant of pharmacokinetic characteristics of a drug
- rate of absorption from the gut, penetration into brain and other tissues, and extent of renal elimination can be predictable
15
Q
how is pH and ionisation involved in absorption
A
- ionised drug formed are much less able to penetrate cell membranes
- ratio of charged drug/uncharged drug concentrations is determined by the pH of the compartment
16
Q
how is lipid solubility involved in absorption
A
- the lipid solubility of uncharged species AH or B depend on the chemical nature of the drug
- for many drugs the uncharged species is sufficiently lipid soluble (except e.g. aminoglycosides0
- bases are neutral and absorbed easily
17
Q
what does ionisation effect
A
- drug permeability across membranes
- ionised drugs show reduced permeability
- steady state distribution of drug molecules between aqueous compartments, in the presence of a pH mechanism
18
Q
what is the pH partition mechanism
A
- qualitative effects of pH changes in different body compartments on the pharmacokinetic of weak acids and bases
- but its not the main determinants of drug absorption from the GI tract
- small intestine has hugely greater surface area of absorption compared to stomach
19
Q
why does aspirin absorption vary
A
- aspirin absorption increased by metoclopramide and decreased by propantheline
- ionised forms are not totally impermeable
- body compartments are rarely at equilibrium in real life
20
Q
what are the consequences of the pH partition mechanism
A
- urinary acidification increases excretion of weak bases and decreased that of weak acids
- urinary alkalisation decreases excretion of weak bases and increases that of weak acids
- increases plasma pH (e.g. sodium bicarbonate) causes extraction of weakly acidic drugs from CNS into plasma
- decreases plasma pH (e.g. acetazolamide) causes weakly acidic drugs to accumulate in the CNS
21
Q
what is the structure of the nephron
A
- distal tubule collecting duct (control Na and H2O balance)
- glomerulus (renal blood flow filtration
- loop of hence (urinary concentration
22
Q
how does urinary alkalinisation increase excretion of aspirin
A
- at normal urinary pH, a proportion of salicylate is unionised and can be absorbed back into the systemic circulation in the nephron
- when urine is alkaline, salicylate is charged, so reabsorption is much reduced
23
Q
what is carrier mediated transport
A
- transport of physiologically important molecules in and out of cells (e.g. sugars, amino acids, neurotransmitters and metal ions)
24
Q
what are the examples of carrier mediated transport
A
- passive transport - move molecules in direction of electrochemical gradient
- active transport - movement against an electrochemical gradient coupled to an energy source
25
what are the properties of carrier mediated transport
- saturable
- can be inhibited
26
why is renal handling of cisplatin important
- cisplatin is very nephrotoxic because it is efficiently taken up into the proximal tubule, but rate of secretion is lower
- higher exposure results in destruction of mitochondria - proximal tubular cell death
- organic solutes lost to urine increased Na loss increases H2O loss - falcon syndrome
27
what are the carried mediated transport systems
- levodopa - transported by the carrier responsible for phenylalanine
- fluorouracil - transported by carrier for natural pyrimidine - thymine and uracil
- iron - carrier system in jejunum
- calcium - vitamine D dépendent carrier system
28
where in the body are carrier mediated transporters essential
- the renal tubule
- the biliary tract
- the blood brain barrier
-the GI tracts
- p-glycoprotein - transporter responsible for MDRa
29
where else are carrier mediated transporters present
- renal tubular brush border
- membranes, in bile canaliculi, in astrocyte foot processes in brain micro vessels and in GI tracts
30
what are the other factors affecting drug pharmacokinetics (distribution and elimination)
- binding to plasma protein
- partition into body fat and other tissues
31
what are the routes of drug administration
oral
sublingual
rectal
application to other epithelial surface (e.g. skin, cornea vagina and nasal mucosa)
inhalation
injection
32
what are the types of injection
subcutaneous
intramuscular
intravenous
intrathecal
33
factors affecting GI absorption of drugs
GI motility
splanchnic blood flow
particle size and formulation
physiochemical factors
34
what does gastrointestinal motility have an effect on
migraine, diabetic neuropathy
malabsorption states and GI diseases
coeliac disease
drugs
food
35
how does coeliac disease effect gastrointestinal motility
- thyroxine anddigoxin absorption decreased
- propranolol, cotrimoxazole and cephalexin absorption increased
36
what are the physiochemical factors that effect drug GI absorption
- tetracycline and calcium ions
- bile acid binding resins (e.g. cholestyramine) interact with warfarin, thyroxin
37
what are the drugs not absorbed GI
- vancomycine (used for treatment of pseudomembranous colitis caused by clostridium difficile)
- mesalazine (a formulation of 5-aminosalicyclic acid) for treatment of crohns disease)
- olsalazine (a dimer of two 5-aminosalicyclic acid) cleaved by colonic bacteria
38
what is systemic availability
the amount of drug that reaches systemic circulation intact
39
what does the rate of systemic availability rely on
- pharmaceutical factors
- gastrointestinal absorption
- pre systemic metabolism relatively unimportant
40
what does the extent of systemic availability depend on
both the extent of absorption and the extent of pre-systemic metabolism
41
what is cutaneous drug administration
- skin allows permeation of drugs from topically applied creams and ointments in quantities sufficient to produce systemic action
42
what are the problems with transdermal drug delivery
- variability in drug administration through skin
- resulting in lack of precision regarding the real dose absorbed
43
what are the therapeutic objectives of transdermal delivery
- lack of drug dosage a characteristic of topically applied ointment and creams
- ointments containing nitroglycerin require multiple applications per day
- variable amount and duration of drug input
44
why is there variable amount and duration of transdermal drugs
- differences in the area of skin covered with ointment
- thickness of the ointment layer applied
45
what are the new topical dosage forms
- delivery drugs to the blood stream at defined rates over prolonged period of time
- design emphasis on control of systemic input residing in the dosage form rather than in the skin
46
why is transdermal therapy particularly suitable for the rate controlled administration
- as potent, non-irritating, non-allergenic agents with suitable physiochemical properties whose current administration causes some problems
47
what are some of problems of ointments
- troublesome side effects or unreliable therapeutic action with repetitive dosing with conventional dosage forms
- patient compliance difficulaties
- need for frequent dosing in conventional dosage forms because of the drugs short biological half life
- gastric irritations with oral therapy
48
what is the scopolamine system
- application to skin
- drug diffusion in the direction of concentration gradient
- energy source (the difference in the drugs chemical potential between the reservoir and the systems exterior)
- constancy of rate delivered assured as long as drug present in excess in reservoir
49
what is intravenous drug administration
- fastest and most certain route
- single bolus injection with high concentration of drugs
- peak drug concentration reaching tissues is critically dependent on the rate of injection
- steady IV infusion avoids high peak plasma concentrations (e.g. lignocaine, propofol, diazepam)
50
what is the benefits of subcutaneous and intramuscular drug injections
SC and IM injections usually produce faster effects than oral administration
51
what does the rate of absorption of SC and IM injections depend on
- the site of injection
- local blood flow
- drug formulations
52
what are intrathecal injections
injections into subarachnoid space via a lumbar puncture
53
what are the examples of intrathecal injections
- methotrexate for treatments of childhood leukaemia
- local anaesthetics (e.g. bupivacaine)
- opiate analgesics
- baclofen - for treatments of muscle spasm cause by chronic neurological disease
- some antibiotics (ahminoglycosides) - treatments of nervous system infection
54
what diseases require drug administration by inhalation
asthma
bronchitis emphysema
lung cancer
respiratory diseases
55
why are drugs administered by inhalation
rapid action - rapid delivery across mucous membranes of the respiratory tract and pulmonary epithelium
minimise systemic absorption
minimise side effects (beta-agonists, glucocorticoids
56
what is asthma
- inflammation (swelling)
- mucus production (snot)
- bronchospasm (muscle tightness)
57
what are the symptoms of asthma
- shortness of breath
- wheezing
- tightness in the chest
- coughing at night or after physical activity
- waking at night with asthma symptoms
58
what are the indoor triggers of asthma
strong smells
cockroaches
smoke
dusty mites
furry friends
colds
mould
59
what are the outdoor triggers for asthma
cold/hot weather
car exhaust
pollens
exercise
mowed lawn
air pollution
grilling
60
How to treat the symptoms of asthma
- adrenergic agonists (bronchodilators) and glucocorticoids (usually by inhalation)
- beta agonists (e.g. pirbuterol, terbutalines, albuterol and salmeterol)
- relax airway smooth muscle directly
- provide relief for 4-6hours
- little stimulation of alpha or beta1 receptors
61
what is the mechanism of action of glucocorticoids
- inhaled glucocorticoids reduce/eliminate the use of oral glucocorticoids
- no direct effect on airway smooth muscle
- decrease the number and activity of cells involved in airway inflammation
- prolonged inhalation of steroid reduced hyper-responsiveness of the airway smooth muscle
62
what are the pharmacokinetics of inhaled drugs
- a large fraction deposited in the mouth and pharynx or swallowed
- many of the clinically useful corticosteroids (e.g. beclomethasone, triamcilonone) undergo extensive 1st pass metabolism
- therefore a small amount reaches the systemic circulation which minimises adverse effects
- 10-20% of inhaled dose reaches the airway
63
what is theophylline
- a potent bronchodilators
- narrow therapeutic window
- overdosing can lead to seizures and cardiac arrhythmias
- interact with many other prescribed drugs
64
what are the other inhaled drugs for treatment of asthma
- sodium cromoglycate and nedocromil sodium
- effective prophylactic anti-inflammatory agents
- but not useful in managing acute attacks of asthma - not direct bronchodilators
- theophylline
65
what is the main benefit of prolonged release inhalation formulations
- current formulations have short duration of clinical effects therefore advantageous to prolong pharmacological effect
66
what factors affect bioavailability of drugs
- the rate of disintegration of the tablet
- the rate of dissolution of the drug particles in the intestinal fluid
67
what are the physical factors affecting pharmaceutical availability
- tablet compression and excipients - affect the rate of tablet disintegration
- other tablet excipients - affect interaction with aqueous GI juices
- the form of the drug e.g. crystalline or salt form
- particle size - smaller drug particles dissolve more quickly
68
what is bioequivalence
- two formulations of a drug are bioequivalent if they show comparable bioavailability and similar times to achieve peak plasma concentrations
69
what is bioinequivalence
- two formulations of a drug with a significant difference in bioavailability are said to be bioinequivalent
70
what is therapeutic equivalence
two similar drugs are therapeutically equivalent if they have comparable efficacy and safety
71
what is the importance of alterations in pharmaceutical availability
- important for drugs with narrow therapeutic index
- switch from a formulation of low pharmaceutical availability to a formulation of high pharmaceutical availability
72
what are the determining factors for route of administration of drug
- therapeutic objective (slow or fast onset of action)
- properties of the drug
73
how can we control insulin absorption
- physical state - crystalline or non-crystalline
- the zinc or protein content
- the nature and pH of the buffer suspension
74
what are the types of subcutaneous injections of insulin
- insulin BP - soluble and amorphous - rapid onset and short duration of action
- ultralente insulin (large crystals of insulin and high zinc content - suspended in a solution of sodium acetate/sodium chloride - onset of action approx. 7hrs and duration of action of 36hrs
75
what are the examples where the vehicle in which the drug is suspended can affect the drug diffusion rate
- the absorption of drug from IM injection site may be retarded by the use of thick oils which slow down diffusion
- vasopressin tannate in oil - diabetes insidious
- fluphenazine decanoate in oil - schizophrenia
76
what are the examples where plasma drug concentrations differ between IM and oral dosing of the same drug
- phenytoin - plasma drug concentrations after IM injections are half of those after oral dosing
- chloramphenicol - also poorly absorbed after IM injection
77
what is the relation between diabetes insidious and lack of ADH (vasopressin)
- ADH (vasopressin) inserts water pores into collecting duct membrane to enable re-absorption of solute-free water from the collecting duct into the systemic circulation
- vasopressin binds to membrane receptor
- receptor activates cAMP (second messenger system)
- cell inserts AQP2 water pores into apical membrane
- water is absorbed by osmosis into the blood
78
why are local injections used
- some formulations of local anaesthetics contain adrenaline
- vasoconstriction at site of injection
- prevent the drug to be carried away by circulation from site of injection
- prolongs the effect of local anaesthetic
79
why use sublingual, buccal, rectal and transdermal formulations
- avoidance of 1st pass metabolism - resulting in rapid therapeutic effect
- corticosteroids can be given by the rectal route for direct effect on the large bowel
80
what is the example of the benefit of avoidance of 1st pass metabolism
- glyceryl trinitrate 10x less of dose required for therapeutic effect compared to local dosing
81
what are the step that allow corticosteroids to be given by the rectal route
- drug administration via transdermal patches
- controlled release of small amount of drug over a period of time
- e.g. glyceryl trinitrate, hyoscine, oestradiol
82
what are the benefits of controlled release of drug
- reduce the risk of gastric erosions - e.g. quinidine
- ideal for drugs with short duration of action e.g. theophylline and nifedipine
- unconventional formulations are not always useful e.g. beta-antagonists show good duration of effect from conventional formulation
83
what is the criteria that needs to be met for combination products in oral therapy
- the frequency of administration of the two drugs is the same
- the fixed doses in the combination product are therapeutically and optimally effective
84
what are the potential advantages of combination formulations
- improved compliance
- ease of administration
- synergistic or additive effect
- decreased adverse effects
85
What is an example of improve compliance
- antituberculous drugs (rifampicin and isoniazid)
- ferrous sulphate and folate acid (pregnancy)
86
what is an example of ease of administration
triple vaccine (diphtheria, tetanus, pertussis)
87
what are the examples of synergistic or additive effect
- e.g. trimethoprim and sulphonamides (e.g. co-trioxazole)
- amoxycillin and clavulanic acid (co-amoxiclav)
- aspirin and codeine (simples analgesia)
- paracetamol and metoclopramide (migraine)
- combines oral contraceptives (oestrogen and progesterone)
88
what is the example of decreased adverse effects
- e.g. -dopa and decarboxylase inhibitors (Parkinson's)
- diuretics (potassium-wasting and potassium sparing)
89
what are the types of special drug delivery systems
- biologically erodible microspheres - loaded with drugs
- pro drugs - cyclophosphamide, levodopa, zidovudine
- antibody-drug conjugates - cancer chemotherapy
- packaging in liposomes
- gene therapy - viral vector for gene delivery
- implantable devices
90
when are special drug delivery systems used in clinical application
- brentuximab vedotin - directed to the protein CD30, which is expressed in classical Hodgkins lymphoma
- trastuzumab emptansine (Herceptin) - binds to the HER2/neu receptor - treatment of HER2-positive metastatic breast cancer
91
what is packaging in liposomes
- phospholipids loaded with non-lipid soluble drugs
- reticuloendothelial cells in the liver - also concentrated in malignant tumours - selective delivery
- e.g. amphotericin - treatments of mycosis - less nephrotoxic and better tolerated
- Pfizer SARS cov2 vaccine - mRNA packaged in liposome
92
what is drug distribution
process by which a drug reversibly leaves the bloodstream and enters the interstitial (extracellular fluid) and/or the cells of the tissues
93
what does the delivery of a drug from plasma to the interstitial depend on
- blood flow
- capillary permeability
- the degree of binding of the drug to plasma and tissue protein
- the relative hydrophobicity of the drug
94
what does blood flow to tissue capillaries depend on
blood flow to tissue capillaries varied widely as a consequence of unequal cardiac output to various organs
95
what is the direction of blood flow to the organs
- blood flow to the brain, liver, kidney --> skeletal muscle --> adipose tissue
96
what does capillary permeability depend on
- capillary structure
- drug structure
97
what is the capillary structure
large fenestrations allow drugs to exchange freely between blood and interstitium in the liver
98
how do drugs interact with blood-brain barrier
- drugs must pass through the endothelial cells of the capillaries of CNS or be actively transported e.g. levodopa
- lipid soluble drugs readily penetrate the CNS
- ionised or polar drugs unable to pass through the endothelial cells of the CNS
99
how does hydrophobicity affect drug movement
- hydrophobic drugs readily cross cell membranes
- major factor in distribution of hydrophobic drug is blood flow to the area
- hydrophilic drugs must go through slit junction
100
what are the characteristics of binding drugs to proteins
- reversible binding to plasma proteins sequesters drugs in non-diffusible form
- binding it non-selective as to chemical structure
- binding sites for drugs similar to those for bilirubin
- plasma albumin conjugate --> free drug --> metabolism --> excretion
101
what is volume of distribution
- Vd is a hypothetical volume of fluid into which the drugs is disseminated
- has no physiological or physical basis
102
what is the plasma compartment
- drug has a large MW or binds extensively to plasma protein
- too large to move out through slit junctions of capillaries therefore drug trapped within the plasma compartment
- Vd = plasma water
103
how does the low molecular impact drug movement
- drug has a low MW but is hydrophilic
- moves through slit junctions into interstitial fluid
- unable to cross lipid membrane of cells and enter intracellular fluid
- Vd = sum of plasma water and extracellular fluid volume
104
what is the calculation for Vd
plasma water + extracellular + intracellular volumes
105
why are bound drugs pharmacologically inactive
- unable to reach target site and elicit a biological response
- by binding to plasma proteins - the drug is effectively trapped
106
what is the binding capacity of albumin
- binding of a drug to albumin is reversible
- low capacity - one drug molecular per albumin molecule
- high capacity - several drug molecules binding to a single albumin molecule
107
what is the binding affinity of drugs to albumin
- albumin has strongest affinity for anionic and hydrophobic drugs
- most hydrophilic and neutral drugs do not bind to albumin
108
what is the competition for binding between drugs
- drugs with high affinity for albumin can compete for available binding sites
- drugs with high affinity for albumin can be divided into two classes
- dependent on dose of the drug > or < than the binding capacity of albumin
109
what are class I drugs
- dose of drug < binding capacity of albumin
- dose/capacity ratio is low, therefore binding sites in excess of the available drug
- most drug molecules are bound to albumin and concentration of free drug is low
110
what are class 2 drugs
- dose of drug > the number of albumin binding sites
- dose/capacity ratio is high, most albumin molecules contain a bound drug; the concentration of free drug is significant
- a relatively high proportion of drug exists in free state
111
what is the clinical importance of drug displacement
- e.g. interaction between tolbutamide (class I drug: 95% protein bound), and sulphonamide antibiotic (class II drug)
- displacement of class I drug occurs when a class ii drug is administered simultaneously
- rapid increase in free fraction of tolbutamide in plasma
- a new equilibrium will be reached
112
what is the relationship between drug displacement and Vd
- impact of displacement dependent on both Vd and the therapeutic index
- if Vd is large, then change in free drug concentration is insignificant
- if Vd is small, the newly displaced drug does not move into the tissue as much
- increase in plasma drug concentration is more profound when Vd is small
113
what is the Vd when high MW and/or protein bound drugs
trapped in plasma, low Vd
114
what is the Vd with low MW, hydrophilic drugs
Vd = plasma water + interstitial fluid
115
what is the Vd with low MW, hydrophobic drugs
Vd is high
116
what is xenobiotic metabolism
process by which foreign compounds are metabolised in the body to facilitate their elimination
117
what are the sites of metabolism
- liver - main site of metabolism
- GI tract
- kidney
- skin
- lungs
- plasma - hydrolysis
- brain
118
what is the liver function
- detoxicification
- metabolism of carbohydrates, lipids and proteins
- synthesis of plasma proteins
- storage of glycogen, vitamins and minerals
- bile products
119
what are the cell types of the liver
- hepatocytes
- cholangiocytes
- Kupffer cells
- stellate cells
- endothelial cells
- fibroblasts
120
what is the liver structures
liver
liver lobules
lobule
121
what is in the liver lobule
- hepatocytes
- central vein - feeds into hepatic vein
- sinusoid
- branch of hepatic artery
- branch of portal vein
- bile ductile
122
what is liver regeneration
- damaged liver cells can be replaced by liver regeneration
- limited process - repeated damage and exhaustion of regeneration leads to fibrosis and cirrhosis
123
what causes the need for liver regeneration
exposure to xenobiotics can stress and damage the liver - more likely to be damaged buy reactive metabolites
- hepatocyte proliferation
- liver stem/progenitor cell
124
where are microsomal enzymes found
- smooth ER is the major site of drug metabolising enzymes
- smooth ER are high in abundance in hepatocytes
125
where are non-microsomal enzymes found
cytosol
mitochondria
126
what reaction are included phase 1 of drug metabolism
most mediated by cytochrome P450 enzymes
oxidation and reduction reactions
127
what reaction are included in phase 2 drug metabolism
conjugation reactions
128
what happens during phase 1 metabolism
functionalisation reactions where a functional group is introduced normally producing a more polar excitable molecule
129
which molecules require phase 2 metabolism before they can be excreted
esterases
oxidases
epoxide hydrolyses
dehydrogenases
130
what is the structure of cytochrome P450
haem proteins - 57 Human CYPs
distinct but overlapping substrates
131
what reactions are cytochrome P450s involved in
genetic polymorphism
genotype-phenotype relationship
subject to induction and inhibition
drug-drug interactions
132
what is included in cytochrome P450 induction and inhibition
- CYP3A4 - responsible for metabolising 50% of drugs
- wide substrate specificity
- most abundant in liver and gut
133
what is an examples of induction of cytochrome P450
St johns wort - induction of CYP3A4 and P-gp
134
what is an example of CYP450 inhibition
grapefruit juice - irreversible inhibition of CYP3A4
135
what happens during phase 2 metabolism
conjugation reactions which detoxify compounds and prepare them for excretion
136
why do some drugs not require phase 1 metabolsism before phase 2
when suitable functional groups for conjugation are already present on the molecules
137
what are the examples of types of drugs that don't require phase 1 metabolism before phase 2
glucuronyl
sulphase
methyl
acetyl
138
what are the enzymes involved in phase 2 metabolism
UDP-glucuronosyltransferases
sulphontransferases
glutathione S-transferases
N-acetyltransferases
methyltransferases
amino acid conjugating enzymes (e.g. glycine and glutamic acid)
139
what is the relationship between first pass metabolism and bioavailability
first pass metabolism in the liver and gut wall reduces the bioavailability of drugs given PO
140
What are the factors that can affect first pass metabolism
- genetic variations between individuals in the liver and GI
- variations between individuals in the liver and GI blood flow
- gut mitochondria
141
what are the characteristics of prodrugs
increase solubility
high first pass metabolism
instability
poor absorption
target drug to specific sites
taste
pain at site of administration
improve PK
reduce toxicity
142
how are prodrugs administered
administered as an inactive form and require metabolism to become active
143
what are the examples of pro drugs
diacetyl-morphine
codeine
cyclophosphamide
144
what are cytochrome P450 enzymes
- most important family in phase 1 metabolism
- contain single haem molecule as prosthetic group
145
where are cytochrome P450 enzymes found
endoplasmic reticulum
mitochordria
146
what is the function of cytochrome P450 enzymes
forms complex that shows maximum absorbance at 450nm when reduced and CO added
147
what is the cytochrome P450 nomenclature
use root CYP followed by family, subfamily and form number
sometimes include allelic variants
148
what are the functions of cytochromes P450
- xenobiotic metabolism
- steroid, fatty acid and vitamin oxidation
- steroid biosynthesis
149
where does xenobiotic metabolism occur
occurs in endoplasmic reticulum
150
where are the cytochromes P450 for steroid biosynthesis found
mitochondria
151
what are the biochemical properties of cytochromes P450
- monomeric proteins of molecular weight 40 000 to 50 000
- all contain haem as a prosthetic group
- regions of the protein concerned with haem and oxygen binding tend to be conserved but area important for substrate binding vary more in sequence
- crystal structures of most human P450s relevant to xenobiotic metabolism not available
152
what is the cytochrome P450 reaction
DH + NADPH + O2 --> DOH + NADP + H2O
153
What is the function of NADPH in the cytochrome P450 reaction
- NADPH supplies 2 protons and 2 electrons in an electron transfer process
- interacts with cytochrome P450 enzyme by electrostatic interaction involving carboxyl groups on the reductase and amino groups on the cytochrome P450
154
what is the name of the protein used for electron transfer
flavoprotein containing a prosthetic group flavin adenine dinucleotide and flavin mononucleotide
155
what is the name of the protein used for mitochrondrial P450s electron transfer
adrenodoxin
156
what is induction of cytochrome P450
- induction represents an increase in the amount of mRNA and protein present
- different inducers have the ability to induce particular isoforms
- inducers include polycyclic aromatic hydrocarbons and barbiturates
157
what are the effects of cytochrome P450 mediated reactions
- inactivate many drugs and increase their rate of excretion
- activate P450-mediated reactions
- produce toxic molecules from certain harmless drugs e.g. paracetamol
- activate products e.g. cyclophosphamide
158
what is the tissue distribution of cytochrome p450
- found at highest levels in liver
- detectable but generally at lower levels in kidney, lung, intestine, adrenals and brain
- some forms are detected mainly in extra hepatic tissue e.g. CYP1A1, some steroid biosynthetic P450s
159
what are the examples of reactions catalysed by P450s
- N-dealkylation e.f. imipramine, diazepam, codeine, erythromycin, morphine, tamoxifen, theophylline
- O - dealkylation e.g. codeine, indomethacin, dextramethorphan
- aliphatic hydroxylation e.g. tolbutamide, ibuprofen, pentobarbital, meprobamate, cyclosporine, midazolam
- aromatic hydroxylation e.g. phenytoin, phenobarbital, propanol, phenylbutazone, ethinyl estradiol
160
what are the cytochrome P450 isoforms
- each is the product of a different gene
- they tend to show distinct substrate specificities but there is considerable overlap
- normally essential to know which P450 metabolites a new drug before it is given a license for therapeutic use
161
what is the importance of P450s in drug metabolism
- 70-80% of all drugs subject to metabolism are P450 substrates
-almost 90% of these are metabolised by CYP3A4, CYP2D6 and CYP2C9
162
what is the content of p450 isoforms in human liver
CYP3A
CYP2C
CYP1A2
CYP2E1
CYP2A6
CYP2D6
CYP2B6
163
how do you identify specific P450 isoforms responsible for metabolism of a drug
- correlation analysis using human liver microsome bank
- inhibition studies e.g. chemical inhibition or antibody inhibition
- studies using purified or expressed enzymes
164
what is CYP2D6
- position of oxidation is usually 5 to 7 A from the basic nitrogen
- some of the population lack this enzyme
- most substrates are either cardiovascular agents, antipsychotics or antidepressants
- hydroxylation reactions common
- not inducible
165
what is CYP2C9
- substrates have areas of strong hydrogen bond forming potential or ion pair formations 5 to 10 A from the site of metabolism
- substrates include a variety of different drug types but especially NSAISd
- subject to genetic polymorphism - some individuals show low activity due to amino acid substitutions
- inducible by barbiturates and rifampicin
166
what is CYP3A4
- highly inducible by glucocorticoids, rifampicin and other compounds
- considerable structural diversity in substrates unlike CYP2D6 ad CYP2C9. binding site can accomodate large molecules and majority of binding seems to involve hydrophobic interactions
- N-dealkylation reactions particularly common but also see aromatic hydroxylation
- effects of genetic polymorphism more limited than for many other P450s
167
what are the other P450 isoforms important for drug metabolism
CYP2C19
CYP1A2
CYP2E1
CYP2B6
CYP2C8
CYP2A6
168
what are the factors determining metabolism by specific P450 isoforms
- topography of active site of enzyme
- degree of steric hindrance of access of Fe-O complex to possible sites of metabolism in substrate
- ease of electron or hydrogen abstraction from various carbon or heterozygous atoms of the substrate
169
what is the role of CYP1A1
major role in activation of polycyclic aromatic hydrocarbons. this enzyme is found mainly outside the liver and is induced by some substrates
170
what is the role of cyp2e1
contributes to ethanol metabolism - auto induction
171
what is the role of CYP1A2
can activate aryl amine compounds to carcinogens and has an important role in caffeine metabolism
172
what are the non P450 reactions involved in phase 1 metabolism
- oxidation reactions e.g. flavin-linked monooxygenases, prostaglandin H-synthase-dependent co-oxidation, amine oxidases, oxidoreductases
- hydrolysis e.g. esterases, epoxide hydrolases
173
what are flavin linked monooxygenases
- smaller but similar to the cytochrome P450 family
- requirement for NADPH and oxygen
- overlap in substrate specificity with certain P450 enzymes but often yield distinct metabolites
174
what are the properties of FMO
- oxidises nucleophilic nitrogen, phosphorus or sulphur centres
- contain the flavin as prosthetic group
- located in the endoplasmic reticulum fraction
- 5 different isoforms
- most isoforms are thought to be non-inducible, but an exception is FMO5, which is induced by rifampicin in human hepatocytes
175
which FMO species is found in the human liver
FMO3
176
which enzymes are associated with peroxidase activity
- prostaglandin H-synthase
- myeloperoxidase
- lactoperoxidase
177
what is prostaglandin H-synthase
- involve co-oxidation of arachinodonic acid to a prostaglandin and a xenobiotic to its oxidised metabolites
- has cyclooxygenase activity
- has peroxidase activity
- co-oxidation of xenobiotic to metabolites
178
what is the oxidation reaction of PGHS
arachidonic acid --> PGG2
179
what is the reduction reaction of PGHS
PGG2 --> PGH2
180
what makes PGHS different to P450/FMO
O atom transferred from xenobiotic is not derived directly from o2 and NADPH is not a cofactor
- for examples high doses of paracetamol can be converted to the toxic N-acetylquinoneminie in the kidney
181
what is monoamine oxidase (MAO)
- a mitochondria enzyme which oxidises by a FAD-dependent pathway
- main role of MAO is in metabolism of neurotransmitters but also oxidises some drugs
182
what is the reaction mechanism of MAO
- RCH2NH2 + FAD --> RCH=NH + FADH2
- RCH=NH + H2O --> RCHO + NH3
- RADH2 + O2 --> FAD + H2O2
183
How dos propranolol a MAOA
propranolol --> N-desisopropyl propranolol --> aldehyde intermediate --> naphthoxylactic acid
184
what are the enzyme examples of oxidoreductases (dehydrogenases)
- alcohol dehydrogenase
- aldehyde dehydrogenase
- carbonyl reductase
- NAD(P)H quinone oxidoreductase
they are all cytosolic except aldehyde dehydrogenase
185
what are the reactions of alcohol and aldehyde dehydrogenases
- RCH2OH + NAD+ --> RCHO + NADH + H+
- RCHO + NAD+ --> RCOOH + NADH + H+
186
what happens during alcohol dehydrogenase
normal method of ethanol detoxification
- CYP2E1 uses excess ethanol as a substrate
NAD+ required as a co-factor
coverts alcohols to carbonyls
187
what is an example of alcohol dehydrogenase
ethanol --> acetyaldehyde
188
what is the use of carbonyl reductase
catalyses the reduction of a wide variety of carbonyl compounds including
- quinones
- prostaglandins
- menadione
- various xenobiotics
189
what is the general reaction form of carbonyl reductase
R-CHOH-R' + NADP+ --> R-CO-R' + NADPH + H+
190
what happens during quinone oxidoreductase 1
- two electron reductase, catalyses the reduction of a broad range of substrates
- reduced quinones directly to hydroquinones, without semiquinoane as an intermediate
- important in detoxification of quinones, quinone-mines and ago compounds
- scavenges ROS directly
191
where are esterase's found
found in plasma as well as liver and other tissues - cytosolic and microsomal forms
192
what are the different forms of esterase's
- butryrylcholinesterase - metabolises aspirin
- carboxylesterases 1 - metabolises cocain and heroin
- carboxylesterases 2 - metabolises procaine
- paraoxonase - metabolises aryl esters, oxon form of organophosphorus compounds
193
what are carboxylesterases
- catalyse the hydrolysis of a wide range of chemicals (esters, thiesters and amides)
- CE1 prefers substrate with a small alcohol group, large acyl group
- CE2 prefers a large alcohol group, small acyl group
194
what are butrylcholinesterases
- ChE or BChE
- major contributor to hydrolysis of aspirin to salicylate in plasma
195
what are epoxide hydrolyses
- specialised type of esterase that in the presence of water cleaves epoxides
- two difference forms-microsomal and cytosolic with difference substrate specificities
- important role in benzo[a]pyrene activation and metabolism of a few drugs
196
what is paroxonase
- multifunctional enzyme with arylesterases, lactose and paroxonase activities (also hydrolyses other organophosphorus compounds)
- synthesised mainly in the liver, also found in plasma
- has a role in degradation of oxidised lipids
197
what is the role of paroxonase in the degradation of oxidised lipids
protects LDL and HDL from lipid peroxidation
198
what is the purpose of conjugation reactions
- compounds have greater molecular weight and are more water soluble
- less able to pass through cell membranes
- easier to excrete
199
what is glucuronidation
- most common phase 2 reaction
- carried out by the uridine diphosphate - glucuronosyltransferase
- UDP-glucuronic acid required as cofactor
- endogenous compounds such as steroids, vitamins, bile acids and bilirubin also undergo conjugation by UDPGTs
200
What are the properties of UDP-GTs
- founds only in endoplasmic reticulum
- monomeric proteins of molecular weight 50 to 60 000
- no prosthetic group
- highest levels found in liver but also present in kidney, lung, small intestine, skin and adrenal gland
- larger number of different isoforms
201
what are the effects of glucuronidation
- promotes excretion and results in loss of biological acivity
- some glucuronides have biological activity - morphine glucuronide
- some drug glucuronides are reactive and can bind irreversibly to cellular proteins
- can trigger an immune response
202
what is the difference between UGT1 and UGT2
there are different UGT1 isoforms but all products of the same gene whereas UGT2 isoforms are all products of separated genes
203
what is the drug substrate specificity of main UGT isoforms
- UGT1A1 - bilirubin, ethinyl estradiol
- UGT1A4 - imipramine, amitriptyline, chloropromazine
- UGT1A6 - paracetamol
- UGT1A6 - valproate, naproxen
- UGT2B7 - morphine, ibuprofen
204
how are glucuronidated compounds
- glucuronide conjugates of MW>400 tend to be excreted in the bile whereas those <400 are mainly excreted in urine
- glucuronides excreted in bile may undergo enterohepatic recirculation, which may potentiate the pharmacological activity of the drug
205
what is the glucuronidation reaction
UDP-glucuronic acid reacts with alcohol, carboxylic acids, amines and amides and thiols
206
what is the induction of UDP-GT
- expression of some UDPGT isoforms can be increased by exposure to certain xenobiotics, though induction is modest compared to CYPs
- family 1 enzymes induced by both phenobarbitone and polycyclic hydrocarbons, but these inducers are not UDP-GT specific
- more specific inducers operate through the antioxidant response element
207
what are sulphotransference
- soluble cytosolic enzymes with two subunits of MW each approx. 34 000
- wide tissue distribution
-
208
what are the sulphotransferases important in xenobiotic metabolism
SULT1A1 - main liver isoform
- simple phenolic compounds, paracetamol, N-hydroxyl PhIP
SULT1A3 - high in intestins
- simple phenol, 1-hydroxymethylpyrene
SULT1C2, IC4 - fetal liver, kidney, stomach
- simple phenols, N-hydroxy-2-acetylaminofluorene
209
what are the examples of sulphating
- sulphate esters much more soluble - anionic
- usually pharmacologically inactive
- can lead to reduced availability of drugs
210
why is sulphating used instead glucuronidation for conjugating reactions
- sulphate tends to be important at low substrate concentrations due to sulphate availability being limited
- PAPs is an expensive commodity
- although most drugs are inactivated by sulphation, minoxidil used in the treatment of baldness and hypertension required sulphating for activity
211
how are sulphotransferases regulated
- the metabolites of nuclear aryl hydrocarbons receptor ligands such as dioxins are substrates for SULTS
- AhR ligands have negative effects on SULT activity in mice, but nor in humans
- evidence is emerging for a role for orphan nuclear receptors such as constitutive androstane receptor and pregnant X receptor
212
what happens during amino acid conjugation
- requirement for carboxylic acid
- occurs in mitochondria
- in humans, glycine, taurine and glutamine are the major conjugating amino acids
- reaction proceeds by activation of the carboxyl group to its acyl-coenzyme A derivative followed by amide formation with the amino group of the conjugating amino acids
213
what is the reaction during amino acid conjugation
- initial reaction usually carried out by mitochondrial xenobiotic medium chain fatty acid - CoA ligases
- at least 2 mitochondrial glycine N-acyltransferases occur - one conjugates both benzoic acid and salicylic acid
214
what is acetylation
- acetylation of compounds containing amino, hydroxyl and sulfhydrul groups carried out by acetyltransferases
- acetyl CoA donates acetyl group
- NAT1 and NAT2 have been detected in human cytosol
215
why are so few xenobiotics conjugated to amino acids
- believed that many xenobiotics are substrates for MACsd
- N-acyltransferase is selective; not all CoA thirsters formed by MACs are conjugated
- salicyl CoA is extensively conjugated
216
what is inactivation of isoniazid by NAT2
- capacity for N-acetylation was the cause of individual variation in metabolism
- slow acetylation phenotype was recessive trait
- isoniazid toxicity was associated with slow acetylator phenotypes
217
what are the properties of acetyltransferases
- both the main human NATs isoforms found in cytosol
- small proteins which are products of adjacent genes
- NAT1 expressed in most tissues but NAT2 mainly in liver and intestines
- both enzymes show distinct but overlapping substrate specificities
- not inducible
218
how does monomorphic and polymorphic acetylation different in NATs
- NAT1 shows some genetic polymorphism but less than NAT2
- NAT1 is sometimes referred to as the monomorphic NAT
219
what are the examples of NAT drug substrates
- isoniazid (NAT2)
- sulphamethoxazole (NAT1)
220
what are the effects of acetylation
- results in the masking of amine group with acetyl group decreasing solubility
- acetylation may activate certain procarcinogens
- some acetylated compounds can undergo deactivation by a specific esterase enzyme
221
what is the relationship between NATs and cancer
- NATs catalyse inactivation of drugs and aryl amine carcinogens
- but also activate some heterocyclic and aromatic aryl amines in well cooked food
- activation by NATs produces reactive intermediated that initiate carcinogens
222
what is glutathione
- tripeptide important in maintaining reduced environment within the cell
223
what is glutathione S-transferase (GST)
- mainly soluble enzymes found in cytosol
- consist of homo or heterodimers of subunits of Mr approx. 25 000
- conjugate reduced glutathione to electrophilic compounds through nucleophilic cysteine thiol groups
- found in most human tissues
- large number of different isoforms
224
which are the most important forms of GST in drug metabolism
- alpha class (GSTA1-A4)
- mu class (GSTM1-M5)
- pi class (GST-P1)
- theta class (GST-T1, GST-T2)
225
which classes of GST are inducible
- alpha and mu
- range of inducers include polycyclic aromatic hydrocarbons barbiturates and also antioxidants
- GST induction thought to be protective against some carcinogens
226
what is the reaction pathway of glutathione conjugation
- glutathione bonds through a nucleophilic cysteine thiol group
- very reactive with electrophilic substance
- nucleophilic substitution of acetates, sulphates, nitrate and epoxides
- conjugates lose glutamic acid and glycine
- cysteine is N-acetylated to give stable mercapturic acid derivatives
227
which drugs are conjugated by GST
- anti-cancer drug - cyclophosphamide
- vasodilator - nitroglycerine
- analgesic - paracetamol
- diuretic - ethacrynic acid
228
which groups of substrates does methylation act on
amine
thiols
alcohols
229
which groups of substrates does acetylation act on
amines
amides
hydrazines
230
what are the different methyltransferase families
- O-methyltransferases (catechol and phenol)
- N-methyltransferases (histamine, nicotinamide)
- S-methyltransferases (thipurine, thiol)
231
what happens during methyltransferase reactions
all methyltransferases use S-adenosylmethionine as co-factor
232
what are the mechanism of induction of xenobiotic metabolising enzymes
- increased levels = induction
- decreased levels = repression
233
what is the klingenberg experiment
- spectrophotometry - reduced microsomes vs reduced microsomes + CO
- identified the presence of a haemoprotein
- Fe3+ - yellow oxidised
- Fe2+ - red reduced
234
how does induction increase gene expression
- DNA makes RNA makes protein
- the complement of the negative strand is synthesised to give a copy of the gene
- pre-mRNA is spliced to mrna
- translation
- mRNA is continuously transcribed and translated. it is continuously broken down
235
what is the use of northern blotting
measuring the amount of a mRNA
236
what is the use of promoter-reporter expression
measuring the amount of transcription
237
what are the approaches for confirming signalling mechanism
- knock out or knock in - known in the human PXR but the mouse PXR is still present
- knock out and knock in approach - take PXR mice and knock in hPXR
238
what are the nuclear receptors that bind steroid
glucocorticoid receptor
mineralocorticoid receptors
androgen receptor
oestrogen receptors
239
what are the nuclear receptors that bind other ligands
retinoid X receptor
retinoid acid receptor
thyroid hormone receptor
vitamin D receptors
240
what are the nuclear receptors that bind bile acids
pregnant X receptors
constitutive activated receptor
241
what are the specific response elements that bind nuclear receptors
two half sites related to
- AGGTCA
- steroid hormone receptor
- bind as homodimers
- imperfect palindromic sequences
- 3bp spacing
242
what are the classes of inducers
- CYP1A - AhR
- CYP2B - CAR
- CYP3A - PXR
- CYP4A - PPARa
- CYP8A - LXR, FXR
243
what are CYP1A
polyaromatic hydrocarbon
tryptophan derived products
244
what are CYP2B
phenobarbitone drugs
bile acids
245
what are CYP3A
many drugs
bile acids
246
what are CYP4A
fibrate drugs
fatty acids
247
what are CYP8A
cholesterol
bile acids
248
what are some of the examples of the pharmacological consequences of induction
- rifampicin and contraceptive pill may result in decreased protection against pregnancy
- barbiturate tolerance
- enhanced metabolism of certain drugs in smokers and those who eat a lot of barbecued food
249
what are the characteristics of non-specific CYP inhibitors
- often bind haem prosthetic group
- all active CYPs require haem/O2 for activity
- B-13 cells have a disrupted CYP1A2 gene
- no message
- no protein
- engineered to express hCYP1A2
- MROD activity - inhibited by furafylline
250
what are the different CYP450 inhibition
- competitive inhibition by a non-substrate
- competitive inhibition by another substrate
- mixed competitive/uncompetitive inhibition
- irreversible inhibition - suicide substrate
251
what are the clinical examples of CYP3A4 inhibition
- azalea antifungal agent
- macrolide antibiotic
- grapefruit juice can result in cardiac arrhythmias
252
what is a clinical example of therapeutic inhibition of CYP metabolism
disulphiram
- inhibits aldehyde dehydrogenase
- this causes build up of acetaldehyde if an individual drinks alcohol
253
when can inhibition become a problem
- two drugs are metabolised by the same enzyme
254
how does age effect xenobiotic metabolism
- in the foetus and neonates levels of drug metabolising enzymes are low
- ion in the foetus and neonate levels of drug metabolising enzymes are low
255
how do CYPs affect xenobiotic metabolism
- the isozyme CYP1A2 is involved in the metabolism of aromatic amines, acetaminophen, imipramine, warfarin, caffeine and theophylline
- in the metabolism of caffeine and theophylline - CYP1A2 is involved in all demethylations as well as in ring hydroxylation although other isozymes also contribute to these reaction
256
how do differences in species affect xenobiotic metabolism
- animal species can vary in their ability to metabolise xenobiotics
- coumarin is found in a wide variety of plants, microorganisms and in some animal species
257
why was the use of coumarin banned
- the finding of hepatotoxic effects in rats and dogs fed coumarin in the diet
258
how is coumarin metabolised in humans
coumarin --> CYP2A6 --> 7HC
259
how is coumarin metabolised in rats
coumarin --> cyp1a/cyp2e --> 3,4 epoxide --> oHPA
3,4 epoxide is toxic and carcinogenic
260
coumarin in flavouring
- it is still present naturally
- not listed as an authorised flavouring in the EU
- CYP2A6 polymorphism
- not an in vivo genotoxin
- non-genotoxic mechanism - therefore has a threshold
261
what is the reaction of glucuronul transferases
- conjugation of sulphate to glucuronic acid to substrates
- IDP-glucuronic acid + X-O-H --> (GT) X-G + UDP
262
what is the reaction of sulphotransferases
3' phosphoadenosine-5'phosphosulphate + X-O-H --> (SULT) X-S + PAP
263
how do species difference effect strains
- different strains of rodents may show differences in metabolism usually due to genetic deficiency
- for example - female DA rats lack rat equivalent of CYP2D6 and is unable to hydroxylate debrisoquine
- gunn rate - unable to synthesise certain phenol glucuronides
264
how does AhR differ in different mouse strains
CYP1A1 - paradigm for the transcriptional regulation of drug metabolising genes
265
what are the hormones affecting P450 levels in rats
growth hormones
oestrogen
progesterone
testosterone
insulin
thyroid hormone
glucocorticoids
266
what are the consequence of differences in CYP isoforms
- sex differences due to sex hormones (testosterone and oestrogen) and sex differences in growth hormone secretion
267
which CYP enzymes effect growth and sex in rats
- growth hormones - specific effects on CYP2C enzymes
- levels increase at puberty and up-regulate expression of CYP2C7, 2C11, 2C12 and 2C22
- female rats-continuous high levels - induce CYP2C7 and 2C12
- male rats - intermittent levels
268
what is the effect of increased hepatic expression of CYP2E1 in rat models of diabetes
glucocorticoids - synthetics analogues known to induce enzymes of CYOP3A family but natural forms may inhibit drug metabolism
268
what are the effects of liver disease on drug metabolism
alcoholic liver disease
cirrhosis
porphyria
269
how does alcohol effect drug metabolism
- may get inhibition of certain drug metabolising enzymes due to changes in NAD(P)H/NAD(P) ratio
- may get direct inhibition of certain drug metabolising enzymes - CYP2E1
- competitive metabolism of other drugs e.g. paracetamol
- induction of certain P450 enzymes resulting in increased metabolism of some drugs
270
what is porphyria
- impaired synthesis of haem and accumulation of toxic precursors
- levels of cytochrome P450 enzymes may be lower due to limitation in supply of haem
- drugs such as barbiturates which induce P450s will trigger increased synthesis of haem precursors triggering a clinical attack
271
what are genetic polymorphisms
- can be base substitution, insertion or deletion
-functional polymorphisms often due to amino acid substitution, splice site change or effect on transcription factor binding
272
how can we analyse phenotyping
measure enzyme activity directly by giving a probe drug and measuring metabolites or direct enzyme assay
273
how can we analyse genotyping
- look directly for presence of mutation in individuals DNA
- need to know gene responsible for the defect and what mutation to scene for
274
what are the characteristics of CYP2D6
- 9 exons
- metabolism of 1/4 of all drugs
- highly polymorphic
- >100* alleles reported
275
what are the common losses of activity of CYP2D6 polymorphisms
- CYP2D6*3 alleles - A deletion
- CYP2D6*4 allele - different digestion patterns with restriction enzyme BstNI
- CYP2D6*5 alleles - entire CYP2D6 gene is deleted
276
how is CYP2D6 an ultra rapid metaboliser
- individuals can have extra copies of the CYP2D6 gene adjacent to the wild type CYP2D6
- between 2-13copies
- gene duplication denotes by an xN following the * allele
- extra genes result in more enzymes and faster drug metabolism
277
what are the consequences of CYP2D6 polymorphism
- failure to metabolise drugs/active metabolites and greater risk of toxicity
- inability to activate prodrugs
- poor response to certain antidpresseants due to fast metabolism
- may suffer toxicity to prodrugs
278
how are CYP2D6 and codeine related
- opiate prodrug
- metabolism of codeine to morphine dependent on CYP2D6
- severe respiratory depression in UM children
- increased morphine breast milk of UM BF mothers
279
what is N-actylation polymorphism
- NAT2 acetylates a variety of xenobiotics including isoniazid
- individuals with normal activity are fast acetylators
- slow acetylators have 2 mutated copies of the gene
280
what is the effect of the NAT2 variation isoniazid
- slow acetylators more likely to have increased risk of hepatotoxicity
- fast acetylators more likely to show poor response to intermittent dosing
281
what is the effect of NAT2 variation hydralazine
slow acetylators and autoimmune systemic lupus erythematous
282
what causes toxicity of drugs
- biotransformation is a major cause of toxicity
- direct biotransformation of parent drug to toxic metabolite
metabolites which are subsequently metabolised to toxic metabolites
283
what is reaction phenotyping
to identify the specific enzymes responsible for the metabolism of a specific drug
284
what are hepatocytes
- parenchymal cells of the liver
- ability to assess whether compound passes through membrane
- immediate cryopreservation is import - can still result in loss of enzyme function
- when cultured over long period loses functionality
285
when are microsomes used
- primarily useful for CYPs and UGTs
- microsomes can easily be derived from any organ
- cryopreserved without loss of enzyme function
286
why do activity levels vary in a bank of human liver microsomes
- genetic variation
- lifestyle differences
- different personal lifetime exposure to inducers
287
why do we use a human liver microsome bank to study metabolising activity
- can measure various P450s in these microsomes using enzyme assays and immunoblotting
- with new compounds, measure levels of activity of interest in each microsome preparation
288
why do we use S9 fractions
- the liver S9 fractions contain both cytosolic and microsomal fractions
- almost same as hepatocytes for phase 1 and phase 2 enzymes
- s9 fractions are therefore a more representative compared with microsomes and cytosolic fractions
- presence iof soluble co-factors but may be diluted
289
what is the general requirement for P450 expression
need full length cDNA for the isoform of interest available
290
what are the characteristics of transient expression
- disappears over time
- cDNA in nucleus but does not achieve genomic integration
- gene not reproduced
- short period of expression
- good for short term experiments
291
what are the characteristics of stable expression
- expression maintained through cell lineages
- genomic integration of cDNA
- gene inherited through mitosis
- daughter cells express
- good for long term experiments/libraries
292
what are the P450 expression systems
- ecoli - requires optimisation
- yeast - widely used system
293
how does e coli works as a P450 expression system
- cDNA N-terminal sequence modification
- change residue immediately after initial met to Ala
- increase AT content of 5' end
add his residues at 3' end
294
what are the outcomes of e coli as a P450 expression system
- amount of active enzyme very variable
- can remove part of N-terminus making P450 soluble
295
how does yeast work as a P450 expression system
- endogenous p450 oxidoreductase
- some strains express human oxidoreductase
- perform more post translational modifications
- membrane organelles
- can isolate P450 containing microsomes --> downstream application
- cheap and scalable
296
what are the issues with P450 expression in E.coli
- no internal membrane system
- P450s have nothing to anchor into
- express/purify/reconstitute/investigate
297
what are the issues with P450 expression in yeast
- yeast already express own P450s
- difficult to isolate/investigate introduced P450 of interest
298
why would you use insect cell P450 expression
- carry out more complex post-translational modification than bacteria or yeast
- co-transfect P450 and oxidoreductase
- baculoviral vectors are technically demanding to work with
- higher cost, longer duration
- microsome isolation
- supersedes commercially available
299
what is the expression of non-P450 metabolic enzymes
- sulphotransferases, GSTs and several other families can be readily expressed in e.coli
- UGT and FMO more difficult but like P450s now expressed in insect cells using baculovirus
300
what are the benefits of studying P450 expression in mammalian cells
- particularly useful for toxicity studies
- relatively cheap and easily accessible, infinite supply
- more human relevant and related intracellular environment
- ensure proper protein folding, post-translational modification and localisation
- similar control pathways
301
what are COS cells
monkey kidney cells that have little endogenous P450 but adequate P450 oxidoreductase
302
why use COS cells for transient expression
- can get P450 expression simply by cloning cDNA into vector with strong promoter and transfecting cells
- plasmid has SV40 origin of replication, COS cells express SV40 T antigen, plasmid is replicated within the cell
- transient expression a major limitation for toxicological studies
303
why do we study expression in human lymphoblastoid cells
- G0 --> G1 stimulation = immortalisation
- transfect with P450 cDNA
- treat with compound
- isolate metabolites
- investigate toxicity and mutagenicity
304
what is chemical inhibition
isoform specific inhibitors should be added to the incubation mix of human liver microsomes and drug substrates
305
what are the examples of chemical inhibitor use
- furafyllrin - CYP1A2
- sulfaphenazole - CYP2C9
- quinidine - CYP2D6
- troleandomycin - CYP3A4
306
what is antibody inhibition
- polyclonal or monoclonal antibodies known to affect activity of certain isoforms/raised against specific isoforms
- pre incubate antibody with human liver microsomes before adding substrates
307
what is high throughput CYP screening
- use of fluorogenic substrates
- high-throughput
- less labour intensive vs HPLC/LCMS
- could give initial idea of which CYPs are involved to study further using marker reactions/probe substrates
308
why used humanised mice
- knock in specific CYPs after knocking out equivalent mouse genes
- can also transplant human liver/hepatocytes into immunodeficient mice after destruction mouse liver
309
why use hepatic cell lines
- easier to maintain and culture for experiments
- human hepatoma cell line hEPg2 is the liver cell line most commonly studied
- metabolic activity of tumour lines is significantly lower than that of primary hepatocytes
310
what are in silicon computational methods used for
predict regioselectivity
predict metabolites
predict interactions of drugs with metabolising enzymes
predict toxicological effects of metabolites
311
how can we calculate amount excreted (tubular handling)
amount excreted = amount filtered - amount reabsorbed + amount secreted
312
how can we calculate amount filtered
when the left untouched by nephron amount excreted = amount filtered (insulin creatine)
313
how can we work out filtered then reabsorbed
amount excreted < amount filtered (e.g. glucose, amino acids, Na+)
314
how can we work out filtered and secreted
amount excreted > amount filtered (PAH, drug molecules, metabolic end products)
315
what is the urinary excretion of PAH
filtered and secreted in tubules
amount in urine always more than amount filtered
316
what is the urinary excretion of insulin
filtered amount in urine - proportion to Insulin x filtration rate
317
what is the urinary excretion of glucose
- filtered then reabsorbed - amount in urine less than amount filtered
- at low concentrations all filtered, glucose reabsorbed
318
what is the calculation for renal excretion
total renal clearance = CL by filtration + CL by secretion - retention by reabsorption
319
what is the relationship between drug metabolism and renal excretion
- drugs (active) metabolised mainly to inactive metabolites. renal function does not greatly affect elimination of active compound
- drug (active) and/or active metabolites excreted in the kidneys
- change in renal function affect elimination of active compounds
320
what is the mechanism of glomerular filtration
- glomerular capillaries allow molecules with MW < 20 000 to diffuse into filtrate
- plasma albumin held back
- drugs enter the filtrate
321
how does protein bind affect filtration
- if drug bound to plasma proteins --> concentration of drug in filtrate = free unbound drug therefore clearance by filtration reduced
- filtration directly proportional to GFR and the fraction of unbound drug in plasm
322
what is the rate of clearance by filtration
fu x GFR
GFR = 120ml/min
323
what is the filtration of creatinine
- creatinine clearance rate is used as a measure of elimination rate
- creatinine is filtered but neither reabsorbed or secreted so CL=GFR
324
what is the clearance of passive tubular reabsorption
- if CLr < fu x GFR, then renal absorption is taking place
- Car significantly affected by changes in urine flow rate
325
how are ionisation and passive reabsorption dependent on the pH in relation to pKa of drug
- if the drug is ionised at the pH of tubular fluid, reabsorption will be much lower
- if the drug is unionised, reabsorption will be higher, as the unionised form of the drug can diffuse through the proximal tubule cell membrane
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how well can weak acids be reabsorbed
weak acids with pKa <7.5 (e.g. aspirin) more highly ionised and less well reabsorbed in alkaline urine
- alkalisation of urine in aspirin overdose reduced tubular reabsorption and increases excretion
327
how well can weak bases be reabsorbed
weak bases with pKa > 7.5 (e.g. amphetamine) reabsorption decreases and CLr increases by an acidic urine
328
what is required for active tubular secretion
- if the CLr > fu x GFR, then the drug filtered at the glomerulus is also cleared by active tubular secretion
329
what are the two independent and relatively non-selective carrier systems for active tubular secretion
- acidic drugs and endogenous compounds
- organic bases
- carrier systems transport drugs against an electrochemical gradient
330
what are the drugs that causes hyperuricaemia and gout
- diuretic and Low dose aspirin
- uricosuric drugs - sulphinpyrazone and probenecid
- allopurinol - first line therapy for gout
331
what are the main determinants for renal drug excretion
- active tubular secretion and passive reabsorption
- some drugs are not metabolised therefore rate of renal elimination is the main factor in determining duration of drug action
332
what can influence renal clearance of drugs
glomerular filtration
reabsorption
secretion in the kidney tubule
333
which drugs can affect dosages due to modified renal function and drug elimination
digoxin
ACE inhibitors
aminoglycosides
antibiotics
class 1 anti arrhythmic aagents
cytotoxic agents
334
what is the calculation for creatinine clearance
CLcr - rate of urinary excretion of creatine (mg/min)/serum concentration of creatinine (mg/ml)
335
how can CLcr be a measure of GFR
- 1st order renal elimination the relationship is linear
- e.g. 50% reduction in renal clearance = 50% reduction in elimination of drug
- drug dosage must be adjusted accordingly
336
what are the implications of renal failure
alters passive reabsorption indirectly by alteration in urine flow rate and pH
active tubular secretion is also impaired --> renal clearance of drug affected
337
what is the relationship between renal function and age
- in neonates both GFR and renal tubular function are immature
- takes ~6months to reach adult levels
- therefore, drugs and active metabolites excreted by kidneys accumulate in neonates and young infants
338
what is the relationship between renal excretion and old age
- GFR decreases with increasing age (60-70ml/min)
- tubular function also declines with age
- reduction in dosage for drugs or active metabolites mainly excreted by kidneys
339
what is the relationship between diet and drug excretion
- renal elimination of certain drugs can be influenced by fasting or starvation
- e.g. sulfisoxazole (excretion decreases during fasting)
- drugs eliminated by GFR are affected by nutrition
- protein loads increase GFR
- paternal or enteral nutrition may enhance renal elimination of drugs
340
how does pregnancy affect drug excretion
- GFR increases by 70% in pregnancy
- therefore drugs mainly eliminated by renal excretion will be cleared more quickly
341
how can NSAIDs affect renal functions
- on an acute basis, prostaglandin important for maintenance of renal function
- acute renal failure may develop
- fluid and electrolyte retention can all occur
- may cause hypertension
342
what are the two main other routes of drug excretion
biliary excretion
enterohepatic circulation
343
how are drug excreted by biliary excretion
drugs transported from liver to bile by transport systems similar to those of renal tubules and involve P-glycoprotein and other transporters
drugs with MW > 300 excreted in bile (especially polar drugs/conjugates)
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what are the stages of biliary excretion
- some drugs conjugates (particularly glucuronides) concentrated in bile and delivered to intestine
- drug conjugate hydrolysed
- free drug
- drug reabsorbed and the cycle repeated ' enterohepatic circulation'
- can prolong drug actions
345
when is pulmonary elimination required
- main route of elimination and uptake of volatile anaesthetics
- medico-legal significance - ethanol concentrations in expired air
346
how can saliva be used for drug excretion
- dependent on lipid solubility and pKa, molecular weight
- useful for therapeutic drug monitoring
- analgesics - paracetamol, salicylate
- anticonvulsants - carbamazepine, phenytoin
- cardiovascular agents - propranolol
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what are the factors influencing passage of compounds across cell membranes
- lipid solubility affects passive diffusion
- degree of ionisation with uncharged molecules cross more efficienctly than charged
- pH difference between cellular compartments can affect transport of foreign compounds
348
what is the role of drug transporters in absorption
role in intestine in both facilitating entry of some drugs but also in preventing access by some xenobiotics including drugs
349
what is the role of drug transporters in distribution
role in allowing entry in target organs for activity but also liver for metabolism
350
what is the role of drug transporters in excretion
role in both renal and biliary excretion
351
what are the main drug transporter proteins
- ABC proteins (ATP dependent) e.g. MRP family, MDR, BSEP, BCRP
352
What are the names of the SLC family drug transporters
OAT - anion transporter
OATP - anion transporter
OCT - cation transporter
MATE - cation transporter
PEPT - mostly peptides but also penicillins
353
what are SLCs
solute carrier
52 different gene families that all code for membrane proteins but these proteins have a variety of functions including roles in normal physiology
354
what are the two general types of SLC proteins
- facilitative transporters - allow substrates to flow downhill with their electrochemical gradients
- secondary active transporters - substrates can flow uphill against their electrochemical gradients by coupling transport to that of a co-substrtate
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what are the properties of the OATP family
- organic anion-transporting polypeptide
- families 1 and 2 are the most important in relation to drug disposition
- important in drugs transport across sinusoidal membrane in liver
- OAT1B1, OATP1B3, OATP2B1 most important in liver
- kideny also has OATPs present but more limited role in renal excretion
356
what are the properties of the OAT protein
- members of SLC22A subfamily
- key role in renal excretion but also expressed elsewhere
- OAT1, OAT2 OAT3 found on basolateral membrane of proximal tubule cells facing blood vessels
- OAT4 on apical membrane of proximal tubules, facing urine
357
what is anionic drug transport in renal proximal tubules
OAT1/2/3 are coupled to dicarboxylic acid transport
- OAT4 reabsorbed drugs from urine and may be bidirectional
358
what are OAT substrates
- likely to be sulphate or glucuronide conjugates
- OAT1 - tetracyclines
- OAT2 - AZT, diclofenace, diclofenac glucuronide
-OAT3 - oestrone sulphate, benzylpenicillin, rosuvastatin
359
what are OCTs
- organic cation transporters
- members of SLC22A so some sequence homology of OATs
- OCT1/2 are the most important cationic transporters in human drug disposition
360
what are OCT1
- important transporter on sinusoidal face of liver though found elsewhere in the body also
- substrates include metformin and cisplatin
361
what are OCT2
- high levels on basolateral membrane of kidney
- substrates include metformin and cisplatin also cimetidine
362
what are PEPT transporters
- peptide transporters encoded by SLC15a subfamily
- both show similar substrate specificities with penicillins, ACE inhibitors and valacyclovir
- proton-dependent transporters
- in kidney PEPT1/2 contribute to drug reabsorption from urine within proximal tubules
363
where is pept1 expressed
intestine and kidney
364
where is pept2 expressed
mainly in kidney
365
what are MATE transporters
- multidrug and toxin extrusion family
- members of SLC47 family
- export pumps which contribute to biliary and renal excretion of cations
- transport is proton dependent
366
where is MATE1 expressed
liver and kidney
367
where is MATE2-K expressed
kidney only
368
what are the transporter proteins in the intestines
- ASBT - Na dependent bile salt transporter
- OST - organic solute transporter
- MCT1 - monocarboxylate transporter
369
what is the role of transporter proteins in the intestines
compounds need to enter enterocytes through brush border membrane and then cross basolateral membrane into hepatic portal vein
370
what is the role of transporter proteins in the liver
- xenobiotic often need to be transported into hepatocytes across sinusoidal membrane for both metabolism and to reach targets
- compounds usually following metabolism need to be transported out either across canalicular membrane or sinusoidal membrane
371
what is the importance of the basolateral membrane in kidney tubules
- compounds excreted by tubular secretion need to enter
- those being reabsorbed need to return to circulation
372
what is the importance of the brush border membrane in kidney tubules
- compounds need to cross this membrane to enter lumen for final renal excretion
- reabsorption may occur
373
what is the role of diclofenac excretion in liver and kidney
- diclofenac undergoes metabolism by CYP2C9 and UGT2B7
- UGT2B7 produces acylglucuronide (DF-AG)
- OAT2 and OAT4 important in renal excretion of DF-AG
374
Which drug interactions can inhibit OAT transport
penicillin and probenecid
375
which drug interactions can inhibit ABCB1 transports
digoxin and erythromycin or statins
376
which transporters are inducible by PXR
abcb1
abcc2
slco1b1
377
which drugs can interact causing induction via PXR
rifampicin, cyclosporin, digoxin
378
what are the pharmacogenetics of OATP1B1
- genetic polymorphism associated with higher plasma levels of some statins due to impaired ability to enter hepatocytes
- higher plasma levels may lead to toxic levels of statin in muscle cells
379
what are the pharmacogenetics of OCT2
- linked to nephrotoxicity with anti-cancer drug cisplatin
- may also involve MATE2
- drug accumulated in tubule
380
380
what is the renal handling of cisplatin
-
381
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