Pharmacokinetics + Ligand Binding Flashcards
Spare receptors/Receptor reserve
Shown by ‘parallel shifting’ on a graph of response vs agonist concentration, when increasing amounts of NON-competitive antagonist is added (e.g. BCM which irreversibly alkylates muscarinic receptor). That means maximum response can still be reached in the presence of a NON-competitive antagonist if there are enough spare receptors.
Remember that partial agonist acts as a competitive antagonist, NOT a non-competitive antagonist.
With INCREASING doses of antagonist, maximum response will eventually be no longer attainable.
By measuring the dose ratio at the point at which the maximum response is just attainable, it is possible to estimate the percentage of receptors that are ‘spare’.
Existence of spare receptors increases the sensitivity of the tissue to the agonist.
Why dose ratio works
Makes no assumptions about the relationship between response and receptor occupancy. It only supposes that equal responses are produced by equal agonist occupancies.
EC50
Concentration of agonist that causes 50% of the maximum response.
Potency
Concentration of an agonist causing a particular magnitude of response.
E.g. A is more potent than B, A needs half the concentration of B to cause 20% of maximum response.
Note that potency is usually compared by comparing EC50 levels.
Stereoisomers can vary greatly in potency.
Competitive antagonist
With a FIXED concentration of COMpetitive antagonist, log agonist concentration vs response curve (which is SIGMOID) shifts to the right. Antagonist is SURMOUNTABLE simply by adding lots of agonist to compete off the antagonist.
Overcoming blockade by NON-competitive antagonist requires spare receptors, not simply more agonist.
Note that agonist concentration (NO LOG) vs response curve is hyperbolic.
Structure activity series
Determines the relative potencies (usually relative EC50 values) of structurally related compounds.
This aids receptor classification.
Dissociation constant (Kd)
Units M (mol/L). Ka units is M^-1 (L/mol). Kd=Bmax/2 So Kd is the concentration of agonist that produces 50% of the maximum specific binding/occupancy (maximum specific binding means all spare receptors are occupied).
Radioligand binding and Bmax
Binding of a radioligand (total binding) consists of a saturable component that - hyperbolic line (specific binding to the receptors) and an effectively linear non-saturable component (non-specific binding to non-receptor material).
In the presence of a large excess of unlabelled ligand the specific binding of the radioligand is almost completely abolished, but non-specific binding is almost unaffected.
So to find specific binding, use total binding minus non-specific binding.
Maximum specific binding (a.k.a binding site density) is Bmax. So Kd = agonist concentration at Bmax/2.
Affinity of competitive antagonist ‘K2’
In the presence of a FIXED concentration of competitive antagonist, a higher concentration ‘[D]2’ of agonist will be needed to occupy the same fraction of receptors and hence produce the SAME response.
So if the question gives you ‘response’ e.g. 50mm contraction and use of antagonist, then think dose ratio.
Affinity of a competitive ligand in radioligand experiment
FIXED concentration of radioligand is added, and then VARYING concentrations of unlabelled drug is added to displace the radioligand.
IC50 = Concentration of unlabelled drug that displaces 50% of SPECIFICALLY bound radioligand. When [U] = IC50, α=0.5 (fraction of receptor bound by unlabelled ligand).
Graph of bound radioligand vs LOG concentration of unlabelled ligand is sigmoid.
Partial agonist
On its own, it’s a weak agonist, usually with a low affinity constant. But this is not always true.
Partial agonist ALWAYS have a lower EFFICACY than full agonist.
When presented together with a full agonist, partial agonist acts as a COMPETITIVE antagonist (which can be competed off by high agonist concentrations - surmountable inhibition).
Hill slope
Does NOT equate to number of binding site per receptor. But it does suggest that there’s 1 binding site per receptor, since if there’s more than one binding site, binding sites are cooperative and Hill slope will be more than 1. (from supervision)
Equal to the number of molecules that must bind a receptor in order for it to open.
3 types of ligand binding question that can be asked
(1) Dose ratio question - NO LABELLED stuff. Look for changing amounts of COMPETITIVE antagonist in one column and changing amounts of agonist needed to produce SAME RESPONSE.
(2) IC50 question - Adding variable amounts of unlabelled drug to compete off a fixed amount of labelled drug. Look for column showing CHANGING AMOUNTS OF UNLABELLED DRUG.
(3) Scatchard plot - increasing amount of labelled drug and a fixed LARGE amount of unlabelled drug to remove non-specific binding. Look for column showing CHANGING AMOUNTS OF LABELLED DRUG and a LARGE amount of unlabelled drug.
Note on Bmax
Bmax can be given in mol of binding sites/gram of protein (see mock exam example).
Can then convert into mol/cell (question will give amount of protein in a cell).
Can also convert to number of binding sites/cell. Need to multiply by Avogardro’s constant (6.022x10^23).
First order kinetics/Linear elimination
Rate of elimination is directly proportional to plasma concentration.
Renal elimination by filtration ALWAYS follows first order kinetics, since it’s not enzyme dependent and CAN’T be saturated. But secretion via transporters can get saturated.
In 1st order kinetics, CLEARANCE IS CONSTANT. Also, route of administration will NOT affect clearance (e.g. take grapefruit juice and then inject by I.V. or take pill. Clearance for both should be the same).
Single compartment model
Absorption and distribution ignored.
Volume of distribution constant throughout.
Two compartment model
Absorption ignored.
Volume of distribution increases as distribution occurs, and plasma concentration, hence rate of elimination falls.
We need to measure terminal phase data - data collected after distribution has occurred.
Single oral dose
First pass metabolism cause loss of drug. So need to take into account fractional bioavailability.
Distribution phase can be ignored since rate of absorption is usually similar to rate of distribution.
Zero order kinetics
Elimination rate is CONSTANT and NOT dependent on plasma concentration. Zero order kinetics occur when liver metabolism become saturated.
If we do constant IV infusion, there will be NO Css. Plasma concentration continue to increase linearly.
For first order kinetics, Css = Rin/CL. So if we increase Rin, then Css increases linearly. For zero order kinetics, if we increase Rin, then plasma concentration increases EXPONENTIALLY.
Vd reference values
3L if a drug is confined to plasma
12L if a drug is confined to interstitial fluid.
42L if a drug can distribute throughout all body compartments. (TBW = 42L)
>42L if a drug is extensively dissolved in fat or bound to TISSUE proteins, or adsorbed to bone (drugs with heavy metal).
Binding to PLASMA proteins DECREASES apparent Vd. This is because ‘plasma concentration’ used to measure Vd = C(free) + C(bound).
Renal clearance values
125ml/min = GFR
Drug elimination can be entirely renal. Drug is filtered but not reabosrbed or secreted.
625ml/min = Renal plasma flow
Drug elimination can be entirely renal and drug is filtered + secreted, but not reabsorbed. Example: PAH para-aminohippuric acid
Over 625ml/min: Some drug must be metabolised by the liver.
Small overall clearance value could suggest:
(1) Drug is largely plasma protein bound and does not get filtered.
(2) Drug is readily filtered but is reabsorbed.
Question where other drug affects another drug
Drug decreases or increases amount of hepatic enzyme, affecting metabolism of another drug.
Two drugs compete for same transporter protein in the kidney for secretion into tubules.
Freely filtered drugs at the kidney
FREE drug can be filtered in the glomerulus, NOT those bound to plasma proteins.
So filtration rate of drug = GFR x free concentration of drug.
Filtration at the glomerulus will NOT change concentration of free drug in the blood, since water and drug are filtered in proportion. Also, there’s NO change in amount of bound drug arriving at glomerulus and leaving the glomerulus.
For drugs that are only filtered, an increase in plasma protein binding DECREASES renal clearance of the drug.
Secreted drugs in the kidney
OATs transport ACIDIC drugs in their negatively charged anionic form, endogenous acids like uric acid, glucuronide conjugates, sulphide conjugates.
Probenecid and penicillin both use OATs. Probenecid prolongs action of penicillin by reducing its tubular secretion.
OCTs transport organic bases in their protonated cationic form (e.g. morphine).
Concentration of free drug in blood decreases, since water does not leave together with secreted drug. This causes drug to be released from plasma proteins, and this ‘new’ free drug can also be secreted. Thus increase in plasma protein binding has NO effect on renal clerance.
Reabsorption in the kidney
Lipid soluble drugs are poorly excreted as they’re readily reabsorbed. Lipid soluble drugs are also poorly filtered as they’re more likely to bind to plasma proteins. Opposite for polar drugs.
Urine pH is acidic. This favours excretion of basic drugs in their charged (BH+) form, and reabsorption of acidic drugs in their neutral (HA) form.
To increase excretion of acidic drugs, e.g. following an overdose of aspirin or barbiturates, increase the pH of urine by adding sodium bicarbonate.
How hepatic metabolism increases efficiency of renal excretion
(1) Conjugated drugs are in a more polar form, which is more readily filtered at glomerulus.
(2) More polar also means less likely to be reabsorbed.
(3) Conjugated drugs can be secreted by OATs.
Biliary excretion
Drugs and conjugates secreted from plasma into bile using by hepatocytes same transporters as in renal tubules. Bile travels to SI. In SI drug can be:
(1) Excreted in faeces.
(2) Reabsorbed.
(3) Conjugates can be hydrolysed, especially glucuronides, and may reactivate the drug. Hydrolysed drug can either be excreted or reabsorbed. If drug is reactivated and reabsorbed, this prolongs the lifetime of a drug.
Reabsorbed drugs is called enterohepatic circulation.
Since faeces contains drug, coprophagy complicate dosing patterns for some animals.
Note that this is all talking about hepatic clearance of a drug. Concerning first pass metabolism, liver can only decrease bioavailability of a drug, can’t increase it, so all this here is irrelevant.
Meyer-Overton correlation
Positive correlation between anaesthetic potency and its solubility in olive oil. Why this is wrong:
(1) Maximum molecule size cut-off
(2) Some lipid soluble molecules DIDN’T cause anaesthesia.
(3) Setereospecificity of some anaesthetics like isoflurane.
General anaesthetics targeting GABA(A)
Pentameric receptor. Usually 2 alpha, 2 beta, 1 gamma subunits.
Barbiturates is a GABA(A) PAM.
Ethanol also acts on GABA(A) receptor.
Propofol and etomidate act on beta subunits. ‘PEB’
Volatile anaesthetics act on alpha AND beta subunits.
Halothane, nitrous oxide, isoflurane activate two pore domain K+ channels. ‘HNI’
Nitrous oxide and xenon compete with glycine for NMDA receptor and hence prevents its activation.
IV general anaesthetics
Proprofol, etomidate, thiopental ‘PET’ can be injected by I.V.
Thiopental is lipophilic and acts in 10s.
Consciousness rapidly recovered after a single I.V. injection. Because initially drug accumulates in high blood flow tissues, but over time distributes to low blood flow tissues, decreasing plasma concentration in high blood flow tissues to sub-anaesthetic levels.
Though drug is at sub-anaesthetic concentration in blood, ALL lipophilic drugs (HIGH oil:water partition coefficient) like halothane and thiopental causes ‘hangover’ effect. It will accumulate in fat, and only slowly equilibrates back into the blood from fat, leading to a slow decrease in plasma concentration.
Drugs with a high oil:water partition coefficient are said to be more POTENT.
Danger is giving multiple injections that would cause the plasma concentration at the end of redistribution to be HIGHER than minimum concentration for anaesthesia.
Propofol has more rapid redistribution and more rapid metabolism (not lipophilic) than thiopental. Propofol is also an anti-emetic.
Etomidate has a wider therapeutic window but causes more vomiting than propofol.
Volatile anaesthetics
Rate of induction depends on rate of equilibration between P(insp) and P(CNS) which depends on separate equilibria, the slowest of which is P(insp) - P(alv) equilibrium.
P(insp) - P(alv) equilibrium depends on blood:gas partition coefficient. If this coefficient is HIGH, more gas from alveoli enters blood and time to get to equilibrium increases. Induction rate is SLOW.
HIGH CO SLOWS induction rate since more gas is removed during each breath. Effect of CO on induction rate greater when blood:gas coefficient is high.
LOW alveolar ventilation rate SLOWS induction rate since less anaesthetic is delivered to alveoli. Effect of ventilation rate on induction rate greater when blood:gas coefficient is high.
HIGH blood:gas partition coefficient causes SLOW recovery of consciousness.
Pharmacokinetics bits and pieces of info
Almost all drug absorption happens in SI. Exception is tetracyclines which get absorbed in the stomach as it’s soluble at acidic pH but insoluble at neutral pH.
Acid/base effects (stomach acidic, SI basic) also affects absorption of drugs in stomach/SI. Hydrophobic form is more easily absorbed.
Peptide drugs like insulin CAN’T be ingested orally.
Heparin is restricted to plasma compartment. No specific transporters for it.
Local inflammation decreases tissue pH, LAs, which are weak bases, turns into BH+ charged form, delaying induction of anaesthesia.
Plasma binding proteins
(1) Binding to PLASMA proteins DECREASES apparent Vd.
(2) Binding to plasma proteins decreases rate of renal clearance by filtration (NOT secretion).
(3) When binding sites become saturated, an increase in dose will cause a disproportionate increase in C(free).
Albumin binds all kinds of drugs - acidic, neutral, basic.
Basic drugs like beta-blockers and antidepressants can bind alpha-1-glycoprotein.
Sulphonamides
Anything with ‘-amide’ inside + glipizide + furosemide.
They compete with bilirubin to bind to plasma proteins, displacing bilirubin and leading to jaundice.
Phase 1 hepatic drug metabolism
‘Functionalisation’ - Catabolic (in general)
Introduces or unmasks a reactive functional group. No effect on water solubility.
Drug can be inactivated by phase 1, or activated if the original drug is a pro-drug.
Paracetamol and codeine do not require phase 1.
Phase 2 hepatic drug metabolism
‘Conjugation’ - Anabolic (in general)
Adds a molecule to drug which increases its water solubility. Usually drug gets inactivated but morphine-6-glucuronide is still active.
CYP Enzymes
Do phase 1 reaction. Catalyse oxidation reactions.
Need to use O2, haem, H+ supplied from NADPH.
Located at ER membrane.
Little selectivity hence slow metabolic rate.
CYP2D6
Inhibited by fluoxetine and quinidine.
Functions:
Turns pro-drug codeine into active morphine.
Metabolises pro-drug tamoxifen.
Metabolises beta blockers.
CYP3A4
Ketoconazole inhibits it.
Grapefruit juice inhibits intestinal wall and hepatic CYP3A4. But in pharmacokinetics questions, usually grapefruit juice doesn’t do anything to hepatic CYP3A4. This is because grapefruit juice is taken orally and when it reaches hepatic CYP3A4, concentration is too low to have an effect. Grapefruit juice itself has a low bioavailability. Also, intestinal wall CYP3A4 is more important than hepatic CYP3A4.
PXR or CAR heterodimerise with RXR to bind XRE to upregulate CYP3A4. Phenobarbital, rifampicin, St John’s Wort upregulates PXR.
FMO3
Use FAD instead of haem. Mutation causes fish odour syndrome.
Metabolises clozapine, amphetamine, ranitidine. ‘CAR’
