Pharmacology and prescribing Flashcards
what is the FP10? who can issue them?
a prescription that can be issued by GPs, nurses and pharmacist prescribers, supplementary prescribers or hospital doctors in England
what is the colour of the GP FP10?
green
what is the colour of the FP10D?
yellow, issued by dentists
what is the colour of the FP10MDA?
blue, used for drugs such as methadone
what is the colour of the FP10P, PN, SP, or CN?
purple or green, used by prescribers such as nurses or pharmacists
what should be included on the FP10 prescription?
- prescriber’s signature
- prescriber’s address: practice address, usually already printed on the FP10. contain a number to identify the prescriber.
- date
- patient’s details
- information about the product supplied
what does the patient do with the FP10 and what payment is made?
- can be taken to any chemist/pharmacy in England
- pay a prescription charge for each which is currently £8.60 unless exempt
what are the protein targets for drug action on mammalian cells?
- receptors
- ion channels
- enzymes
- transporters (carrier molecules)
what is colchicine used for? what is the mechanism of its action?
- used to treat arthritic gout attacks
- interacts with the structural protein tubulin
what is an example of an immunosuppressive drug? how do most of them work?
- ciclosporin
- bind to cytosolic proteins known as immunophilins
how can therapeutic antibodies work?
act by sequestering cytokines
what are targets for chemotherapeutic drugs?
- aim is to suppress invading microorganisms or cancer cells
- targets include DNA and cell wall constituents, and other proteins
what are receptors?
sensing elements in the system of chemical communications that coordinates the function of all different body cells and chemical messengers
what are orphan receptors?
the mediator is still unknown
when are endogenous mediators usually discovered?
years before the receptor was characterised pharmacologically and biochemically
how can receptors be characterised? what are examples of receptors that were identified later?
- on basis of pharmacological and molecular characteristics
- cannabinoid and opioid receptors were discovered later
what are ion channels?
gateways in cell membranes that selectively allow the passage of particular ions, and are induced to open or close by many mechanisms
what are types of ion channels?
- ligand gated channels
- voltage gated channels
what are ligand gated channels?
- open only when one or more agonist molecules are bound
- properly classified as receptors, since agonist binding is needed to activate them
what are voltage gated channels?
ion channels which are gated by changes in the transmembrane potential rather than by agonist binding
in what ways can drugs affect ion channel function?
- by binding to the channel protein itself, either to the orthosteric or allosteric sites
- indirect interaction, involving G protein and other intermediaries
- by altering the level of expression of ion channels on the cell surface
what are orthosteric and allosteric sites?
- orthosteric: ligand-binding site of ligand-gated channels
- allosteric: other, non ligand-binding site
what is an example of drug molecules plugging the channel physically?
- action of local anaesthetics on the voltage-gated sodium channel
- drug molecule plugs the channel physically, blocking ion permeation
what are examples of drugs that bind to allosteric sites on the channel protein and thus affect channel gating?
- benzodiazepine tranquilisers: bind to a region of the GABA(a) receptor-chloride channel complex (ligand gated channel) that is distinct from the GABA binding site and facilitates the opening of the channel by GABA
- vasodilator drugs of the dihydropyridine type, which inhibit opening of L-type calcium channels
- sulfonylureas, used in treating diabetes, which act on ATP-gated potassium channels of pancreatic beta cells, enhancing insulin secretion
what is the effect/action of benzodiazapene transquilisers?
bind to a region of the GABA(a) receptor-chloride channel complex (ligand gated channel) that is distinct from the GABA binding site and facilitates the opening of the channel by GABA
what is the effect/action of dihydropyridine type drugs?
- vasodilation
- inhibit opening of L-type calcium channels
what is the effect/action of sulfonylureas?
used in treating diabetes; act on ATP-gated potassium channels of pancreatic beta cells, which enhances insulin secretion
what does indirect action between a drug and an ion channel involve?
G protein and other intermediaries
what is the action of gabapentin?
reduces the insertion of neuronal calcium channels into the plasma membrane
what effect do agonists/inverse agonists have on receptors?
- direct: ion channel opening/closing
- transduction mechanisms: enzyme activation/inhibition, ion channel modulation, DNA transcription
what effect do antagonists have on receptors?
- no effect
- endogenous mediators blocked
what effect do blockers have on ion channels?
permeation blocked
what effect do modulators have on ion channels?
increased or decreased opening probability
what effect do inhibitors have on enzymes?
normal reaction inhibited
what effect do false substrates have on enzymes?
abnormal metabolite produced
what effect do prodrugs have on enzymes?
active drug produced
what effect does an inhibitor have on transporters?
transport blocked
what effect does a false substrate have on transporters?
abnormal compound accumulated
what is an example of a drug molecule acting as a competitive inhibitor of the enzyme?
captopril, acting on ACE
what is an example of irreversible and non-competitive binding to an enzyme?
aspirin, acting on cyclo-oxygenase
what is a false substrate?
drug molecule undergoes chemical transformation to form an abnormal product that subverts the normal metabolic pathway
what is an example of a false substrate acting on an enzyme?
- fluorouracil, anticancer drug
- replaces uracil as an intermediate in purine biosynthesis but cannot be converted into thymidylate, thus blocking DNA synthesis and preventing cell division
what is a prodrug?
drugs may require enzymatic degradation to convert them from an inactive form to an active form
what is an example of prodrug conversion?
enalapril is converted by esterases to enalaprilat, which inhibits ACE
what does drug toxicity often result from?
- enzymatic conversion of the drug molecule to a reactive metabolite
what are ABC transporters?
- ATP-binding cassette transporters
- hydrolysis of ATP provides energy for transport of substances against their electrochemical gradient
what are MDR transporters?
multi-drug resistance transporters
- eject cytotoxic drugs from cancer and microbial cells, conferring resistance to these therapeutic agents
what are symports and antiports?
- symports: transport of organic molecules is coupled to the transport of ions in the same direction
- antiports: transport of organic molecules is coupled to the transport of ions in the opposite direction
what are the four receptor types?
- ligand-gated ion channels
- G protein coupled receptors (metabotropic receptors or 7 transmembrane receptors)
- kinase-linked and related receptors
- nuclear receptors
what is the basic function, time scale and examples of ligand-gated ion channels (ionotrophic receptors)?
function: influx of ions -> hyperpolarisation/depolarisation -> cellular effects
time scale: milliseconds
examples: nicotinic ACh receptor
what is the basic function, time scale and examples of G protein coupled receptors (metabotrophic)?
function: binding of ligand to receptor leads to increase/decrease in second messenger production or ion influx. Second messengers lead to Ca2+ release, protein phosphorylation or other. ion influx -> change in excitability -> cellular effects
time scale: seconds
example: muscarinic ACh receptor
what is the basic function, time scale and example of kinase-linked receptors?
function: binding of ligand to receptor/enzyme -> protein phosphorylation -> gene transcription -> protein synthesis -> cellular effects
time scale: hours
example: cytokine receptors
what is the basic function, time scale and example of nuclear receptors?
function: binding of ligand to receptor within the nucleus -> gene transcription -> protein synthesis -> cellular effects
time scale: hours
example: oestrogen receptor
what are characteristics of receptors within a given family (heterogeneity)?
- generally occur in several molecular varieties/subtypes
- similar architecture
- significant differences in their sequences and pharmacological properties
where is the nicotinic acetylcholine receptor located?
skeletal NMJ
what are cys loop receptors?
receptors that have a large intracellular domain between transmembrane domains 3 and 4 containing multiple cystein residues
what are examples of cys-loop receptors?
- GABA
- glycine receptors
- 5-HT receptors
- nicotinic ACh receptor
what are types of ligand-gated ion channels?
- nicotinic ACh receptors
- ionotropic glutamate receptors
- purinergic P2X receptors
what is the molecular structure of the nicotinic ACh receptor?
- pentameric assembly of different subunits (2 x alpha)
- alpha, beta, gamma and delta subunits, each of molecular weight 40-58 kDa
- subunits show marked sequence homology, each containing four membrane-spanning alpha-helices
- two ACh binding sites, each at the interface between one of two alpha subunits and its neighbour
- each subunit spans the membrane four time, so there are 20 membrane-spanning helices surrounding a central pore
how must nicotinic ACh receptors be activated?
both binding sites must bind ACh molecules
where do the two ACh binding sites on nicotinic ACh receptors lie?
- at interface between one of two alpha subunits and its neighbour
- on the extracellular N-terminal region of the two alpha subunits
what forms the lining of the ion channel in nicotinic ACh receptors? what forms the constriction?
- one of the transmembrane helices (M2) from each of the five subunits forms the ion channel
- five M2 helices that form the pore are sharply kinked inwards halfway through the membrane, forming a constriction
what conformational change occurs when ACh molecules bind to a nicotinic ACh receptor?
- conformation change occurs in the extracelllar part of the receptor
- twists the alpha subunits, causing the kinked M2 segments to swivel out of the way
- this opens the channel
what does the channel lining of the nicotinic ACh receptor contain?
series of anionic residues, making the channel selectively permeable to cations
- primarily Na+ and K+, sometimes Ca2+
what is the location, effector, coupling, examples and structure of ligand-gated ion channels?
- membrane
- ion channel
- direct
- nicotinic ACh receptor, GABA(a) receptor
- oligomeric assembly of subunits surrounding the central pore
what is the location, effector, coupling, examples and structure of G-protein coupled receptors?
- membrane
- channel/enzyme
- G protein or arrestin
- muscarinic ACh receptors, adrenoreceptors
what is the location, effector, coupling, examples and structure of receptor kinases?
- membrane
- protein kinases
- direct
- insulin, growth factors, cytokines
- single transmembrane helix linking extracellular receptor domain to intracellular kinase domain
what is the location, effector, coupling, examples and structure of nuclear receptors?
- intracellular
- gene transcription
- via DNA
- steroid receptors
- monomeric structure with receptor and DNA binding domain
what is the structure of ionotropic glutamate receptors?
tetrameric
- pore is built from loops rather than transmembrane helices
what is the structure of P2X receptors?
trimeric
- each subunit has only two transmembrane domains
what is positive cooperativity?
binding of one agonist molecule to one site increasing the affinity of binding at the other site
what are examples of GPCRs?
- muscarinic AChRs
- adrenoceptors
- dopamine receptors
- 5-HT receptors
- opioid receptors
- peptide receptors
- purine receptors
how many GPCRs are encoded for by GPCRs?
~ 400
what was the first GPCR to be characterised?
beta adrenoceptor, which was cloned in 1986
what is the structure of GPCRs?
- single polypeptide chain, usually of 350-400 residues
- can be up to 1100 residues
- 7 transmembrane alpha helices
- extracellular N-terminal domain of varying length, and an intracellular C-terminal domain
what are the families of GPCRs?
A: rhodopsin family
B: secretin/glucagon receptor
family
C: metabotropic glutamate receptor/calcium sensor family
what receptors are in family A of GPCRs? what are their structural features?
- largest group
- receptors for most amine neurotransmitters, many neuropeptides, purines, prostanoids, cannabinoids
- short extracellular (N-terminal) tail
- ligand binds to transmembrane helices (amines) or to extracellular loops (peptides)
what receptors are in family B of GPCRs? what are their structural features?
- receptors for peptide hormones, including secretin, glucagon, calcitonin
- intermediate extracellular tail incorporating ligand-binding domain
what receptors are in family C of GPCRs? what are their structural features?
- small group
- metabotropic glutamate receptors, GABA(b) receptors, Ca2+ sensing receptors
- long extracellular tail incorporating ligand-binding domain
what is a fourth distinct family of GPCRs?
includes many receptors for pheromones but no pharmalogical receptors
what is the site of the molecule that couples to the G protein?
third cytoplasmic loop
- experiments show that deletion/modification of this section results in receptors that will still bind ligands but cannot associate with G proteins/produce responses
what can result in receptor desensitisation?
phosphorylation of serine and threonine residues on the C-terminal tail and other intracellular domains by intracellular kinases
where is the ligand-binding domain of small molecules in class A receptors?
buried in the cleft between the alpha helical segments within the membrane
- similar to the slot occupied by retinal in the rhodopsin molecule
where do peptide ligands bind?
e. g. substance P
- bind more superficially to the extracellular loops
what are protease-activated receptors? how can they be activated?
- four types identified
- many proteases e.g. thrombin, can activate PARs by snipping off the end of the extracellular N-terminal tail of the receptor to expose 5 or 6 N-terminal residues that bind to receptor domains in the extracellular loops
what is PAR-2 activated by?
- activated by a protease released from mast cells
- expressed on sensory neurons
- may play a role in inflammatory pain
how many times can a PAR molecule be activated?
only once
- because cleavage cannot be reversed, so continuous resynthesis of receptor protein is necessary
how is PAR inactivated?
- further proteolytic cleavage that frees the tethered ligand
- by desensitisation involving phosphorylation
- receptor is internalised and degraded, then replaced
what are G proteins? what is their role?
- family of membrane-resident proteins
- function is to recognise activated GPCRs and passes on the message to effector systems that generate a cellular response
- interact with guanine nucleotides, GTP and GDP
what subunits do G proteins consist of?
three: alpha, beta and gamma
what happens to G proteins during binding?
- guanine nucleotides bind to the alpha subunit, which has enzymatic (GTPase) activity, catalysing the conversion of GTP to GDP
- beta and gamma subunits remain together as a complex
what is the structure of the gamma subunit in the G protein?
anchored to the membrane through a fatty acid chain
- coupled to the G protein through a reaction known as prenylation
are G proteins diffusible?
they are freely diffusable in the plane of the membrane, so a single pool of G protein in a cell can interact with several receptors and effectors
what is the state of the resting G protein?
alpha-beta-gamma trimer
- may or may not be precoupled to the receptor
- GDP occupies the site on the alpha subunit
what happens to the G protein when a GPCR is activated?
- when activated by an agonist molecule, there is a conformational change involving the cytoplasmic domain of the receptor
- high affinity interaction of the trimer and the receptor within 50 ms
- bound GDP dissociates and is replaced with GTP
- this causes dissociation of the trimer, releasing alpha GTP and beta-gamma subunits
what are the active forms of the G protein?
- alphaGTP
- beta-gamma
what are the actions of the active forms of the G protein?
diffuse in the membrane and can associate with various enzymes and ion channels, causing activation of the target
what does G protein activation lead to?
- amplification: a single complex can activate several G protein molecules, and each can remain associated with the effector enzyme for long enough to produce many molecules of product (often second messenger)
what is often the product of G protein activation?
second messenger
when is signalling of G proteins terminated? what happens after this?
when the hydrolysis of GTP to GDP occurs through the GTPase activity of the alpha subunit
- resulting alphaGDP dissociates from the effector, then reunites with beta-gamma
what are RGS? how can they affect GPCR?
- regulators of G protein signalling proteins
- possess a conserved sequence that binds specifically to alpha subunits to increase their GTPase activity, thus hastening the hydrolysis of GTP and inactivating the complex
- inhibitory effect on G protein signalling
how many RGS proteins are there?
~ 20 cellular proteins
what causes increased GTPase activity of the alpha subunit?
binding of the target protein by alpha subunit and/or RGS proteins
what are the Galpha protein subtypes?
Ga(s) Ga(i) Ga(o) Ga(q) Ga(12)
what are associated receptors and functions of Ga(s) subunits? what is it activated/blocked by?
associated receptors
- many amine and other receptors
- e.g. catecholamines, histamine, serotonin
functions
- stimulates adenylyl cyclase, increasing cAMP formation
activated by cholera toxin, which blocks GTPase activity, thus preventing inactivation
what are associated receptors and functions of Ga(i) subunits? what is it activated/blocked by?
associated receptors
- amine and other receptors (e.g. catecholamines, histamine, serotonin)
- opioid/cannabinoid receptors
functions
- inhibits adenylyl cyclase, decreasing cAMP formation
blocked by pertussis toxin, which prevents dissociation of the alpha-beta-gamma complex
what are associated receptors and functions of Ga(o) subunits? what is it activated/blocked by?
associated receptors
- amine and other receptors (e.g. catecholamines, histamine, serotonin)
- opioid/cannabinoid receptors
functions
- ?
- limited effects of alpha subunit (effects mainly due to beta-gamma subunits)
blocked by pertussis toxin
occurs mainly in nervous system
what are associated receptors and functions of Ga(q) subunits? what is it activated/blocked by?
associated receptors
- amide, peptide and prostanoid receptors
functions
- activates phospholipase C, increasing production of second messengers of inositol triphosphate and diacylglycerol
what are the associated receptors and functions of Gbeta-gamma subunits?
associated receptors
- all GPCRs
functions
- activate K+ channels
- inhibit voltage-gated calcium channels
- activate GPCR kinases
- activate mitogen-activated protein kinase cascade
- interact with some forms of adenylyl cyclase and phospholipase Cbeta
how many subtypes of the alpha subunit of G proteins are there?
more than 20
what subtypes stimulate and inhibit adenylyl cyclase?
Gs and Gi, respectively
what do cholera toxin and pertussis toxin do to G proteins?
- catalyse a conjugation reaction (ADP ribosylation) on the alpha subunit of G proteins
- cholera toxin acts only on Gs and causes persistent activation
- pertussis toxin specifically blocks Gi and Go by preventing dissociation of the G protein trimer
- symptoms of cholera, e.g. excessive secretion of fluid from the GI epithelium are due to uncontrolled activation of adenylyl cyclase
what are the main targets of G proteins, through which GPCRs control aspects of cell function?
- adenylyl cyclase: responsible for cAMP formation
- phospholipase C: responsible for inositol phosphate and diacylglycerol formation
- ion channels, particularly Ca2+ and K+
- Rho A/Rho kinase, a system that regulates the activity of many signalling pathways controlling cell growth and proliferaton, smooth muscle contraction etc
- mitogen activated protein kinase, a system that controls many cell functions, e.g. cell division
what is cAMP?
cyclic 3’,5’ adenosine monophosphate
- nucleotide synthesised synthesised within the cell from ATP by adenylyl cyclase
how is cAMP produced/inactivation?
- produced continuously and inactivated by hydrolysis to 5’-AMP by phosphodiesterases
what acts on GPCRs to increase/decrease the catalytic activity of adenylyl cyclase?
- drugs, hormones and neurotransmitters
- change concentration of cAMPs
how many molecular isoforms are there of adenylyl cyclase?
10 different molecular isoforms
what is the action of cAMP?
activation of protein kinases, esp. protein kinase A
what does cAMP do in cardiac myocytes?
increased activity of voltage-gated calcium channels
- phosphorylation of these channels increases the amount of Ca2+ entering the cell during the action potential, thus increasing the force of contraction of the heart
what does cAMP do in smooth muscle?
- phosphorylates (and inactivates) myosin light chain kinase, which is required for contraction
- accounts for smooth muscle relaxation produced by many drugs that increase cAMP production in smooth muscle
what are examples of Gi receptors?
- certain types of mAChR (e.g. M2 receptor of cardiac muscle)
- a2 adrenoceptors in smooth muscle
- opioid receptors
what is an example of a drug that activates adenylyl cyclase directly?
forskolin
how many subtypes of PDEs are there?
11
- some (e.g. PDE3 and PDE4) are cAMP selective
- others (e.g. PDE5) are cGMP selective
what are weak inhibitors of PDEs?
methylxanthines (e.g. theophylline and caffeine)
what drug is selective for PDE4?
- rolipram (asthma)
- PDE4 is expressed in inflammatory cells
what drug is selective for PDE5?
- milrinone, used for heart failure
- PDE5 is expressed in heart muscle
what drug is selective for PDE6?
- sildenafil (Viagra)
- enhances the vasodilator effects of NO and drugs that release NO, whose effects are mediated by cGMP
how does cAMP regulate energy metabolism?
- increased lipolysis
- reduced glycogen synthesis
- increased glycogen breakdown
how does cAMP increase lipolysis?
protein kinase phosphorylates/activates lipase
how does cAMP reduce glycogen synthesis?
protein kinase phosphorylates/inactivates glycogen synthase
how does cAMP increase glycogen breakdown?
protein kinase phosphorylates/activates phosphorylase kinase -> activated phosphorylase b -> conversion of glycogen to glucose-1-phosphate
what is PIP2? what is its structure?
- phosphatidylinositol (4,5) bisphosphate
- member of the membrane phospholipid class of phosphoinositides
- additional phosphate groups attached to the inositol ring
what is PIP2 a substrate for? what can it break up into?
- membrane bound enzyme, phospholipase Cbeta
- splits it into diacylglycerol and inositol (1,4,5) triphosphate
what is DAG and IP3?
diacylglycerol
inositol (1,4,5) triphosphate
what mediates the activation of phospholipase Cbeta (PLCb)?
Gq
how is PIP2 reformed?
- DAG is phosphorylated to form phosphatidic acid
- IP3 is dephosphorylated and then recoupled with phosphatdic acid to form PIP2
what is IP3? what is its role?
- water-soluble mediator that’s released into the cytosol and acts on a ligand-gated calcium channel present on the membrane of the endoplasmic reticulum
- controls the release of Ca2+ from intracellular stores
what can IP3 be converted into?
inositol (1,3,4,5) tetraphosphate
- role is unclear but it may play a role in controlling gene expression
what is the action and behaviour of DAG?
- activates protein kinase C, which catalyses the phosphorylation of intracellular proteins
- highly lipophilic, remains within the membrane
- binds to a specific site on the PKC molecule, causing it to migrate from the cytosol to the cell membrane (activation)
how many subtypes are there of PKC?
- at least 10
- distinct cellular distributions and phosphorylate different proteins
- several activated by DAG and raised intracellular Ca2+
what can PKCs be activated by?
- DAG and raised Ca2+ levels
- phorbol esters
- arachidonic acid (generated by action of phospholipase A2 on membrane phospholipids)
what are phorbol esters?
highly irritant, tumour promoting compounds produced by certain plants
what do the various PKC isoforms act on?
different functional proteins
- ion channels
- receptors
- enzymes
- transcription factors
- cytoskeletal proteins
how do G proteins target ion channels?
- control ion channel function directly by mechanisms not involving second messengers
- done by beta-gamma subunits of Gi and Go proteins
- controls K+ and Ca2+ channels
what are examples of G proteins acting directly on ion channels?
in cardiac muscle: mAChRs enhance K+ permeability which hyperpolarises the cells and inhibits electrical permeability
in neurons: many inhibitory drugs reduce excitability by opening G protein-activated inwardly rectifying K+ channels or by inhibiting voltage activated N and P/Q type Ca2+ channels
what is the Rho/Rho kinase system?
- activated by certain GPCRs which couple to G proteins of the G12/13 type
- free Galpha subunit interacts with a guanosine nucleotide exchange factor, which facilitates GDP-GTP exchange at another GTPase, Rho
what are the active/inactive forms of Rho?
Rho-GDP (resting form) is inactive
when GDP-GTP exchange occurs, Rho is activated, which activates Rho kinase
what is the action of Rho kinase?
- phosphorylates many substrate proteins
- controls cellular functions, e.g. smooth muscle contraction/proliferation, angiogenesis and synaptic remodelling
- enhances hypoxia-induced pulmonary artery vasoconstriction, which is importnat in pathogenesis of pulmonary hypertension
what is an example of a Rho kinase inhibitor?
fasudil
what is the MAP kinase system?
- involves several signal transduction pathways that are activated by cytokines and growth factors acting on kinase-linked receptors and by ligands activating GPCRs
- coupling of GPCRs to familes of MAP kinases can involve alpha and beta-gamma subunits and Src and arrestins (GPCR desensitisation)
- controls processes involved in gene expression, cell division, apoptosis and tissue regeneration
what is homologous desensitisation?
restricted to the receptors activated by the desensitising agonist
what is heterologous desensitisation?
affects other GPCRs
what processes are involved in GPCR desensitisation?
- receptor phosphorylation
- receptor internalisaton (endocytosis)
how can GPCRs be phosphorylated?
- residues (serine and threonine) mainly in the C terminal cytoplasmic tail can be phosphorylated by specific membrane-bound GPCR kinases and PKA/C
how do specific GPCR kinases act?
- on receptor activation, GRK2 and GRK3 are recruited to the plasma membrane by binding to free G protein beta-gamma subunits
- GRKs then phosphorylate the receptors in their activated state
how does phosphorylation lead to homologous desensitisation?
- phosphorylated receptor serves as a binding site for arrestins
- arrestins are intracellular proteins that block the interaction between the receptor and G protein, leading to selective homologous desensitisation
what is arrestin? what is its function?
- intracellular protein which blocks the interaction between the receptor and G proteins
- produces homologous desensitisation of GPCRs
- arrestin binding targets the receptor for endocytosis in clathrin-coated pits
what happens to an internalised GPCR?
- dephosphorylated and reinserted into the plasma membrane (resensitisation)
- trafficked to lysosomes for degradation (inactivation)
what can phosphorylation by PKA and PKC at residues different from those targeted by GRKs lead to?
- impaired coupling between the activated receptor and the G protein; agonist effect is reduced
- leads to homologous or heterologous desensitisation
- receptors phosphorylated by second messenger kinases are probably not internalised and are reactivated by dephosphorylation by phosphatases when the agonist is removed
what are the subtypes of the GABAb receptor?
2 subtypes of the GPCR, encoded by different genes
- functional receptor consists of a heterodimer of the two
what do most GPCRs exist as?
- some are functional as monomers
- most can exist as homomeric or heteromeric oligomers
what are the targets of receptors and G proteins?
receptors -> guanylyl cyclase and G proteins
G proteins -> adenylyl cyclase, phospholipase C and ion channels
what is the effect of guanylyl cyclase?
produces cGMP
what is the effect of adenylyl cyclase?
produces cAMP
what is the effect of phospholipase C?
produces IP3 and DAG
what is the effect of cGMP?
- activates protein kinase G
- ion channels
what is the action of PKG?
affects contractile proteins and ion channels
what is the effect of cAMP?
- activates protein kinase A
- ion channels
what is the action of PKA?
- activates enzymes, transport proteins etc
- ion channels
what is the action of IP3?
increased [Ca2+], leads to:
- activated enzymes, transport proteins etc
- activated contractile proteins
- activated ion channels
what is the action of DAG?
activates arachidonic acid and protein kinase C
what do arachidonic acids produce?
eicosanoids
- released as local hormones
- affect ion channels
what does PKC do?
activates enzymes, transport proteins etc, contractile proteins and ion channels
what does increased free intracellular Ca2+ lead to?
initiates many events, including contraction, secretion, enzyme activation and membrane hyperpolarization
how do G proteins affect ion channels?
- opening potassium channels, leading to membrane hyperpolarisation
- inhibiting calcium channels, thus reducing neurotransmitter release
what forms arachidonic acid and eicosanoids?
phospholipase A2 (controlled by G proteins)
how can constitutively active receptors act?
- GPCRs may be spontaneously active in the absence of any agonist
- seen in beta adrenoceptor, where mutations in the third intracellular loop/overexpression of the receptor, results in constitutive activation
- native GPCRs can show constitutive activity when expressed in vitro
- histamine H3 receptor shows constitutive activity in vivo
what is the process of receptor coupling to MAP kinases?
receptor activation -> RAF1 -> MEK1 -> ERK1/2 -> altered gene expression
receptor activation -> ASK1 -> MKK4/7 + MKK3/6 -> JNK1-3 + p38 (respectively) -> altered gene expression
what is the process of arrestin coupling to MAP kinases? how can ERK be activated?
GRK acts on two Ps on receptor -> binding of ARR
ERK is activated by:
- binding of Src at the plasma membrane then binding of ERK to Src
- direct activation after internalisation of the receptor/arrestin complex; JNK3 can also be activated like this
what is the process of desensitisation and trafficking of GPCRs?
- on prolonged agonist activation of the GPCR, selective GRKs are recruited to the plasma membrane and phosphorylate the receptor
- arrestin binds and trafficks the GPCR to clathrin-coated pits for internalisation into endosomes in a dynamin-dependent process
- GPCR is dephosphorylated by a phosphatase (PP2A)
- either recycled and reinserted to the plasma membrane or trafficked to lysosomes for degradation
what is biased agonism?
ligand-dependent selectivity for certain signal transduction pathways relative to a reference ligand at the same receptor
what are RAMPs? how were they discovered?
receptor activity-modifying-proteins
- family of membrane proteins that associate with some GPCRs and alter their functional characteristics
- discovered in 1998 when the functionally active receptor for the neuropeptide calcitonin gene-related peptide consisted of a complex of a GPCR that by itself lacked activity, with another membrane protein RAMP1
- when CRLR was coupled with RAMP2, it was activated by adrenomedulin
what are modern characteristics of GPCRs?
- one gene, through alternative splicing, RNA editing etc, can give rise to more than one receptor protein
- one GPCR protein can associate with others, or with proteins e.g. RAMPs, to produce more than one type of functional receptor
- different agonists may affect the receptor in different ways and elicit qualitatively different responses
- the signal transduction pathway does not invariably require G proteins, and shows cross-talk with tyrosine kinase-linked receptors
what can kinase-linked receptors be activated by?
- protein mediators: growth factors and cytokines
- hormones: insulin and leptin (at level of gene transcription)
what is the general structure of kinase-linked receptors?
- large proteins
- single chain of up to 1000 residues with single membrane-spanning helical region
- links a large extracellular ligand-binding domain to an intracellular domain of variable size and function
- over 100 have been cloned
what are roles of kinase-linked receptors?
controlling cell division, growth, differentiation, inflammation, tissue repair, apoptosis and immune responses
what are the main types of kinase-linked receptors?
- receptor tyrosine kinases
- receptor serine/threonine kinases
- cytokine receptors
what are examples of receptor tyrosine kinase receptors?
- receptors for many growth factors, e.g. epidermal growth factor and nerve growth factor
- TLRs
- insulin receptor (more complex, dimeric structure)
what are examples of receptor serine/threonine kinases? what do they do?
- smaller class than RTKs
- phosphorylate serine and/or threonine residues rather than tyrosine
- example: transforming growth factor
what are examples of cytokine receptors? what do they do?
- lack intrinsic enzyme activity
- when occupied, they activate various tyrosine kinases, such as Jak (the Janus kinase)
- ligands include cytokines such as interferons and CSFs
how is phosphorylation and dephosphorylation of proteins accomplished?
by kinases and phosphatases, respectively
- enzymes which are subject to regulation dependent on their phosphorylation status
what happens when ligands bind to kinase linked receptors?
in many cases, dimerisation occurs
- association of two intracellular kinase domains allows a mutual autophosphorylation of intracellular tyrosine residues to occur
what is the function of phosphorylated tyrosine residues?
serve as high-affinity docking sites for other intracellular proteins that form the next stage in the signal transduction cascade
what is the growth factor pathway of kinase-linked receptors?
Ras/Raf/mitogen-activated protein (MAP) kinase pathway
what are the steps in the growth factor pathway of kinase-linked receptors?
- growth factor binds to receptor domain which is lined to an intracellular tyrosine kinase domain through a transmembrane alpha helix
- conformation change and dimerisation occurs
- tyrosine autophosphorylation occurs. SH2-domain protein (Grb2) binds to intracellular tyrosine kinase domain
- phosphorylation of Grb2
- activation of Ras GDP/GTP exchange
- activation of Raf, which phosphorylates Mek
- Mek phosphoryaltes MAP kinase
- MAP kinase phosphorylates various transcription factors, which leads to gene transcription
what is the cytokine pathway of kinase-linked receptors?
Jak/Stat pathway
what are the steps in the cytokine pathway of kinase-linked receptors?
- cytokine binds to receptor, which is linked via a transmembrane alpha helix to an intracellular Jak domain
- dimerisation and conformational change; activation of Jak
- phosphorylation of receptor and Jak
- binding and phosphorylation of SH2-domain protein
- dimerisation of Stat
- gene transcription
what are SH2 domain proteins? what do they do?
Src homology proteins; first identified in the Src oncogene product
- highly conserved sequence of about 100 amino acids
- form a recognition site for the phosphotyrosine residues of the receptor
- individual SH2 domain proteins bind selectively to particular receptors, so the pattern of events triggered is specific
what happens when the SH2 domain protein binds to the phosphorylated receptor?
- varies according to the receptor involved
- many SH2 domain proteins are enzymes
- some growth factors activate a specific subtype of phospholipase C (PLCgamma), causing phospholipid breakdown, IP3 formation and Ca2+ release
- may couple phosphotyrosine-containing-proteins with other functional proteins
- end result is to activate or inhibit, by phosphorylation, transcription factors that migrate to the nucleus and suppress/induce expression of genes
what are Stats?
- family of transcription factors
- SH2-domain proteins that bind to phosphotyrosine groups on the receptor-Jak complex
- are phosphorylated themselves
- when activated, Stat migrates to the nucleus and activates gene expression
what is PI3 kinase? what are they activated by? what does it do?
phosphatidylinositol-3-kinase
- enzyme family activated by GPCRs and RTKs
- attach a phosphate group to position 3 of PIP2 to form PIP3
- protein kinases, esp. PKB, have recognition sites for PIP3 and are thus activated, controlling apoptosis, differentiation, proliferation and trafficking, and NO synthase activation in the endothelium
what does guanylyl cyclase respond to?
binding of natriuretic peptides
- activated by dimerisation
- similar to RTKs
what is CaM kinase?
Ca2+/calmodulin-dependent kinase
what target proteins do kinase cascades affect?
- enzymes
- receptors
- ion channels
- transporters
- transcription factors
- contractile proteins
- secretory mechanisms
what are responses to phosphorylation of target proteins by kinases?
- physiological responses
- immune response
- apoptosis
- malignant transformation
- growth
- differentiation
what is the NR family?
nuclear receptor family
- receptors for hormones/substances present in the cytoplasm of cells and translocate into the nucleus after binding with ligand
- includes orphan receptors (receptors with no known well-defined ligands)
what are examples of nuclear receptors?
- receptors for steroid hormones e.g. oestrogen and clucocorticoids
- thyroid hormone T3
- fat soluble vitamins D and A (retinoic acid)
what is an example of an orphan receptor?
- retinoid X receptor (first to be described in the 1990s)
- cloned on the basis of its similarity with the vitamin A receptor
what does the vitamin A receptor bind?
vitamin A derivative 9-cis-retinoic acid
what are adopted orphan receptors?
those which have found binding partners
where can nuclear receptors be located?
- in cytosol and then translocate to nucleus
- in nuclear compartment
how many members are there of the nuclear receptor family?
48
what is the structure of nuclear receptors?
- heterogenous N terminal domain harbours the AF1 (activation function 1) site
- core DNA binding domain with ‘zinc fingers’
- hinge region
- ligand-binding domain, AF2 co-activator domain and HSP binding
- C-terminal extension
what does the heterogenous N terminal domain of the nuclear receptor do?
- displays most heterogeneity
- harbours the AF1 site
- AF1 site binds to other cell-specific transcription factors in a ligand independent way and modifies the binding or regulatory capacity of the receptor itself
- alternative splicing of genes may yield several receptor isoforms each with different N-terminal regions
what does the core domain of nuclear receptors do?
- highly conserved
- consists of structure responsible for DNA recognition and binding
- comprises two zinc fingers: cysteine rich loops in the amino acid chain held in place by zinc ions
- recognise and bind to hormone response elements in genes
- regulates receptor dimerisation
what are zinc fingers?
cysteine rich loops in the amino acid chain held in place by zinc ions
- in the core DNA binding domain of nuclear receptors
what are HREs?
hormone response elements
- located in genes that are regulated by nuclear receptors
- short (4/5 base pairs) sequences of DNA to which NRs bind to modify gene transcription
what does the hinge region in nuclear receptors do?
- highly flexible
- allows it to dimerise with other NRs
- dimerisation can produce molecular complexes with diverse configurations, able to interact differently with DNA
what does the C-terminal do?
- contains the ligand-binding module
- specific to each class of receptor
what does the AF2 region do?
- important in ligand-dependent activation
- generally highly conserved although absent in Rev-erbAalpha and Rev-erbAbeta, which regulate metabolism as mart of a circadian rhythm
where are HREs usually present?
usually symmetrically in pairs or half-sites
- may be arranged together in different ways (e.g. simple or inverted repeats)
how do HREs affect gene transcription?
- ligand-bound receptor recruits large complexes of other proteins including co-activators/repressors to modify gene expression through its AF1 and AF2 domains
what are examples of co-activators that are recruited by ligand bound nuclear receptors?
- enzymes involved in chromatin remodelling, e.g. histone acetylase/decetylase: regulate unravelling of DNA to allow access by polymerase enzymes
what are examples of co-repressors that are recruited by ligand bound nuclear receptors?
histone decetylase and other factors that cause the chromatin to become tightly packed, preventing further transcriptional activation
what are the classifications of nuclear receptors?
two main classes (I and II) and two other minor groups (III, IV)
what is class I of nuclear receptors?
endocrine steroid receptors
- glucocorticoid and mineralocorticoid receptors (GR and MR)
- oestrogen, progesterone and androgen receptors (ER, PR and AR)
what is the mechanism of action of class I nuclear receptors?
- after diffusion/transportation into the cell from blood, ligands bind their NR partner with high affinity
- form homodimers and translocate to the nucleus
- transactivate or transrepress genes by binding to positive or negative HREs
- once bound, the NR recruits other proteins to form complexes that promote transcription of multiple genes
where are class I NRs located?
in absence of their ligand, they’re predominantly located in the cytoplasm, complexed with head shock and other proteins and possibly reversibly attached to the cytoskeleton/other structures
what are the ligands for AR, ERalpha/beta, GRalpha, PR and MR nuclear receptors?
AR: testosterone ERalpha/beta: 17beta oestradiol GRalpha: cortisol, corticosterone PR: progesterone MR: aldosterone
what drugs are there for class I NRs?
- all natural and synthetic glucocorticoids, mineralocorticoids and sex steroids
- together with their antagonists (e.g. raloxifine, 4-hydroxy-tamoxifen and mifepristone)
what are class II NRs? what does this group include and what are their functions?
ligands are generally lipids already present to some extent within the cell
- peroxisome proliferator-activated receptor (PPAR): recognises fatty acids
- liver oxysterol receptor (LXR): recognises and acts as a cholesterol sensor
- farnesoid receptor (FXR)
- xenobiotic receptor (SXR): recognises foreign substances/drugs
- constitutive androstane receptor (CAR): recognises steroid androstane and some drugs
what is responsible for metabolising most prescription drugs?
CYP3A metabolises 60%
what is the action of class II NRs when they bind ligands?
- induce drug metabolising enzymes when they sense foreign material
- bind some prostaglandins, non steroidal drugs and the antidiabeteic thiazolidinediones and fibrates
what is the ligand binding/operation of class II NRs?
almost always operate as heterodimers with the retinoid X receptor (RXR)
what types of heterodimers can be formed by class II NRs?
- non-permissive heterodimer: can be activated only by RXR ligand itself
- permissive heterodimer: can be activated by retinoic acid itself or by its partner’s ligand
how do class II NRs affect gene transcription?
- generally bound to co-repressor proteins
- dissociate when the ligand binds and allows recruitment of coactivator proteins
- leads to changes in gene transcription
- tend to mediate positive feedback effects
what do class III NRs do?
- form homodimers
- can bind to HREs, which don’t have an inverted repeat sequence
what do class IV NRs do?
- function as monomers or dimers
- only bind to one HRE half site
what are ion channels?
consist of protein molecules designed to form water-filled pores that span the membrane, and can switch between open and closed states
what is the rate and direction of ion movement through the pore governed by? what is this a function of?
electrochemical gradient for the ion, which is a function of its concentration on either side of the membrane, and of the membrane potential
what are ion channels characterised by?
- selectivity for particular ion species, determined by the size of the pore and nature of its lining
- gating properties (the nature of the stimulus that controls the transition between open and closed states of the channel)
- molecular architecture
what are the main cation-selective channels selective for?
- Na+, Ca2+ or K+
- or non-selective and permeable to all three
what are anion channels mainly permeable to?
Cl-
what is an example of a ligand-gated ion channel that doesn’t respond to neurotransmitters?
responds to changes in the local environment instead
- TRPV1 channel on sensory nerves mediates pain-producing effect of chilli pepper ingredient capsaicin
- responds to extracellular protons when tissue pH falls in inflamed tissue, and to heat
what ligand-gated channels in the plasma membrane respond to intracellular signals rather than extracellular ones?
- calcium-activated potassium channels: open and hyperpolarise the cell when Ca2+ increases
- calcium-activated chloride channels
- ATP-sensitive potassium channels: open when intracellular ATP concentration falls because the cell is short of nutrients
- arachidonic-acid sensitive potassium channels
- DAG sensitive calcium channels
what are the functions of calcium-activated chloride channels?
expressed in excitable and non-excitable cells
- epithelial secretion of electrolytes and water
- sensory transduction
- regulation of neuronal and cardiac excitability
- regulation of vascular tone
where are ATP-sensitive potassium channels located?
- many nerve and muscle cells
- insulin-secreting cells
what are calcium release channels? what do they do?
- IP3 and ryanodine receptors
- class of ligand-gated calcium channels that are present on the endoplasmic or sarcoplasmic reticulum rather than the plasma membrane
- control the relase of Ca2+ from intracellular stores
where can calcium also be released from, apart from the endoplasmic and sarcoplasmic reticulum? what controls this?
- from lysosomal stores
- by nicotinic acid adenine dinucleotide phosphate activating two pore domain calcium channels
what are store operated calcium channels? what is their function?
- located in the plasma membrane
- open when intracellular Ca2+ stores are depleted; through interaction of a Ca2+ sensor protein in the ER membrane with a dedicated Ca2+ channel in the plasma membrane
- opening of channels allows cytosolic free Ca2+ conc to remain elevated even when intracellular stores are low
what are examples of cation channels?
6T1P
2T1P
4T2P
what are examples of 6T1P channels?
voltage gated K+ channels, TRP channels
what are examples of 2T1P channels?
inward-rectifying K+ channels, acid-sensing ion channels, epithelial Na+ channel, degenerins
what are examples of 4T2P channels?
resting K+ channels
what factors modulate the gating and permeation of both voltage-gated and ligand-gated ion channels?
- ligands that bind directly to various sites on the channel protein
- mediators and drugs that act indirectly, mainly by activation of GPCRs
- intracellular signals, particularly Ca2+ and ATP/GTP
what are drug-binding domains of voltage-gated sodium channels?
altered gating: GPCR ligands -> phosphorylation of channel
channel block (extracellular side): tetrodotoxin, saxitoxin, conotoxins
block of inactivation: veratridine, batrachotoxin, scorpion toxins, DDT, pyrethroids
channel block: local anaesthetics, antiepileptic drugs, antidysrythmic drugs
how are receptors regulated in the short and long term?
short term regulation: desensitisation
long term regulation: increase or decrease of receptor expression
what are the principal mechanisms in receptor malfunction?
- autoantibodies directed against receptor proteins
- mutations in genes encoding receptors, ion channels and proteins involved in signal transduction
what is the pathway of adrenaline and noradrenaline synthesis?
tyrosine -> dopa -> dopamine -> noradrenaline -> adrenaline
- final step only occurs in the adrenal medulla
what was the timeline of ANS discovery?
1860s: muscarine was shown to mimic the actions of nervous activation and atropine to oppose these actions
1905: Langley showed nicotine activated the NMJ and curare opposed this activation
1920s: vagal nerve stimulation slowed the heart and released a transferrable chemical that could slow (frog) hearts
what is the role of the ANS?
- peripheral ANS divides into sympathetic and parasympathetic branches (opposing effects)
- ANS conveys all outputs from the CNS to the body, except for skeletal muscular control
- regulatory roles in vascular, airway and visceral smooth muscle, exocrine secretions, control of heart rate, energy metabolism in the liver, links to immune system
what is the structure of the ANS/SNS?
somatic nervous system: neurone comes from the CNS to innervate muscle
ANS: two nerves in series, the pre- and post-ganglionic fibres
- parasympathetic ganglia are near their targets with short post-ganglionic nerves
- sympathetic ganglia are near the spinal cord with longer post-ganglionic fibres
what are effects of the parasympathetic division of the ANS?
- constricts pupil
- stimulates tear glands
- strong stimulation of salivary flow
- slows heart rate
- constricts bronchi
- stimulates digestive juice secretion
- stimulates intestinal motility
- contracts bladder
- stimulates erection
what are the effects of the sympathetic division of the ANS?
- dilates pupil
- tear glands maintain eye moisture
- inhibition of excess salivary secretion
- accelerates heart rate and constricts arterioles
- dilates bronchi
- inhibits stomach motility and secretion
- inhibits pancreas and adrenals
- inhibits intestinal motility
- relaxes bladder
- stimulates ejaculation
what neurotransmitters are released in sympathetic neurones? what receptors do they act on?
preganglionic neuron: cholinergic, ACh released at autonomic ganglion, acts on nicotinic receptor of postganglionic neuron
postganglionic neuron: adrenergic, norepinephrine (NE) released at target tissue which has adrenergic alpha or beta receptors
what neurotransmitters are released in parasympathetic neurones? what receptors do they act on?
preganglionic neuron: cholinergic, ACh released at autonomic ganglion, acts on nicotinic receptor of postganglionic neuron
postganglionic neuron: cholinergic, ACh released at target tissue which has cholinergic receptors (muscarinic)
what cranial nerves carry parasympathetic signals?
III (oculomotor), VII (facial), IX (glossopharyngeal) and X (vagus)
what are examples of dual parasympathetic and sympathetic innervation with opposing roles?
gut, bladder, heart
what is innervated only by sympathetic nerves?
sweat glands, blood vessels
what is innervated only by parasympathetic nerves?
bronchial smooth muscle
what do post-ganglionic sympathetic fibres release when innervating sweat glands?
ACh to stimulate muscarine receptors
what are NANCs? what are they used by?
non-adrenergic, non-cholinergic autonomic receptors
- used by enteric nervous system, parasympathetic and sympathetic system
what are examples of NANC use in parasympathetic and sympathetic systems?
parasympathetic: NO and vasoactive intestinal peptide
sympathetic: ATP and neuropeptide Y
what does nicotine stimulate?
all autonomic ganglia via specific ganglionic nicotinic receptors, activating both parasympathetic and sympathetic nervous systems
what does muscarine stimulate?
- from poisonous mushroom
- activates muscarinic receptors of the parasympathetic nervous system
- amenable to drug targeting
what are muscarinic receptors?
M1-5, GPCRs
where is M1 located?
mainly in the brain
where is M2 located?
- mainly in the heart
- activation slows the heart, so we can block these
what are examples of M2 blockers?
atropine for life-threatening bradycardias and cardiac arrest
where is M3 located? what does it cause?
- glandular and smooth muscle
- causes bronchoconstriction, sweating, salivary gland secretion
where is M4/5 located?
mainly in the CNS
what is an example of a muscarinic agonist? what could it be used to treat?
pilocarpine
- stimulates salvation: useful after radiotherapy, or in Sjorgren’s syndrome (PNS)
- contracts iris smooth muscle: used to treat glaucoma by facilitating drainage of aqueous humour (PNS)
what is a side effect of Pilocarpine/muscarinic agonists?
slow the heart
what can hyoscine be used for?
in palliative are, to antagonise parasympathetic driven secretions
where is atropine from?
deadly nightshade
how can muscarinic antagonists be used to treat bradycardia?
- prevent bradycardia and BP drop, dry secretions perioperatively
- treat bradycardias induced by excessive doses of beat blockers
- treat bradycardia in cardiac arrest
how can muscarinic antagonists be used to treat bronchoconstriction? what are examples of short/long acting ones?
- in the airway, drugs block the M3 receptor (called anti-cholinergics or anti-muscarinics)
- short acting: ipratropium bromide (atrovent)
- long acting: LAMAs e.g. tiotropium, glycopyrrhonium
how is selectivity of muscarinic antagonists in treatment of bronchoconstriction controlled?
- selectivity by drug delivery mechanisms (inhalers)
- receptor selectivity (e.g. tiotropium relatively selective for M3 receptors)
what other muscle roles do anticholinergics have?
- treating overactive bladders (e.g. solifenacin)
- short acting ones open up the pupil to allow eye examination
- act on parasympathetic fibres in intestinal colic and spasm: IBS (mebeverine) and palliative care (hyoscine)
what roles do antimuscarinics and anticholinergics have outside the autonomic system?
- ACh signalling involved in memory: anticholinergics worsen memory, and acetylcholinesterase inhibitor may treat dementia
- anti-emetic actions (e.g. hyoscine for travel sickness)
how can anticholinergics affect memory?
worsen memory
- acetylcholinesterase inhibitors may treat dementia
what is muscarine the active principle of?
the posionous mushroom Amanita muscaria
what do larger doses of ACh produce after muscarinic effects have been blocked by atropine?
nicotinic-like effects, including:
- stimulation of all autonomic ganglia
- stimulation of voluntary muscle
- secretion of adrenaline from the adrenal medulla
- initial rise in BP due to stimulation of sympathetic ganglia and consequent vasoconstriction
- secondary rise in BP due to secretion of adrenaline
what do small and medium doses of ACh produce?
transient fall in BP due to arteriolar vasodilation and slowing of the heart: muscarinic effects that are abolished by atropine
what effect does ACh have on vascular endothelial cells?
release NO, which relaxes smooth muscle to cause generalised vasodilation
what do the muscarinic actions of ACh correspond to? are there any exceptions?
to those of ACh releaesd at postganglionic parasympathetic nerve endings
exceptions:
- ACh causing generalised vasodilation even though most blood vessels have no parasympathetic innervation
- ACh evokes secretion from sweat glands, which are innervated by cholinergic fibres of the sympathetic nervous system
what do the nicotinic actions of ACh correspond to?
to those of ACh acting on autonomic ganglia of the sympathetic and parasympathetic systems, the motor endplate of voluntary muscle and the secretory cells of the adrenal medulla
what are the main classes of nicotinic ACh receptors?
muscle, ganglionic and CNS types
what is the structure of nAChRs?
- pentameric
- five subunits are similar in structure
what are the different routes of administration for drugs?
oral (po) intravenous (iv) rectal (pr) subcutaneous (sc) intramuscular (im) intranasal (in) topical (top) sublingual (sl) inhaled (inh) nebulised (neb)
how are the 8 week immunisations administered?
mainly intramuscular (im) rotavirus is administered orally (po)
what are the vaccines that are given at 8 weeks?
- 5 in 1 vaccine (diphtheria, tetanus, whooping cough (pertussis), polio and haemophilus influenza type b)
- pneumococcal vaccine
- rotavirus vaccine
- Men B vaccine
how can paracetamol be administered?
orally (po), per-rectum (pr), intravenous (iv)
what are the members of the nAChRs subunit family?
- five subunits that form the receptor-channel complex are similar in structure
- so far 17 different members have been identified and cloned, designated alpha (10 types), beta (4 types), gamma, delta and e (one of each)
what is the structure of nAChR subunits?
each possess 4 membrane spanning helical domains
- one of these helices (M2) from each subunit defines the central pore
what do nAChR subtypes generally contain?
both alpha and beta subunits, the exception being the homomeric (alpha7)5 subtype found in the bran
where do the two binding sites for ACh on nAChRs reside?
- interface between the extracelluar domain of each of the alpha subunits and its neighbour
what is the muscle type nAChR?
(α1)2β1δε
what is the ganglion type nAChR?
(α3)2(β4)3
what is the CNS type nAChR?
(α4)2(β2)3
(α7)5
what are the main synaptic locations of nAChR subtypes?
(α1)2β1δε: skeletal NMJ, mainly postsynaptic
(α3)2(β4)3: autonomic ganglia, mainly postsynaptic
(α4)2(β2)3: many brain regions; pre- and postsynaptic
(α7)5: many brain regions; pre- and postsynaptic
what are the membrane responses of nAChR subtypes?
(α1)2β1δε: excitatory; increased cation permeability, mainly Na+ and K+
(α3)2(β4)3: excitatory; increased cation permeability, mainly Na+ and K+
(α4)2(β2)3: pre and postsynaptic excitation; increased cation permeability, mainly Na+ and K+
(α7)5: pre and postsynaptic excitation; increased cation permeability; produces large Ca2+ entry, evoking transmitter release
what are the agonists of nAChR subtypes?
(α1)2β1δε: acetylcholine, carbachol, succinylcholine
(α3)2(β4)3: acetylcholine, carbachol, nicotine, epibatidine, dimethylphenylpiperazinium
(α4)2(β2)3: nicotine, epibatidine, acetylcholine, cytosine, varenicline
(α7)5: epibatidine, dimethylphenylpiperazinium, varenicline
what are the antagonists of nAChR subtypes?
(α1)2β1δε: tubocurarine, pancuronium, atracurium, vecuronium, alpha-bungarotoxin, alpha-conotoxin
(α3)2(β4)3: mecamylamine, trimetaphan, hexamethonium, alpha-conotoxin
(α4)2(β2)3: mecamylamine, methylaconitine
(α7)5: alpha-bungarotoxin, alpha-conotoxin, methylaconitine
what do muscarinic receptors couple with? what do they activate?
- odd numbered muscarinic receptors (M1, M3 and M5) couple with Gq to activate the IP3 pathway
- even numbered receptors (M2 and M4) open potassium channels causing membrane hyperpolarisation and acting through Gi to inhibit adenylyl cylcase and reduce intracellular cAMP
- both groups activate the MAP kinase pathway
what is the location of M1 muscarinic receptors?
- autonomic ganglia (including intramural ganglia in stomach)
- glands: salivary, lacrimal, etc
- cerebral cortex
what is the location of M2 muscarinic receptors?
heart: atria
CNS: widely distributed
what is the location of M3 muscarinic receptors?
exocrine glands: gastric (acid-secreting parietal cells), salivary etc
smooth muscle: GI tract, eye, airways, bladder
blood vessels: endothelium
what is the location of M4 muscarinic receptors?
CNS
what is the location of M5 muscarinic receptors?
CNS: very localised expression in substantia nigra
salivary glands and iris/ciliary muscle
what is the cellular response to M1 activation?
- increased IP3, DAG depolarisation
- excitation (slow epsp)
- decreased K+ conductance
what is the cellular response to M2 activation?
- decreased cAMP
- inhibition
- decreased Ca2+ conductance
- increased K+ conductance
what is the cellular response to M3 activation?
- increased IP3
- stimulation
- increased [Ca2+]
what is the cellular response to m4 activation?
- decreased cAMP
- inhibition
what is the cellular response to M5 activation?
- increased IP3
- excitation
what is the functional response to M1 activation?
- CNS excitation
- improved cognition?
- gastric secretion
what is the functional response to M2 activation?
- cardiac inhibition
- neural inhibition
- central muscarinic effects
what is the functional response to M3 activation?
- gastric, salivary secretion
- GI smooth muscle contraction
- ocular accommodation
- vasodilation
what is the functional response to M4 activation?
enhanced locomotion
what are non-selective M1 agonists?
- acetylcholine
- carbachol
- oxotremorine
- pilocarpine
- bethanechol
what are selective agonists for M1 and M3?
M1: McNA343
M3: Cavimeline
what are non-selective antagonists for M1?
- atropine
- dicycloverine
- tolterodine
- oxybutynin
- ipratropium
what are selective agonists for M1, M2, M3 and M4?
M1: Pirenzepine and Mamba toxin MT7
M2: Gallamine
M3: Darifenacin
M4: Mamba toxin MT3
what is Cevimeline used for?
- selective M3 agonist
- used to improve salivary and lacrimal secretion in Sjogren’s syndrome
what is Sjogren’s syndrome?
an autoimmune disorder characterised by dryness of the mouth and eyes
what has pirenzepine been used for?
peptic ulcer disease
what is darifenacin used for?
urinary incontinence in adults with detrusor muscle instability
what is Gallamine used for?
was used as a neuromuscular blocking drug
what are the classic muscarinic antagonists?
atropine and hyoscine
what is the process of acetylcholine synthesis?
- choline is taken up into the nerve terminal by a specific transporter
- free choline within the nerve terminal is acetylated by a cytosolic enzyme, choline acetyltransferase (CAT), which transfers the acetyl group from acetyl-CoA
what is the concentration of choline in the blood and body fluids? how does this change as it gets closer to cholinergic nerve terminals?
- normally 10 micromol/l
in immediate vicinity of - cholinergic nerve terminals: 1 mmol/l
- more than 50% of hydrolysed released ACh is recaptured by the nerve terminals
what is the rate limiting process in ACh synthesis?
choline transport, which is determined by the extracellular choline concentration and is linked to the rate at which ACh is released
what does ACh act on?
- acts postsynaptically on a nicotinic ACh receptor controlling a cation channel
- acts presynaptically on a nicotinic receptor that acts to facilitate ACh release during sustained synaptic activity
what does acetylcholinesterase break ACh down into?
choline and acetate
what inhibits AChE?
anticholinesterases e.g. neostigmine
what does inhibition of AChE cause?
- amount of free ACh and rate of leakage of ACh via the choline carrier is increased
how is choline transferred back into the presynaptic nerve?
choline carrier, which is coupled with ACh leaving
what inhibits the choline carrier?
hemicholinium
what inhibits ACh carrier formation?
vesamicol
what inhibits exocytosis of ACh containing vesicles?
presynaptic toxins, e.g. botulinum
what stimulates exocytosis of ACh containing vesicles?
recycling of ACh via presynaptic nicotinic ACh receptor
what inhibits the action of ACh on pre and postsynaptic nicotinic ACh receptors?
non-depolarising blocking agents, e.g. tubocurarine
what stimulates the action of ACh on postsynaptic nicotinic ACh receptors?
depolarising blocking agents, e.g. suxamethonium
what is the concentration of ACh in synaptic vesicles?
100 mmol/l
what triggers ACh release from vesicles?
Ca2+ entry into the nerve terminal
what is accumulation of ACh coupled to? what is it blocked by?
the large electrochemical gradient for protons that exists between acidic intracellular organelles and the cytosol
- blocked selectively by the experimental drug vesamicol
how many synaptic vesicles does a single nerve impulse release?
300 synaptic vesicles (around 3 million ACh molecules) supplying a single muscle fibre
how long do ACh molecules remain bound to receptors for?
around 2 ms, and are quickly hydrolysed after dissociating so they can’t combine with a second receptor
what modulates presynaptic release of ACh?
- ACh acts on presynaptic receptors and stimulates activity
- at postganglionic parasympathetic nerve endings, inhibitory M2 receptors participate in autoinhibition of ACh release
- noradrenaline inhibits the release of ACh
- at the NMJ, presynaptic nAChRs facilitate ACh release
what does ACh cause at the postsynaptic membrane of a nicotinic (neuromuscular or ganglionic) synapse?
- causes a large increase in its permeability to cations, particularly to Na+ and K+, and to a lesser extent Ca2+
- inflow of Na+ depolarises the postsynaptic membrane
what is the ACh-mediated depolarisation of nicotinic receptors called in skeletal muscle fibres and at the ganglionic synapse?
endplate potential in skeletal muscle fibre
fast excitatory postsynaptic potential at the ganglionic synapse
what is the effect of tubocurarine?
- blocks postsynaptic ACh receptors
- reduces the amplitude of the fast epsp until it no longer initiates an action potential
- cell is still capable of responding when stimulated electrically
what is integrative action?
most ganglion cells are supplied by several presynaptic axons, and require simultaneous activity in more than one to make the postganglionic cell fire
at the NMJ, what supplies each muscle fibre?
only one nerve fibre
what is a slow ipsp?
- lasts 2-5 s
- this mainly reflects a M2 receptor mediated increase in K+ conductance, but other transmitter, e.g. dopamine and adenosine also contribute
what is a slow epsp?
- lasts for 10s
- produced by ACh acting on M1 receptors, which close K+ channels
what is a late slow epsp?
- lasts for 1-2 min
- may be mediated by a peptide co-transmitter, which may be substance P in some ganglia, and a gonadotrophin-releasing hormone-like peptide in others
when does depolarisation block occur?
- at cholinergic synapses when the excitatory nAChRs are persistently activated
- results from a decrease in the electrical excitability of the postsynaptic cell
why is there a loss of electrical excitabillity during a period of maintained depolarisation?
voltage-sensitive sodium channels become inactivated (refractory) and are no longer able to open in response to a brief depolarising stimulus
what happens when nicotine is applied to a sympathetic ganglion?
- activation of nAChRs, causing a depolarisation of the cell, which initiates action potential discharge
- after a few seconds, this discharge ceases and the transmission is blocked
when/why does secondary, non-depolarising block occur?
- after nicotine has acted for several minutes, the cell partially repolarises and its electrical excitability returns
- transmission remains blocked
- occurs at the NMJ if repeated doses of depolarising drug suxamethonium are used
what is the clinical name for the secondary non-depolarising block?
phase II block
what is the speed of transmission at muscarinic receptors?
much slower, synaptic structures are less defined
- ACh functions as a modulator rather than as a direct transmitter
what are the main mechanisms of pharmacological block in cholinergic transmission?
- inhibition of choline uptake
- inhibition of ACh release
- block of postsynaptic receptors or ion channels
- persistent postsynaptic depolarisation
what are the types of drugs that affect cholinergic transmission?
- muscarinic agonists
- muscarinic antagonists
- ganglion-stimulating drugs
- ganglion-blocking drugs
- neuromuscular-blocking drugs
- anticholinesterases and other drugs that enhance cholinergic transmission
what are the only muscarinic agonists used clinically?
bethanechol, pilocarpine and cevimeline
what is bethanechol used for clinically?
treatment of bladder and GI hypotonia
what is pilocarpine used for clinically?
glaucoma
what is cevimeline used for clinically?
Sjogren’s syndrome (to increase salivary and lacrimal secretion)
what are muscarinic agonists?
- acetylcholine
- carbachol
- methacholine
- bethanechol
- muscarine
- pilocarpine
- oxotremorine
- cevimeline
what are the cardiovascular effects of muscarinic agonists?
- cardiac slowing and decrease in in cardiac output
- due to reduced heart rate and a decreased force of contraction of the atria
- generalised vasodilation (mediated by NO)
- sharp fall in arterial pressure
what is an example of adsorption?
paracetamol binding to activated charcol
what is pharmacodynamics?
effect of a drug on the body
what is pharmacokinetics?
effect of the body on a drug
what are types of regulatory protein that are used as protein targets for drug binding?
- receptors
- enzymes
- carrier molecules (transporters)
- ion channels
what is a drug?
a chemical applied to a physiological system that affects its function in a specific way
what is a receptor?
a component of a cell whose function is to recognise and respond to endogenous chemical signals
- interacts with a specific ligand (exogenous or endogenous) and initiates a change of biochemical events leading to the ligand observed effects
what are drug targets?
macromolecules with which drugs interact to produce their effects
what is the difference between agonists and antagonists?
agonists: activate the receptors
antagonists: combine at the same site without causing activation, and block the effect of agonists on that receptor
what is drug specificity?
- drugs must act selectively on particular cells and tissues
- must show a high degree of binding site specificity
what is ligand specificity?
proteins that function as drug targets show a high degree of ligand specificity; they bind only molecules of a certain precise type
what are qualitative and quantitative aspects of pharmacodynamics?
qualitative: receptors, enzymes, selectivity
quantitative: dose response, potency, therapeutic efficacy, tolerance
what are factors affecting pharmacokinetics?
time course of drug concentration: drug passage across membranes, order of reaction, plasma half life and steady-state concentration; therapeutic drug monitoring
individual processes: absorption, distribution, metabolism, elimination
drug dosage: dosing schedules
chronic pharmacology: consequences of prolonged drug administration and drug discontinuation syndromes
what are examples of individual or biological variations that affect drug metabolism?
- pharmacogenomics: variability due to inherited influences
- variability due to environmental and host influences
- drug interactions: outside the body, at site of absorption, during distribution, directly on receptors, during metabolism, during excretion
what is pharmacogenomics?
variability due to inherited influences
how do drugs act on processes within or near the cell?
- enzyme inhibition
- inhibition or induction of transporter processes that carry substances into, across and out of cells
- incorporation into larger molecules
- altering metabolic processes unique to microorganisms, or by showing quantitative differences in affecting a process common to both humans and microbes
how do drugs act outside the cell?
- direct chemical interaction
- osmosis
what is down-regulation?
when tissues are continuously exposed to an agonist, the number of receptors decreases
what is tachyphylaxis?
loss of efficacy with frequently repeated doses
- can be caused by down-regulation
what is up-regulation?
prolonged contact with an antagonist leads to formation of new receptors
what are agonists? how do they act on receptors?
- drugs that activate receptors
- they resemble the natural transmitter or hormone
- value in clinical practice often rests on their greater capacity to resist degradation and to act for longer than the endogenous ligands they mimic
what are antagonists? how do they act on receptors?
- blockers of receptors
- sufficiently similar to the natural agonist to be recognised by the receptor and to occupy it without activating a response
- block the natural agonist from exerting its effect
what are pure antagonists?
drugs that have no activating effect whatsoever on the receptor
what does ADME stand for?
absorption, distribution, metabolism and excretion
what is ‘ligandability’?
the ability of a protein target to bind small molecules with high affinity
what ligands bind to GPCRs?
light energy, peptides, lipids sugars and proteins
what is the action of tamoxifen?
- treats breast cancer
- acts as a selective estrogen receptor (SERM), or as a partial agonist of the estrogen receptors
what is EC50?
the concentration that gives half the maximal response
what is efficacy/intrinsic activity?
ability of a drug-receptor complex to produce a maximum functional response
what is non-competetive antagonism?
binding to an allosteric (non-agonist) site on the receptor to prevent activation of the receptor
what is the agonist/antagonist for mAChR?
muscarine and atropine
what is the agonist/antagonist for nAChR?
nicotine and curare
how is the H1 receptor (histamine) characterised?
- amino acids: 481
- protein: 56 kDa
- allergic conditions
- GPCR
how is the H2 receptor (histamine) characterised?
- amino acids: 359
- protein: 40 kDa
- gastric acid secretion
how is the H3 receptor (histamine) characterised?
- amino acids: 445
- protein: 49 kDa
- mostly CNS disorders (e.g. narcolepsy, ADHD, Alzheimers, schizophrenia)
- role in obesity, pain and rhinitis
- GPCR
how is the H4 receptor (histamine) characterised?
- amino acids: 390
- protein: 44 kDa
- immune system and inflammatory conditions (e.g. rhinitis, pruritis and asthma)
- inflammatory pain
- GPCR
what is the agonist/antagonist for H2 receptor?
histamine and mepyramine
what is the effect of histamine (agonist) on H2 receptor?
- contraction of ileum
- acid secretion from parietal cells
what is the effect of mepyramine (antagonist) on H2 receptor?
- reversed contraction of ileum
- no effect on acid secretion
what are receptor-related factors governing drug action?
affinity and efficacy
what are tissue-related factors governing drug action?
receptor number and signal amplification
what is affinity? do agonists and antagonists show it?
describes how well a ligand binds to the receptor
- shown by agonists and antagonists
what is efficacy? do agonists and antagonists show it?
describes how well a ligand activates the receptor
- shown by agonists but not antagonists
what is inverse agonism?
when a drug that binds to the same receptor as an agonist but induces a pharmacological response opposite to that of the agonist
what is tolerance?
- reduction in agonist effect over time
- continuously, repeatedly high concentrations
what is an enzyme inhibitor? how does it work?
a molecule that binds to an enzyme and decreases (normally) its activity
- prevents the substance from entering the enzyme’s active site and prevents it from catalysing its reaction
what are the types of enzyme inhibitors? how do they act?
- irreversible inhibitors: usually react with the enzyme and change it chemically (e.g. via covalent bond formation)
- reversible inhibitors: bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind to the enzyme, the enzyme-substrate complex or both
what is an example of enzymes themselves acting as drugs?
streptokinase: a clot buster
what are statins?
- HMG-CoA reductase inhibitors
- class of lipid-lowering medications that reduce the levels of bad cholesterol
what is the mechanism of statins? what is their effect?
- block the rate limiting step in the cholesterol pathway
- reduce cardiovascular disease and mortality in those at high risk
what is the rate limiting step in the cholesterol formation pathway? what is it catalysed by?
3-hydroxy-3-methyglytaryl-CoA (HMG-CoA) -> mevalonic acid
- catalysed by HMG-CoA reductase
what is HMG-CoA?
3-hydroxy-3-methylglutaryl-CoA
what do ACE inhibitors do?
- RAAS increases blood pressure by inceasing the amount of salt and water the body retains
- inhibition of ACE reduces the conversion of angiotensin I to angiotensin II
what are the effects of angiotensin II?
stimulates:
- sympathetic activity
- tubular Na+ Cl- reabsorption and K+ excretion; water retention
- aldosterone secretion
- arteriolar vasoconstriction; increase in BP
- ADH secretion -> H2O absorption in collecting duct
what is the overall effect of the RAAS
- water and salt retention
- increased effective circulating volume
- increased perfusion of the juxtaglomerular apparatus
- increase BP
- eventually inhibits renin production by kidney (negative feedback)
what is angiotensinogen produced by? what happens to it?
- liver
- renin converts it to angiotensin I
where is ACE located?
surface of pulmonary and renal endothelium
what are examples of ACE inhibitors?
captopril (1st gen) and enalaprilat (2nd gen)
what are symptoms of Parkinson’s disease?
- hypokinesia (decreased motor movement)
- tremor at rest
- muscle rigidity, motor inertia
- cognitive impairment
- degenerative disease of the basal ganglia
- early degeneration of dopaminergic neurons in the nigrostriatal pathway leading to autonomic dysfunction and dementia
what is L-DOPA produced from?
amino acid L-Tyrosine
what is the process of dopamine synthesis and action?
- L-DOPA crosses the BBB
- DOPA decarboxylase acts on L-DOPA to produce dopamine
- dopamine acts on D1 and D2 receptors
what is the action of a peripheral DDC inhibitor?
blocks DDC in the periphery, which generates more for the CNS pathway
- leads to improved Parkinsonoid symptoms
what are examples of a peripheral DDC inhibitor?
- carbidopa
- co careldopa
- benserzide
how is L-DOPA broken down, and what into? what catalyses this?
L-DOPA -> 3-methyl DOPA
catalysed by COMT: catechol-O-methyl transferase
what are the actions of a peripheral COMT inhibitor?
prevents breakdown of L-DOPA, generating more for the CNS pathway
- leads to improved Parkinsonoid symptoms
what are examples of peripheral COMT inhibitors?
- tolcapone
- entacapone
what is the action of central COMT inhibitors?
function within the CNS to prevent dopamine breakdown by COMT and thus keep dopamine levels up - improved Parkinsonoid symptoms
what is an example of a central COMT inhibitor?
tolcapone
what is DOPAC?
3,4-Dihydroxyphenylacetic acid
what catalyses the breakdown of dopamine into DOPAC?
mono amine oxidase B (MAO-B)
what is the action of mono amine oxidase B inhibitors?
prevent dopamine breakdown and increases availability
- improved parkinsonoid symptoms
what are examples of MAO-B inhibitors?
selegiline and rasagiline
what is the action of central dopamine receptor agonists?
antagonise dopamine receptors (not enzyme inhibitors)
- improved Parkinsonoid symptoms
what are examples of central dopamine receptor agonists?
- bromocryptine
- pergolide
- pramipexole
- ropinirole
- rotigotine
what is transport?
when molecules move across a cell membrane
what are the types of protein ports in cell membranes?
- uniporters: use energy from ATP to pull molecules in
- symptorters: use the movement in of one molecule to pull in another molecule against a concentration gradient
- antiporter: one substance moves against its gradient, using energy from the second substance moving down its gradient
what is the action of Furosemide?
- loop diuretic
- used for hypertension and edema
- inhibits luminal NKCC co-transporter in the thick ascending limb of the loop of Henle
- binds to the NKCC
- causes sodium, chloride and potassium loss in urine
what are examples of ion channels in dysfunctioning and causing disease?
epithelial (sodium): heart failure
voltage-gated (calcium, sodium): nerve, arrhythmia
metabolic (potassium): diabetes
receptor activated (chloride): epilepsy
what is the action of the ENaC channel?
- an apical membrane-bound heterotrimeric ion channel selectively permeable to Na+ ions
- causes reabsorption of Na+ ions at the collecting ducts of the kidneys nephrons
what drugs target the ENaC channel?
- blocked by the high affinity diuretic amiloride (often used with Thaizide)
- Thaizide targets Na+Cl- cotransporter that reabsorbs Na and Cl from tubular fluid
- used as an anti-hypertensive
how do voltage-gated calcium channels exist normally?
at physiologic or resting membrane potential, VDCCs are normally closed
what is an action potential?
a momentary change in electrical potential on the surface of a cell, that occurs when it is stimulated, resulting in the transmission of an electrical impulse
what is a voltage gated calcium channel inhibitor?
amlodipine
what is Amlodipine? what does it do?
an angioselective calcium channel blocker that inhibits the movement of calcium ions into vascular smooth muscle cells and cardiac muscle cells
what is the effect of Amlodipine?
- inhibit contraction of cardiac muscle and vascular smooth muscle cells
- causes vasodilation and a reduced peripheral vascular resistance -> lowered BP
- prevents excessive constriction in the coronary arteries
why is ENaC heterotrimeric?
two sets of three proteins: alpha, beta and gamma; all need to be activated
what are the conformational states of voltage-gated Na+ channels in excitable cells?
close, open and inactivated
what drug blocks voltage gated sodium channels? how does it affect the heart?
lidocaine (anaesthetic) blocks transmission of the action potential which allows the activation gates to open
- blocks signalling in the heart, thus reducing arrhythmia
what is the action of voltage gated potassium channels?
regulate insulin in pancreas: beta islets of Langerhans
- increased glucose goes through glucose transporter and is metabolised to produce ATP
- ATP closes ATP dependent K+ channels
- this leads to membrane depolarisation, which triggers calcium influx
- calcium influx stimulates exocytosis
what drugs affect calcium channels in pancreatic beta cells? how?
- repaglinide
- nateglinide
- sulfonylureal
- lower blood glucose levels by blocking K+ channels to stimulate insulin secretion; treats type II diabetes
what is a neutrotransmitter which opens receptor mediated Cl- channel?
GABA A receptor (gamma aminobutyric acid): major inhibitory neurotransmitter in the CNS
- opens Cl- channel and induces hyperpolarisation
what are drugs that increase permeability of the channel to chloride?
barbituates, e.g. phenobarbitone
what are the sites present in the chloride channel?
- GABA site
- barbituate site
- benzodiazepine site
- steroid site
- picrotoxin site
what is a drug that targets the Na/K ATPase?
- Digoxin was first isolated in 1930 from the foxglove plant, Digitalis Ianata
what is the action of Digoxin? what does this lead to?
- used for AF, atrial flutter and heart failure
- inhibition causes an increase in intracellular Na, resulting in decreased activity of the Na/Ca exchanger and increases intracellular Ca
- lengthens the cardiac action potential, which leads to a decrease in heart rate
what are the actions of proton pump inhibitors?
- block the proton pump in the stomach (acidifies the stomach and activates pepsin)
- most potent inhibitors of acid secretion
what is an example of a proton pump inhibitor?
omeprazole (1st in class)
- irreversible inhibition
- half life 1hour, works for 2-3 days
- metabolised at acid pH by enteric coated granules
what are organophosphates?
irreversible inhibitors of cholinesterase
what are examples of organophosphates?
insecticides (Diazinon) and nerve gases (Sarin)
what are muscarinic, nicotinic and CNS effects of organophosphates?
muscarinic: salivation, defecation, urination, bradycardia, hypotension
nicotinic: twitching, severe weakness, paralysis, diaphragm
CNS: confusion, loss of reflexes, convulsions, coma
what are xenobiotics?
compounds foreign to an organism’s normal biochemistry, such as any drug or poison
what are cytochrome P450s? where are they located?
primarily membrane associated proteins located either in the inner membrane of mitochondria or in the ER of cells
what percentage of drug metabolism is done by CYPs?
75%
what is the action of CYPs?
- drug metabolism
- deactivation and bioactivation of drugs
- metabolism of endogenous and exogenous chemicals
what are examples of types of CYPs?
2A6 2C19 2C8 2C9 3A4 2D6 1A2 2E1 2B6
what are substrates, inhibitors and inducers of CYP 2A6?
- coumarin
- methimazole
- phenobarbitone
what are substrates, inhibitors and inducers of CYP 2C19?
- mephenytoin
- tranylcypromine
- phenobarbitone
what are substrates, inhibitors and inducers of CYP 2C8?
- taxol
- quercetin
- phenobarbitone
what are substrates, inhibitors and inducers of CYP 2C9?
- S-warfarin
- phenytoin
- tolutamide
- sulphapheazole
- rifampicin
what are substrates, inhibitors and inducers of CYP 3A4?
- nifedipine
- cyclosporine
- erythromycine
- terfenadine
- ketoconazole
- rifampicin
what are substrates and inhibitors of CYP 2D6?
- debrisoquine
- metoprolol
- dextromethorphan
- quinidine
what are substrates, inhibitors and inducers of CYP 1A2?
- caffeine
- phenacetin
- theophylline
- furafylline
- omeprazole
what are substrates, inhibitors and inducers of CYP 2E1?
- chlorzoxazone
- disulfiram
- ethanol
- isoniazid
what are substrates and inhibitors of CYP 2B6?
- ifosphamide
- orphenadrine
what are substrates of CYP 1A2?
caffeine, theophylline, phenacetin, clomipramine, clozapine, thioridazine
what are inhibitors of CYP 1A2?
omeprazole, nicotine, cimetidine, ciprofloxacin
what are inducers of CYP 1A2?
phenobarbital, fluvoxamine, venlafaxine, ticlodipine
which drugs does avocado and brassicas affect? how?
- CYP450 inducer
- warfarin
which drugs does grapefruit juice affect? how?
- CYP450 inhibitor
- Ca2+ channel blockers
- cyclosporin
- tacrolimus
- carbemazepine
- midazolam
- terfenadine
which drugs does soya affect? how?
- CYP450 inhibitor
- clozapine
- haloperidol
- NSAIDs
- warfarin
- phenytoin
what drugs does garlic affect? how?
- increases anti-platelet activity
- anti-coagulants
- aspirin
- NSAIDs
what drugs does ginseng affect?
- anti-coagulants
- aspirin
- NSAIDs
what drugs does Ginko Biloba affect? how?
- strong inhibitor of platelet aggregation
- warfarin
- aspirin
- NSAIDs
what drugs does Hypericum perforatum (St Johns Wort) affect? how?
- CYP450 inducer
- warfarin
- digoxin
- cyclosporine
- phenytoin
- antiretrovirals
what drugs does ephedra affect? how?
- receptor level antagonist
- MAOIs
- CNS stimulants
what drugs does ginger affect? how?
- inhibits thromboxane synthetase
- anticoagulants
which CYP does grapefruit juice affect? how?
CYP3A4: increases bioavailability
what does renal excretion of drugs depend on?
- pH dependent
- weak bases: cleared faster if urine acidic
- weak acids: cleared faster if urine alkali
what are examples of drugs that are weak acids?
aspirin, ibuprofen, ampicillin, paracetamol, phenobarbital, warfarin, theophylline, phenytoin, levodopa
what are examples of drugs that are weak bases?
amphetamine, atropine, hydralazine, propranolol, amiloride, chlorpheniramine, diazepam, reserpine, salbutamol
how can a low-efficacy agonist act as an antagonist?
a receptor occupied by a low-efficacy agonist is inaccessible to a subsequent dose of a high efficacy agonist
what is partial agonist activity?
some drugs, in addition to blocking access of the natural agonist to the receptor, are capable of some activation
what is an example of partial agonists? what is an alternative name for it?
beta-adrenoceptor antagonists pinodolol and oxprenolol have partial agonist activity
- this is called intrinsic sympathomimetic activity
what is an example of a pure antagonist?
propanolol
what are inverse agonists? what is an example?
- some substances binding to the same receptor are specifically opposed to those of the agonist
- e.g. benzodiazepines acting on the benzodiazepine receptor in the CNS produces sedation, anxiolysis, muscle relaxation and controls convulsions; beta-carbolines, also binding to this receptor, cause stimulation, increased muscle tone and convulsions
- modulate effects of GABA
what is competetive antagonism?
an agonist and antagonist compete to occupy the receptor according to the law of mass action
what is the equilibrium dissociation constant Kd?
concentration of a radioligand that occupies half of a particular receptor population
- measures the affinity of a drug for a receptor/the strength of the ligand-receptor interaction
what is Bmax?
maximum receptor number/saturation
when does high/low affinity binding occur?
high affinity binding occurs at low drug concentrations; vice versa
how is Bmax used to determine Kd?
half of Bmax is used to extrapolate Kd on the x axis
what is efficacy?
the ability of a drug to produce a response
what is potency?
- the dose range over which a response is produced
- dose of drug that produces 50% of the maximum response (ED50)
- maximum response itself is a crude measure of efficacy
what are the types of antagonism?
- competetive (reversible, surmountable)
- non-competetive (irreversible, insurmountable)
how can the action of a competitive antagonist be overcome?
by increasing the dose of the agonist (i.e. the block is surmountable)
what does increasing the dose of the agonist have on the dose-response curve of an agonist and competitive/non-competitive antagonists?
competetive agonist: shifts to the right
- maximum response remains unchanged
- degree of rightward shift is related to the affinity of the antagonist and the dose used
- the higher the affinity of the antagonist, the greater the right shift
non-competitive antagonist: the binding site may be the same as the agonist but it’s reversible so cannot be displaced nor overcome
- depresses the maximum response
what are mixed agonists-antagonists?
where subtypes of receptors occur, a single ligand may have agonist and antagonist properties
what are inverse agonists?
a drug that binds to the same receptor as an agonist but induces a pharmacological response opposite to that of the agonist
what is physiological (functional) antagonism?
a second/another drug overcomes the pharmacological effect, by a different physiological mechanism
- produces counteracting effects to another substance using a mechanism that doesn’t involve binding to the same receptor
what is an example of irreversible inhibition?
occurs with organophosphorus insecticides and chemical warfare agents which combine covalently with the active site of acetylcholinesterase
- recovery of cholinesterase activity depends on formation of new enzyme
how can selectivity of drug action be obtained?
- modification of drug structure
- selective delivery (drug targeting)
- stereoselectivity
how do drug molecules move around the body?
- bulk flow (i.e. in the bloodstream, lymphatics or CSF)
- diffusion (i.e. molecule by molecule, over short distances)
what is the diffusion coefficient? what does the rate of diffusion depend on?
- diffusion coefficient is inversely proportional to the square root of molecular weight
- rate of diffusion of a substance depends mainly on its molecular size
what determines where and for how long a drug will be present in the body after administration?
movement between compartments, involving penetration of non-aqueous diffusion barriers
what is an example of cell membranes making it very difficult for drugs to pass between aqueous compartments in the body?
CNS and placenta have tight junctions between endothelial cells, and is encased in an impermeable layer of periendothelial cells (pericytes)
- prevents potentially harmful molecules from penetrating to brain or fetus
what controls the formation of fenestrated endothelium?
EG-VEGF
what are the four main ways by which small molecules cross cell membranes?
- diffusing directly through the lipid
- combination with a solute carrier (SLC) or other membrane transporter
- diffusing through aquaporins that traverse the lipid
- by pinocytosis
what can block aquaporins?
mercurial reagents such as para-chloromercurobenzene sulfonate
what is the role/importance of diffusion through aquaporins in pharmacokinetic mechanisms?
- important in transfer of gases e.g. CO2
- pores are too small in diameter (0.4nm) to allow most drug molecules (more than 1nm usually) to pass through
- drug distribution not notably abnormal in patients with genetic diseases affecting aquaporins
what is the importance/mechanism of drug absorption by pinocytosis?
- involves invagination of part of the cell membrane and the trapping within the cell of a small vesicle containing extracellular constituents
- vesicle contents released within cell or extruded from other side
- transport of some macromolecules, e.g. insulin across the BBB
- not for small molecules
what is the permeability coefficient, P?
the transport flux of material through the membrane per unit driving force per unit membrane thickness
what physiochemical factors contribute to the permeability coefficient?
- solubility in the membrane: can be expressed as a partition coefficient for substance distributed between the membrane phase and aqueous environment
- diffusivity: measure of the mobility of molecules within the lipid, expressed as a diffusion coefficient
how well does a lipid-soluble drug diffuse across a membrane from an extracellular compartment to an intracellular one, compared to a low lipid soluble drug? why?
- lipid soluble drug is subject to a much larger transmembrane concentration gradient than a lipid insoluble drug
- diffuses more rapidly, even though the aqueous concentration gradient is same in both cases
what do drugs that are weak acids or bases exist as? how does this affect membrane permeation?
- exist in both unionised and ionised forms, the ratio of both varying with pH
- ionised species, BH+ or A-, has very low lipid solubility and can’t permeate membranes
- lipid solubility of the uncharged species depends on chemical nature of the drug
how are weak acids and weak bases distributed between aqueous compartments? give examples
- gastric juice is acidic, plasma neutral and urine alkaline
- ionisation of weak acid, e.g. aspirin, is greatest at alkaline pH
- ionisation of weak base, e.g. pethidine, is greatest at acid pH
what is the ratio of ionised to unionised drug in aqueous compartments determined by?
pKa of the drug and pH of the compartment
what is ion trapping?
- ionised species is assumed to not permeate membrane at all, while the unionised species does
- acidic drug is concentrated in the compartment with high pH, vice versa
- can produce high concentration gradients if theres a large pH difference between compartments
why are large concentration gradients produced by ion trapping not achieved in reality?
- assuming total impermeability of the charged species is not realistic, and even a small permeability will change the concentration gradient
- body compartments rarely approach equilibrium
why is pH partition not the main determinant of the site of absorption of drugs from the GI tract?
difference between the large SA of the villi and microvilli in the ileum compared to the smaller SA in the stomach
what is absorption of acidic drugs e.g. aspirin in the GI tract affected by?
- promoted by drugs that accelerate gastric emptying (e.g. metoclopramide)
- retarded by drugs that slow gastric emptying (e.g. propantheline)
what are basic drugs, from strong to weak?
chloroquine, desmethylimipramine, amphetamine, atropine, histamine, propanolol, chlorpromazine, mepyramine, dopamine, noradrenaline, morphine, ergometrine, trimethoprim, chlordiazepoxide, diazepam
what are acidic drugs, from strong to weak?
levodopa, penicillins, probenecid, aspirin, methotrexate, warfarin, sulphamethoxazole, chlorothiazide, phenobarbital, thiopental, phenytoin, ascorbic acid
what are the consequences of pH partition?
- free-base trapping of some antimalarial drug (e.g. chloroquine) in the acidic environment in the food vacuole of the malaria parasite contributes to disruption of the haemoglobin digestion pathway underlying their toxic effect)
- urinary acidification accelerates excretion of weak bases and retards that of weak acids; vice versa for urinary alkalinisation
- increasing plasma pH causes weakly acidic drugs to be extracted from the CNS into the plasma
- reducing plasma pH causes weakly acidic drugs to become concentrated in the CNS, increasing their neurotoxicity
what are the types of carrier-mediated transporters? what do they do?
- solute carrier (SLC) transporters: facilitate passive movement of solutes down their electrochemical gradient
- ATP-binding cassette (ABC) transporters: active pumps fuelled by ATP
what are types of SLCs important in drug distribution?
- organic cation transporters (OCT)
- organic anion transporters (OAT)
what do OCTs do?
- translocate dopamine, choline and drugs including vecuronium, quinine and procainamide
- uniporters: bind one solute molecule at a time and transport it down its gradienet
what is the role of OCT2?
- present in proximal renal tubules
- concentrates drugs e.g. cisplatin in cells, leading to selective nephrotoxicity
- related drugs e.g. carboplatin and oxaliplatin aren’t transported by OCT2 and are less nephrotoxic
- competition with cimetidine for OCT2 offers protection against cisplatin nephrotoxicity
where is OCT1 expressed?
liver
what is the effect of cisplatin on OCT transporters?
- influences activity of OCT2 but not OCT1
- not affected by less nephrotoxic drugs carboplatin and oxaliplatin
- cisplatin accumulates in cells expressing OCT2 and causes cell death
what protects against cisplatin nephrotoxicity towards OCT2?
cimetidine competes with cisplatin for OCT2 and thus protects against cisplatin-induced apoptosis
where are the main sites where SLCs are expressed?
- BBB
- GI tract
- renal tubule
- biliary tract
- placenta
what are P-glycoprotein transporters?
- belong to the ABC transporter superfamily
- responsible for multidrug resistance in cancer cells
where are P-glycoproteins present?
- renal tubular brush border membranes
- bile canaliculi
- astrocyte foot processes in brain microvessels
- GI tract
what are P-glycoproteins often co-located with?
SCL drug carriers
what contributes to individual genetic variation in responsiveness to different drugs?
polymorphic variation in the genes coding SLCs and P-gp
what does OCT1 transfer into hepatocytes?
metformin (used to treat diabetes)
what can influence the effectiveness of metformin?
single nucleotide polymorphisms of OCT1
what additional factors influence drug distribution and elimination?
- binding to plasma proteins
- partition into body fat and other tissues
what is the active form of drug?
unbound to plasma proteins; free in aqueous solution
what is the most important drug in relation to drug binding? what does it bind
albumin
- binds many acidic drugs (warfarin, NSAIDs, sulfonamides)
- binds some basic drugs (tricyclic antidepressants and chlorpromazine)
what does the amount of drug that is bound to a protein depend on?
- concentration of free drug
- affinity for the binding sites
- concentration of protein
how many binding sites are there on albumin? what is its usual concentration in plasma?
- 2
- 0.6 mmol/l
how is the fraction bound calculated?
[DS]/([D] + [DS])
why do few therapeutic drugs affect the binding of other drugs?
because they occupy, at therapeutic plasma concentration, only a tiny fraction of available sites
how can sulfonamides be harmful?
occupy about 50% of protein binding sites at therapeutic concentrations, so can displace other drugs or bilirubin (premature babies)
what limits the accumulation of drugs in body fat?
- low fat:water partition coefficient
- low blood supply
what are examples of drugs that can accumulate in body tissues?
- thiopental accumulates in body fat due to its high fat:water partition coefficient
- chloroquine accumulates in the retina (ocular toxicity) due to its high affinity for melanin
- tetracyclines accumulate slowly in bones and teeth due to their high affinity for calcium
- high concentrations of amiodarone accumulate in the liver and lung during chronic use, causing hepatitis and interstitial pulmonary fibrosis
what are the routes of elimination for drugs?
- urine
- faeces
- milk, sweat
- expired air
what is absorption of a drug?
passage of a drug from its site of administration into the plasma
what are the main routes of administration of a drug?
- oral
- sublingual
- rectal
- application to other epithelial surfaces (e.g. skin, cornea, vagina and nasal muscoa)
- inhalation
- injection (subcutaneous, intramuscular, intravenous, intrathecal and intravitreal)
where does the bulk of drug absorption occur after oral administration?
- small intestine
how does drug absorption occur from the intestine?
- passive transfer at a rate determined by the ionisation and lipid solubility of the drug molecules
- in some cases, carrier-mediated transport is used (e.g. levodopa and fluorouracil)
how long does it take to absorb drugs?
75% of a drug given orally is absorbed in 1-3 hours
what are factors affecting GI absorption of drugs?
- gut content (e.g. fed vs fasted)
- GI motility
- splanchnic blood flow
- particle size and formulation
- physicochemical factors, including some drug interactions
what reduces splanchnic blood flow and thus drug absorption?
hypovolaemia or heart failure
how is particle size and formulation controlled?
- therapeutic drugs are formulated to produce desired absorption characteristics
- capsules may be designed to remain intact for some hours after ingestion to delay absorption
- tablets may have a resistant coating to give same effect
- mixture of slow and fast release particles may be included to produce rapid but sustained absorption
- modified release preparations allow less frequent dosing
what is the absorption/function of vancomycin?
- very poorly absorbed
- administered orally
- eradicates toxin-forming Clostridium difficile from gut lumen in patients with pseudomembranous colitits
what is the absorption/function of mesalazine?
- formulation of 5-aminosalicylic acid in a pH dependent acrylic coat
- degrades in terminal ileum and proximal colon
- treats inflammatory bowel disease
what is the absorption/function of olsalazine?
- prodrug
- dimer of two molecules of 5-aminosalicylic acid
- cleaved by colonic bacteria in the distal bowel
- treats distal colitis
what is bioavailability?
the fraction (F) of an orally administered dose that reaches the systemic circulation as intact drug, taking into account both absorption and metabolic degradation
how is the fraction (F) of a drug measured?
by determining the plasma drug concentration versus time curves in a group of subjects following oral and IV administration
what is AUC?
areas under plasma concentration time curves
what are AUCs used for in relation to F?
AUCoral / AUCintravenous
what can affect bioavailability?
- drug preparation
- variations in enzyme activity of gut wall/liver
- change in gastric pH
- intestinal motility
what is required for a drug to be considered as bioequivalent? what does bioequivalence imply?
AUC(0-infinity), Cmax and Tmax must be between 80-125%
- implies that if one formulation of a drug is substituted for another, no clinically untoward consequences will ensue
when can sublingual administration be useful?
- when a rapid response is required
- drug is unstable at gastric pH
- is rapidly metabolised by the liver
what are examples of drugs that are administered sublingually?
- glyceryl trinitrate
- buprenorphine
what is an important feature of sublingual administration of a drug?
pass directly into the systemic circulation without entering the portal system, so escape first-pass metabolism by enzymes in the gut wall and liver
when is rectal administration of a drug used?
- for drugs that are required to produce a local or systemic effect
- absorption following rectal administration is often unreliable
- useful for patients vomiting or unable to take meds by mouth
- may be used to administer diazepam to children in status epilepticus
when is cutaneous administration used? what is an example?
- when a local effect on the skin is required
- absorption may occur and lead to systemic effects
- local rub on gels of NSAIDs e.g. ibuprofen
what is an example of harmful drugs being absorbed through the skin?
- organophosphate insecticides need to penetrate an insects cuticle to work
- absorbed through skin, accidentally poison some farmers
what are transdermal dosage forms? what are examples? what is their mechanism of action?
- drug is incorporated in a stick-on patch applied to the skin
- oestrogen/testosterone for HRT; fentanyl for breakthrough intermittent pain
- produce a steady rate of drug delivery and avoid presystemic metabolism
how do nasal sprays work? what is an example?
- absorption takes place through mucosa overlying nasal-associated lymphoid tissue (similar to Peyer’s patches, which are also permeable)
- e.g. ADH, GTRH, calcitonin
how do eye drops work? what are some advantages?
- absorption through the epithelium of the conjunctival sac
- desirable local effects within the eye achieved without causing systemic side effects
- some systemic absorption occurs and can lead to unwanted side effects
how are drugs absorbed when administered by inhalation?
- volatile and gaseous anaesthetics
- lung is the route of administration and elimination
- rapid exchange from the large SA and blood flow makes it possible to achieve rapid adjustments of plasma concentration
how are drugs absorbed when administered by injection?
- IV injection is the fastest and most certain route of drug administration
- bolus injection rapidly produces a high concentration of drug, first in the right heart and pulmonary vessels, then in the systemic circulation
- administration by steady IV infusion avoids the uncertainties of absorption from over sites, and high peak plasma concentration
what are the rate-limiting factors in absorption from the injection site?
- diffusion through the tissue
- removal by local blood flow
what increases/decreases absorption from a site of injection?
- increased by increased blood flow and by hyaluronidase
- reduced in patients with circulatory failure (shock), where tissue perfusion is reduced
what are methods for delaying absorption? why may this be done?
to produce a local effect or to prolong systemic action
- addition of adrenaline to local anaesthetic reduces its absorption into general circulation and prolongs the effect
- formulation of insulin with protamine and zinc produces a long acting form
- when injected as an aqueous suspension, procaine penicillin is slowly absorbed and exerts a prolonged action
- esterification of steroid hormones and antipsychotic drugs increases their solubility in oil
- subcutaneous implantation of drug may produce slow and continuous absorption of steroid hormones
what is intrathecal injection? what is an example?
- injection of a drug into the subarachnoid space via lumbar puncture needle
- methotrexate is administered this way to treat childhood leukaemias to prevent relapse in the CNS
what is intravitreal administration? what is an example?
route of administration of a drug where the substance is delivered into the eye
- e.g. ranibizumab (monoclonal antibody fragment that binds to VEGF) to treat wet age-related macular degeneration
what are the main body fluid compartments?
- plasma water (5%)
- interstitial water (16%)
- intracellular water (35%)
- transcellular water (2%)
- fat (20%)
what comprises extracellular fluid?
- blood plasma
- interstitial fluid
- lymph (1.2%)
what is intracellular fluid?
the sum of the fluid contents of all cells in the body
what is included in transcellular fluid?
cerebrospinal, intraocular, peritoneal, pleural and synovial fluids, and digestive secretions, and potentially fetus
what does the equilibrium pattern of distribution between various compartments depend on?
- permeability across tissue barriers
- binding within compartments
- pH partition
- fat:water partition
what can disrupt the integrity of the BBB? how can this be exploited?
- inflammation can disrupt integrity, allowing normally impermeant substances to enter the brain
- penicillin can be given intravenously to treat bacterial meningitis
where can the BBB be leaky? how can this be exploited?
- in some parts of the CNS, including the chemoreceptor trigger zone
- domperidone (antiemetic dopamine-receptor antagonist) can access the chemoreceptor trigger zone; prevents nausea caused by dopamine agonists e.g. apomorphine for Parkinsons
what peptides can increase BBB permeability? how can this be used clinically?
several peptides, including bradykinin and enkephalins
- improve penetration of anticancer drugs during brain tumour treatment
how can stress affect the BBB?
extreme stress renders the BBB permeable to drugs e.g. pyridostigmine, which normally acts peripherally
what is the volume of distribution?
the volume that would contain the total body content of the drug (Q) at a concentration equal to that present in the plasma (Cp)
Vd = Q/Cp
what is the plasma volume?
0.05 l/kg body weight
what drugs are largely confined to the plasma compartment? what happens after repeated dosing?
- molecules that are too large to cross the capillary wall easily; lipid-insoluble drugs e.g. heparin
- retention in the plasma after a single dose reflects strong binding to plasma protein
- following repeated dosing, equilibration occurs and measured Vd increases
what is the total extracellular volume?
0.2 l/kg body weight
what is the Vd for many polar compounds?
0.2 l/kg; vecuronium, gentamicin, carbenicillin
why may drugs be distributed in the extracellular compartment?
- cannot easily enter cells due to low lipid solubility
- don’t traverse BBB or placenta easily
- many macromolecular antibodies e.g. monoclonal antibodies, access receptors on cell surfaces but don’t enter cells
what is the total body water volume?
0.55 l/kg
what are drugs that have a Vd greater than the total body volume? what does this mean?
- morphine, tricyclic antidepressants, haloperidol
- drugs accumulate outside the plasma compartment
- not efficiently removed from body by haemodialysis
what happens to lipid soluble drugs?
reach all compartments and may accumulate in fat
what can drug interactions caused by altered distribution be caused by/lead to?
- a drug may alter the distribution of the other by competing for a common binding site on plasma albumin/tissue protein
- increases concentration of free drug, followed by increased elimination
- leads to a new steady state where total drug concentration in plasma is reduced but free drug concentration is similar to before
what are consequences of drug interactions caused by altered distribution?
- toxicity from the transient increase in conc of free drug before new steady state is reached
- if dose is being adjusted to measurements of total plasma conc, the target therapeutic conc range will be altered by co-administration of a displacing drug
- when the displacing drug additionally reduces elimination of the first, so that the free conc is increased acutely and chronically at the new steady state, severe toxicity may occur
what are some protein bound drugs that are given in large enough dosage to act as displacing agents?
sulfonamides and chloral hydrate: trichloracetic acid, a metabolite of chloral hydrate, binds very strongly to plasma albumin
what can displacement of bilirubin from albumin by sulfonamides and chloral hydrate in jaundiced premature neonates lead to?
- bilirubin metabolism is undeveloped in the premature liver
- unbound bilirubin can cross the immature BBB and cause kernicterus
- causes a distressing and permanent disturbance of movement (choreoathetosis), characterised by inoluntary writhing and twisting in the child
what is kernicterus?
staining of the basal ganglia by bilirubin
what is choreathetosis? what is it caused by?
- caused by kernicterus
- distressing and permanent disturbance of movement characterised by involuntary writhing and twisting movements
what are examples of drugs that alter protein binding also reducing elimination of the displaced drugs?
- salicylates displace methotrexate from binding sites and reduce its secretion into the nephron by competition with the OAT
- quinidine, verapamil and amiodarone displace digoxin from tissue-binding sites while reducing its renal excretion
what are approaches to improve drug delivery and localise the drug to the target tissue?
- prodrugs
- biologically erodible nanoparticles
- antibody-drug conjugates
- packaging in liposomes
- coated implantable devises
what is drug elimiation? by which processes does it occur?
irreversible loss of drug from the body
- metabolism: anabolism and catabolism
- excretion: elimination from the body of drug or drug metabolites
what are the main excretory routes?
kidneys, hepatobiliary system and the lungs
what are examples of drugs that are excreted faecally?
rifampicin (uncharged drug in healthy individuals) and digoxin (normally excreted in urine, but faecally for patients with renal failure)
what drugs are excreted via the lungs?
highly volatile or gaseous agents (e.g. general anaesthetics)
what is chirality?
- more than one stereoisomer
- affects overall metabolism
what steps does drug metabolism involve? what is the aim of them?
- phase 1 and 2
- both decrease lipid solubility, and thus increase renal elimination
what are phase 1 reactions?
- catabolic
- e.g. oxidation, reduction or hydrolysis
- products are often more chemically reactive and sometimes more toxic/carcinogenic than the parent drug
what is involved in phase 1 reactions?
- functionalisation: introduce a reactive group, e.g. hydroxyl, into the molecule
- this reactive group serves as the point of attack for the conjugating system to attach a substituent e.g. glucuronide
where do phase 1 reactions occur?
in the liver
what enzymes are involved in phase 1 reactions? what are they often called?
- hepatic drug-metabolising enzymes, including CYP enzymes, are embedded in the smooth ER
- often called microsomal enzymes
why are hepatic drug-metabolising enzymes and CYP enzymes often called microsomal enzymes?
- because on homogenisation and differential centrifugation, the ER is broken into very small fragments that sediment only after prolonged high-speed centrifugation in the microsomal fraction
what types of reaction occur in phase 1 reactions?
oxidation, hydroxylation, dealkylation, deamination, hydrolysis
what are cytochrome P450 enzymes?
- haem proteins, comprising a large family of related but distinct enzymes
- CYP followed by defining set of numbers and a letter
how do different cytochrome P450 enzymes differ from each other?
- amino acid sequence
- sensitivity to inhibitors and inducing agents
- specificity of reactions that catalyse
how many CYP gene families have been described?
74; CYP1, 2 and 3 are involved in drug metabolism in the liver
what are examples of some P450 isoenzymes?
CYP1A2 CYP2B6 CYP2C8 CYP2C19 CYP2C9 CYP2D6 CYP2E1 CYP3A4,5,7
what are substrate drugs of CYP1A2?
caffeine, paracetamol, tacrine, theophylline
what are substrate drugs of CYP2B6?
cyclophosphamide, methadone
what are substrate drugs of CYP2C8?
paclitaxel, repaglinide
what are substrate drugs of CYP2C19?
omeprazole, phenytoin
what are substrate drugs of CYP2C9?
ibuprofen, tolbutamide, warfarin
what are substrate drugs of CYP2D6?
codeine, debrisoquine, S-metoprolol
what are substrate drugs of CYP2E1?
alcohol, paracetamol
what are substrate drugs of CYP3A4,5,7?
ciclosporin, nifedipine, indinavir, simvastatin
what does drug oxidation by the monooxygenase P450 system require?
- drug (substrate, DH)
- P450 enzyme
- molecular oxygen
- NADPH
- NADPH-450 reductase
what is the outcome of the monooxygenase P450 cycle?
addition of one atom of oxygen (from molecular oxygen) to the drug to form a hydroxylated product (DOH)
- other atom of oxygen is converted to water
what are the stages of monooxygenase P450 cycle?
- P450 containing ferric iron (Fe3+) combines with a molecule of drug (DH)
- receives an electron from NADPH-P450 reductase, which reduces the iron o Fe2+
- combines with molecular oxygen, a proton and a second electron (either from NADPH-P450 reductase or cytochrome b5) to form an Fe2+OOH-DH complex
- this complex combines with another proton to yield water and a ferric oxene (FeO)3+ -DH complex
- (FeO)3+ extracts a hydrogen atom from DH, with the formation of a pair of short-lived free radicals, liberation from the complex of oxidised drug (DOH) and regeneration of P450
what is the glucuronide conjugation reaction?
UDP-alpha-glucuronide -> drug-gluconide
catalysed by glucuronyl transfer
what are sources of inter-individual variation in P450 enzymes?
- genetic polymorphisms
- environmental factors (enzyme inhibitors and inducers are present in the diet and environment)
how can diet and environment affect individual variation in P450 enzymes?
- component of grapefruit juice inhibits drug metabolism
- brussel sprouts and cigarette smoke induce P450 enzymes
- components of the herbal medicine St John’s wort induce CYP450 isoenzymes and P-gp
what are examples of drugs being metabolised without the P450 system?
- plasma (e.g. hydrolysis of suxamethonium by plasma cholinesterase)
- lung (e.g. prostanoids)
- gut (e.g. tyramine, salbutamol)
- ethanol is metabolised by alcohol dehydrogenase as well as CYP2E1
- xanthine oxidase inactivates 6-mercaptopurine
- monoamine oxidase inactivates amines
what is an example of hydrolytic/reduction reactions? give examples
- hydrolysis (e.g. of aspirin) occurs in plasma and many tissues
- ester and amide bonds are susceptible to hydrolytic cleavage
- reduction is less common in phase 1, but warfarin is inactivated by reduction of a ketone to a hydroxyl group by CYP2A6
what are phase 2 reactions? what do they involve?
synthetic (anabolic) and involve conjugation (attachment of a substituent group) which usually results in inactive products
where do phase 2 reactions take place?
mainly in the liver
what groups could be inserted in conjugation?
glucuronyl, sulfate, methyl or acetyl
how does the tripeptide glutathione conjugate drugs?
via its sulfhydryl group, e.g. in the detoxification of paracetamol
what is involved in glucuronidation?
- formation of a high-energy phosphate (donor) compound, UDPGPA, from which glucuronic acid is transferred to an electron-rich atom (N,O or S) or the substrate
- this forms an amide, ester or thiol bond
- UDP-glucuronyl transferase has broad substrate specificity
what is UDPGPA?
uridine diphosphate glucuronic acid
what are the donor compounds for acetylation and methylation in phase 2 reactions?
acetyl-CoA and S-adenosyl methionine, respectively
what are stereoisomers?
their components differ in pharmacological effects and their metabolism, which may follow distinct pathways
what are examples of stereoisomers?
sotalol, warfarin and cyclophosphamide
what is an example of a non-competetive inhibitor of P450 enzymes? how does it function?
drugs such as ketoconazole, which forms a tight complex with the Fe3+ form of the haem iron of CYP3A4, causing reversible non-competitive inhibition
how do mechanism-based inhibitors of P450 function?
require oxidation by a P450 enzymes
- an oxidation product binds covalently to the enzyme, which then destroys itself
what are examples of mechanism-based inhibitors of P450?
oral contraceptive gestodene (CYP3A4)
anthelmintic drug diethylcarbamazine (CYP2E1)
what are examples of substances that induce microsomal enzymes?
drugs: rifampicin, ethanol and carbamazepine
carcinogenic chemicals: benzpyrene, 3-MC
why can enzyme induction increase drug toxicity and carcinogenicity?
because several phase 1 metabolites are toxic or carcinogenic e.g. paracetamol
how is enzyme induction exploited therapeutically?
administering phenobarbital to premature babies to induce glucuronyl transferase, which increases bilirubin conjugation and reducing the risk of kernicterus
what is first pass metabolism?
- some drugs are extracted so efficiently by the liver or gut wall that the amount reaching the systemic circulation is much less than the amount absorbed
- reduces bioavailability, even when a drug is well absorbed
why can first pass metabolism be a problem?
- much larger dose of the drug is needed when it’s taken by mouth than when given parenterally
- marked individual variations occur, in the activities of drug metabolising enzymes and due to variation in hepatic blood flow
what are examples of drugs that become pharmacologically active only after being metabolised (prodrugs)?
- azathioprine (immunosuppressant drug) is metabolised to mercaptopurine
- enalapril (ACE inhibitor) is hydrolysed to its active form analaprilat
what is an example of drug metabolites having different activity to the drug?
aspirin inhibits platelet function and has anti-inflammatory activity; when hydrolysed to salicylic acid, it has anti-inflammatory but not antiplatelet activity
what is an example of drug metabolites having similar activity to the drug?
benzodiazepines
what are examples of drug metabolites being responsible for toxicity?
- bladder toxicity of cyclophosphamide, caused by its toxic metabolite acrolein
- methanol and ethylene are toxic via metabolites formed by alcohol dehydrogenase
what are examples of drugs that undergo substantial first-pass elimination?
aspirin, gluceryl trinitrate, isosorbide dinatrate, levodopa, lidocaine, metoprolol, morphine, propanolol, salbutamol and verapamil
what are prodrugs that are metabolised straight to active metabolites?
- azathioprine
- cortisone
- prednisone
- enalapril
- zidovudine
what are the active metabolites of azathioprine, cortisone, prednisone, enalapril and zidovudine?
azathioprine -> mercaptopurine cortisone -> hydrocortisone prednisone -> prednisolone enalapril -> enalaprilat zidovudine -> zidovudine triphosphate
what is a prodrug that produces an active metabolite which produces a toxic metabolite? what are these?
cyclophosphamide -> phosphoramide mustard -> acrolein
what is an active drug that produces an active metabolite and a toxic metabolite? what are these?
diazepam -> nordiazepam -> oxazepam
what is an active drug that produces just an active metabolite? what is this?
morphine -> morphine 6-glucuronide
what are active drugs that produce just toxic metabolites? what are they?
halothane -> trifluoroacetic acid
methoxyflurane -> fluoride
paracetamol -> N-Acetyl-p-benzoquinoneimine
how can enzyme induction lead to tolerance?
because the inducing agent is often a substrate for the induced enzymes, there is a slow tolerance development
what are examples of drug interactions caused by enzyme induction?
- graft rejection due to loss of effectiveness of immunosuppressive treatment
- seizures due to loss of anticonvulsant effectiveness
- unwanted pregnancy from loss of contraceptive action
- thrombosis or bleeding
what can induction of P450 enzymes lead to?
greatly accelerate hepatic drug metabolism, which can increase the toxicity of drugs with toxic metabolites
- important cause of drug-drug interaction
what does enzyme inhibition lead to?
slows metabolism and increases the number of drugs inactivated by the enzyme
what are examples of drugs that induce drug-metabolising enzymes, and what drugs does this affect?
phenobarbital - warfarin rifampicin - oral contraceptives griseofulvin - corticosteroids phenytoin - ciclosporin ethanol and carbamazepine
what are examples of drugs that inhibit drug-metabolising enzymes, and what drugs does this affect?
allopurinol - mercaptopurin, azathioprine
chloramphenicol - phenytoin
cimetidine - amiodarone, phenytoin, pethidine
ciprofloxacin - theophylline
corticosteroids - tricyclic antidepressants, cyclophosphamide
disulfiram - warfarin
erythromycin - ciclosporin, theophylline
MAO inhibitors - pethidine
ritonavir - saquinavir
what are drugs that exert steroselective inhibition of warfarin metabolism for S isomer?
phenylbutazone metronidazole sulfinpyrazone trimethoprim-sulfamethoxazole disulfiram
what are drugs that exert stereoselective inhibition of warfarin metabolism for R isomer?
cimetidine
omeprazole
what are drugs that exert a non-stereoselective effect on both S and R isomers on warfarin metabolism?
amiodarone
what are the S and R isomers of warfarin?
S: active
R: less active
what is the enterohepatic circulation?
- hydrophilic drug conjugates are concentrated in bile and delivered to the intestine, where they can be hydrolysed
- this regenerates active drug; free drug can be reabsorbed by the liver and the cycle repeated
what is renal clearance?
the volume of plasma containing the amount of substance that is removed from the body by the kidneys in unit time
what is the equation for renal clearance?
CLren = (Cu x Vu)/Cp
- CLren = renal clearance
- Cp = plasma concentration
- Cu = urinary concentration
- Vu = rate of flow of urine
what three fundamental processes account for renal drug excretion?
- glomerular filtration
- active tubular secretion
- passive reabsorption (diffusion from the concentrated tubular fluid back across tubular epithelium)
what are drugs and related substances secreted into the proximal renal tubule by OAT?
p-Aminohippuric acid furosemide glucuronic acid conjugates glycine conjugates indometacin methotrexate penicillin probenecid sulfate conjugates thiazide diuretics uric acid
what are drugs and related substances secreted into the proximal renal tubule by OCT transporters?
amiloride dopamine histamine mepacrine morphine pethidine quaternary ammonium compounds quinine serotonin triamterene
what would prevent a portion of drug from being filtered by the glomerulus?
- binding to albumin, because it’s almost completely impermeant
- only free drug would be filtered
- e.g. warfarin is 98% bound to albumin, so only 2% of it is filtered
- most drugs cross the barrier freely
what are the differences between OAT and OCT carriers?
- OAT transports drugs against an electrochemical gradient and reduce the plasma concentration nearly to 0
- OCT facilitates transport down the electrochemical gradient
what are examples of drugs that are excreted largely unchanged in the urine?
100-75%: furosemide, gentamicin, methotrexate, atenolol, digoxin
75-50%: benzylpenicillin, cimetidine, oxytetracycline, neostigmine
50%: propanthelin, tubocurarine
what are the main mechanisms by which one drug can affect the rate of renal excretion of another?
- altered protein binding and hence filtration
- inhibiting tubular secretion
- altering urine flow and/or urine pH
how are lipid soluble drugs reabsorbed across the tubule?
passively reabsorbed by diffusion across the tubule, so are not efficiently excreted in the urine
what are examples of drugs that lower intraocular pressure? what is their mechanism of action?
- timolol, carteolol: beta-adrenoceptor antagonist
- acetazolamide, dorzolamide: carbonic anhydrase inhibitor
- clonidine, apraclonidine: alpha2-adrenoceptor agonist
- latanoprost: prostaglanding analogue
- pilocarpine: muscarinic agonist
- ecothiophate: anticholinesterase
what are the smooth muscle effects of muscarinic agonists?
- generally contracts in direct response to muscarinic agonists, in contrast to the indirect effect via NO on vascular smooth muscle
- peristatic activity of the GI tract is increased, which can cause colicky pain
- bladder and bronchial smooth muscle contract
what are the effects of muscarinic agonists on sweating, lacrimation, salivation and bronchial secretion?
- stimulate exocrine glands
- increases all of these things
- combined effect of bronchial secretion and constriction can interfere with breathing
what are the effects of muscarinic agonists on the eye? what are the roles of the constrictor pupillae muscle and the ciliary muscle?
- parasympathetic nerves supply constrictor pupillae muscle and the ciliary muscle
- contraction of ciliary muscle due to mAChRs pulls the ciliary body forward and inward, relaxing the tension on the suspensory ligament and reducing the lens’s focal length
- constrictor pupillae adjusts the pupil in response to changes in light intensity and in regulating intraocular pressure
what is glaucoma? how can drugs be used to treat it?
- abnormally raised intraocular pressure leads to glaucoma
- drainage of aqueous humour becomes impeded when the pupil is dilated because folding of the iris tissue occludes the drainage angle, causing the intraocular pressure to rise
- activation of the constrictor pupillae muscle by muscarinic agonists lowers the intraocular pressure
- increased tension in the ciliary muscle may improve drainage by realigning the connective tissue trabeculae
what are effects of muscarinic agonists that are able to penetrate the BBB?
- M1 receptors in brain are activated
- tremor, hypothermia and increased locomotor activity
- improved cognition
what are muscarinic antagonists?
- competitive antagonists whose chemical structures usually contain ester and basic groups in the same relationship as ACh
- bulky aromatic group in place of the acetyl group
what are examples of naturally occurring muscarinic antagonists? where are they found? what are their characteristics?
- atropine and hyoscine (scopolamine)
- alkaloids found in solanaceous plants
- deadly nightshade (Atropa belladonna) contains mainly atropine
- thorn apple (Datura stramonium) contains mainly hyoscine
- tertiary ammounium compounds that are lipid-soluble and absorbable from the gut/conjunctival sac, and can penetrate the BBB
what are some quaternary ammonium compounds? what are their properties/uses?
- peripheral actions similar to atropine
- cannot cross BBB
- lack central actions
- hyoscine butylbromide, propantheline, ipratropium (bronchodilator)
what are tertiary amides with opthalamic uses?
cyclopentolate and tropicamide; developed for opthalamic use and administered as eye drops
what are some M3 selective muscarinic antagonists? what are their uses/effects?
- oxybutyrin, tolterodine and darifenacin
- act on bladder to prevent micturition
- used for treating urinary incontinence
- effects: dry mouth, constipation and blurred vision
what are clinical uses of muscarinic agonists and what are their effects? give examples
- pilocarpine eye drops cause constriction of the pupils and are used for glaucoma
- pilocarpine/cevimeline, selective M3 agonist, can be used to increase salivation and lacrimal secretion in patients with dry mouth or dry eyes
- bethanechol or distigmine are seldom used as stimulant laxatives or to stimulate bladder emptying
what are the effects on secretions by muscarinic antagonists?
- salivary, lacrimal, bronchial and sweat glands are inhibited by very low doses of atropine, producing dry mouth and skin
- slightly reduced gastric secretion
- inhibited mucociliary clearance in the bronchi
what is the effect of muscarinic antagonists on heart rate?
- atropine causes tachycardia through block of cardiac mAChRs
- no effect on sympathetic system, only inhibition of tonic parasympathetic tone
- at very low doses, atropine causes a paradoxical bradycardia, possibly due to a central action
- arterial BP and response of heart to exercise are unaffected
what is the effect of muscarinic antagonists on the eye?
- pupil is dilated and becomes unresponsive to light
- relaxation of the ciliary muscle causes paralysis of accomodation (cyloplegia), so near vision is impaired
- intraocular pressure may rise
what is the effect of muscarinic antagonists on the GI tract?
- GI motility is inhibited by atropine
what is the effect of muscarinic antagonists on other smooth muscle?
- bronchial, biliary and urinary tract smooth muscle are all relaxed by atropine
- reduces incontinence due to bladder overactivity
what is the effect of muscarinic antagonists on the CNS?
- atropine produces mainly excitatory effects on the CNS
- at low doses, there is agitation and disorientation, and in poison, there is hyperactivity, increase in body temp, loss of sweating
- affect the extrapyramidal system, reducing involuntary movement and rigidity in Parkinsons patients
what are ganglion stimulants?
most nAChR agonists act on either neuronal (ganglionic and CNS) nACh receptors or on striated muscle (motor endplate) receptors, but not, apart from nicotine and ACh, on both
what are examples of nicotine agonists? where is their main site and type of action?
- nicotine: stimulates then blocks autonomic ganglia; stimulates CNS
- lobeline: stimulates autonomic ganglia and sensory nerve terminals
- epibatidine: stimulates autonomic ganglia and CNS
- varenicline: stimulates CNS and autonomic ganglia
- suxamethonium and decamethonium: depolarisation block at NMJ
what are the mechanisms by which ganglion block can occur?
- by interference with ACh release at the NMJ
- by prolonged depolarisation
- by interference with the postsynaptic action of ACh
what are examples of nicotine antagonists? what is their main site and type of action?
- hexamethonium and trimetaphan: transmission block at autonomic ganglia
- tubocurarine, pancuronium, atracurium and vecuronium: transmission block at NMJ
what are some cardiovascular and opthalamic clinical uses of muscarinic antagonists?
- treatment of sinus bradychardia by atropine
- to dilate the pupil by tropicamide or cyclopentolate
what are some neurological clinical uses of muscarinic antagonists?
- prevention of motion sickness by hyoscine
- treatmetn of Parkinsonism by benzhexol, benztropine
what are some respiratory and anaesthetic premedication clinical uses of muscarinic antagonists?
- asthma and COPD treatment by ipratropium or tiotropium by inhalation
- to dry secretions by atropine or hyoscine
what are some GI clinical uses of muscarinic antagonists?
- to facilitate endoscopy and GI radiology by relaxing smooth muscle by hyoscine
- as an antispasmodic in IBS or colonic diverticular disease by dicycloverine
what are effects of ganglion-stimulating drugs?
- both sympathetic and parasympathetic ganglia are stimulated
- tachycardia and increased BP
- variable effects on GI motility and secretions
- increased bronchial, salivary and sweat secretions
what are effects of ganglion-blocking drugs?
- block all autonomic and enteric ganglia
- hypotension and loss of CV reflexes
- inhibition of secretions
- GI paralysis
- impaired micturition
how can drugs block neuromuscular transmission?
- by acting presynaptically to inhibit ACh synthesis or release
- by acting postsynaptically
what is the mechanism of action of non-depolarising blocking drugs?
- act as competitive antagonists at he ACh receptors of the endplate
- block facilitatory presynaptic autoreceptors, thus inhibiting release of ACh during repetitive stimulation of the motor nerve
what are examples of neuromuscular-blocking drugs?
tubocurarine pancuronium vecuronium atracurium mivacurium suzamethonium
what are the speeds of onset and duration of action of neuromuscular-blocking drugs?
tubocurarine: slow (>5min) and long (1-2 h)
pancuronium: intermediate (2-3min) and long (1-2h)
vecuronium: intermediate and intermediate (30-40min)
atracurium: intermediate and intermediate (<30 min)
mivacurium: fast (2min) and short (15min)
suxamethonium: fast and short (10min)
what are the main side effects of tubocurarine (neuromuscular blocking drug)?
- hypertension (ganglion block and histamine release)
- bronchoconstriction (histamine release)
what are the main side effects of pancuronium (neuromuscular blocking drug)?
- slight tachycardia
- hypertension
what are the main side effects of atracurium and mivacurium (neuromuscular blocking drug)?
transient hypotension (histamine release)
what are the main side effects of suxamethonium (neuromuscular blocking drug)?
- bradycardia (muscarinic agonist effect)
- cardiac dysrhythmias (increased plasma K+ conc)
- raised intraocular pressure
- postop muscle pain
what is often used to reverse the action of non-polarising drugs postoperatively?
cholinesterase inhibitor, neostigmine
- rapid postop recovery of muscle strength is needed to reduce risk of complications
what are neuromuscular blocking agents mainly used for?
mainly in anaesthesia to produce muscle relaxation
- given intravenously
- most non-depolarising blocking agents are metabolised by the liver
what are the differences in patterns of neuromuscular block produced by depolarising and non-depolarising mechanisms?
- anticholinesterase drugs are effective in overcoming the blocking action of competitive, non-depolarising agents. this is because ACh is protected from hydrolysis and can diffuse further into the cleft, increasing its chance of it finding an unoccupied receptor before hydrolysation
- depolarisation block is unaffected/increased by anticholinesterase drugs
- fasciulations seen with suxamethonium as a prelude to paralysis don’t occur with competitive drugs
- tetanic fade is increased by non-depolarising blocking drugs, compared with normal muscle. does not occur with depolarisation block
what is tetanic fade?
failure of muscle tension to be maintained during a brief period of nerve stimulation at a frequency high enough to produce a fused tetanus
what are the unwanted effects and dangers of suxamethonium?
- bradycardia
- potassium release
- increased intraocular pressure
- prolonged paralysis
- malignant hyperthermia
why does suxamethonium given intravenously only last less than 5 mins?
hydrolysed by plasma cholinesterase
what are factors that reduce the activity of plasma cholinesterase and thus prolong the action of suxamethonium?
- genetic variations of plasma cholinesterase with reduced activity/absence of the enzyme
- anticholinesterase drugs e.g. organophosphates and competing substrates
- neonates may have low plasma cholinesterase activity and show prolonged paralysis with suxamethonium
what is malignant hyperthermia? how can it be treated?
- rare inherited condition due to a mutation of the ryanodine receptor
- intense muscle spasm and dramatic rise in body temp when certain drugs are administered
- 65% mortality rate
- treated by dantrolene
what are examples of drugs that inhibit acetylcholine synthesis? how do they do this?
- hemicholinium (blocks transport of choline into the nerve terminal)
- vesamicol (blocks ACh transport into synaptic vesicles)
what are examples of drugs that inhibit acetylcholine release? how do they do this?
- Mg2+ and aminoglycoside antibiotics e.g. streptomycin and neomycin (inhibit Ca2+ entry)
- botulinum toxin and beta-bungarotoxin (inhibit ACh release)
what are clinical and cosmetic uses of Botulinum toxin?
- blepharospasm, torsion dystonia and spasmodic torticollis
- spasticity
- urinary incontinence
- squint
- hyperhidrosis
- sialorrhoea
- headache prophylaxis
- forehead wrinkles
what are the effects of Botulinum poisoning?
progressive parasympathetic and motor paralysis, dry mouth, blurred vision, difficulty in swallowing and progressive respiratory paralysis
what are catecholamines?
compounds containing a catechol moiety (a benzene ring with two adjacent hydroxyl groups) and an amine side chain
what are the most important catecholamines?
- noradrenaline: a transmitter released by sympathetic nerve terminals
- adrenaline: a hormone secreted by the adrenal medulla
- dopamine: the metabolic precursor of noradrenaline and adrenaline, and also a transmitter/neuromodulator in the CNS
- isoprenaline: a synthetic derivative of noradrenaline, not present in the body
how are alpha and beta receptors defined in terms of agonist potencies?
alpha: noradrenaline > adrenaline > isoprenaline
beta: isoprenaline > adrenaline > noradrenaline
what are subdivisions of alpha and beta receptors? what type of receptor are they?
alpha 1 and alpha 2: alpha1A, alpha1B, alpha1D and alpha2A, alpha2B, alpha2C
beta 1, 2 and 3
- GPCRs
where are alpha1 receptors mainly located?
CVS and lower urinary tract
where are alpha2 receptors mainly located?
neuronal, acting to inhibit transmitter release in the brain and at autonomic nerve terminals in the periphery
what are the secondary messenger systems used by alpha 1 and 2 receptors?
alpha1: coupled to phospholipase C and produce effects mainly by the release of intracellular Ca2+
alpha2: negatively coupled to adenylyl cyclase and reduce cAMP formation and inhibit Ca2+ channels and activate K+ channels
what are the secondary messenger systems used by beta receptors?
all three stimulate adenylyl cyclase
where are beta 1 receptors mainly located?
heart; responsible for positive inotropic and chronotropic effects of catecholamines
what are the main effects of alpha1 receptor activation?
vasoconstriction, relaxation of GI smooth muscle, salivary secretion and hepatic glycogenolysis
what are the main effects of alpha2 receptor activation?
inhibition of transmitter release, platelet aggregation, vascular smooth muscle constriction, insulin release
what are the main effects of beta1 receptor? activation?
increased cardiac rate and force
what are the main effects of beta2 receptor activation?
bronchodilation, vasodilation, relaxation of visceral smooth muscle, hepatic glycogenolysis, muscle tremor
what are the main effects of beta3 receptor activation?
lipolysis and thermogenesis, bladder detrusor muscle relaxation
what is the pathway of catecholamine metabolism? what enzymes catalyse these reactions?
1: tyrosine -> DOPA
2: DOPA -> dopamine
3: dopamine -> noradrenaline
4: noradrenaline -> adrenaline
1: tyrosine hydroxylase
2: DOPA decarboxylase
3: dopamine beta-hydroxylase
4: phenylethanolamine N-methyltransferase
what is a noradrenergic neuron?
postganglionic sympathetic neurons whose cell bodies are situated in sympathetic ganglia
- long axons ending in a series of varicosities along the branching terminal network
what is contained in the synaptic vesicles in noradrenergic neuron varicosities?
- sites of synthesis and release of noradrenaline and co-released mediators
- mediators include ATP and neuropeptide Y
what is the agonist potency order for alpha1 adrenoceptors?
NA > A»_space; ISO
what is the agonist potency order for alpha2 adrenoceptors?
A > NA»_space; ISO
what is the agonist potency order for beta1 adrenoceptors?
ISO > NA > A
what is the agonist potency order for beta2 adrenoceptors?
ISO > A > NA
what is the agonist potency order for beta3 adrenoceptors?
ISO > NA = A
what are selective agonists and antagonists for alpha1 adrenoceptors?
agonists: phenylephrine and methoxamine
antagonists: prazosin and doxazocin
what are selective agonists and antagonists for alpha2 adrenoceptors?
agonists: clonidine
antagonists: yohimbine and idazoxan
what are selective agonists and antagonists for beta1 adrenoceptors?
agonists: dobutamine and xamoterol
antagonists: atenolol and metoprolol
what are selective agonists and antagonists for beta2 adrenoceptors?
agonists: salbutamol, terbutaline, salmeterol, formoterol and clenbuterol
antagonists: butoxamine
what are selective agonists for beta3 adrenoceptors?
mirabegron
what inhibits tyrosine hydroxylase?
- noradrenaline: moment-to-moment regulation of the rate of synthesis
- alpha-methyltyrosine
what affects dopamine-beta-hydroxylase?
- copper chelating agents and disulfiram inhibit it
where is PNMT enzyme located? what affects it?
- located in the adrenal medulla
- adrenal medulla contains a population of adrenaline-releasing (A) cells separate from the smaller population of noradrenaline releasing cell (N)
- production of PNMT is induced by action of steroid hormones secreted by the adrenal cortex
where is most noradrenaline stored?
vesicles in nerve terminals or chromaffin cells
what is the concentration of noradrenaline in vesicles and what maintains this concentration?
- very high (0.3-1 mol/l)
- maintained by vesicular monoamine transporter (VMAT)
what drugs can block noradrenaline uptake into vesicles?
reserpine
what do noradrenaline vesicels contain?
- noradrenaline
- ATP (4/NA molecule)
- chromogranin A
- DBH
reversible complex is formed within the vesicle to reduce the osmolarity of the vesicle contents and to reduce the tendency of NA to leak out of the vesicles in the nerve terminal
what are the different types of noradrenaline transport systems?
- neuronal (NET)
- extraneuronal (EMT)
- vesicular (VMAT)
what is the Km of NET, EMT and VMAT noradrenaline transport systems?
NET: 0.3
EMT: 250
VMAT: -0.2
what is the specificity of NET, EMT and VMAT noradrenaline transport systems?
NET: NA > A > ISO
EMT: A > NA > ISO
VMAT: NA = A = ISO
what are the locations of NET, EMT and VMAT noradrenaline transport systems?
NET: neuronal membrane
EMT: non-neuronal cell membrane
VMAT: synaptic vesicle membrane
what are substrates for NET noradrenaline transport systems?
tyramine
methylnoradrenaline
adrenergic neuron-blocking drugs
amphetamine
what are substrates for EMT noradrenaline transport systems?
noradrenaline
dopamine
5-hydroxytryptamine
histamine
what are substrates for VMAT noradrenaline transport systems?
dopamine
5-hydroxytryptamine
guanethidine
MPP
what are inhibitors of NET noradrenaline transport systems?
cocaine
tricyclic antidepressants
phenoxybenzamine
amphetamine
what are inhibitors of EMT noradrenaline transport systems?
normetanephrine
steroid hormones
phenoxybenzamine
what are inhibitors of VMT noradrenaline transport systems?
reserpine
tetrabenazine
what is the process of feedback control of noradrenaline release?
- presynaptic alpha2 receptor inhibits Ca2+ influx in response to membrane depolarisation by action of betagamma subunits of the G protein on the voltage-dependent Ca2+ channels
how is noradrenaline taken back up after release?
75% released by sympathetic neurons is recaptured and repackaged into vesicles
25% is captured by non-neuronal cells in the vicinity, limiting its local spread
what is uptake of noradrenaline by NET, VMAT and EMT driven by?
NET: act as cotransporters of Na+, Cl- and the amine, using the electrochemical gradient for Na+ as a driving force
VMAT: driven by proton gradient between the cytosol and the vesicle contents
what are endogenous and exogenous catecholamines metabolised by?
two intracellular enzymes: monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT)
where is MAO located?
- two isoforms: MAO-A and MAO-B
- bound to the surface membrane of mitochondria
- abundant in noradrenergic nerve terminals and also in the liver, intestinal epithelium and other tissues
what is the action of MAO?
- converts catecholamines to their corresponding aldehydes
- aldehydes are rapidly metabolised by aldehyde dehydrogenase to the corresponding carboxylic acid (3,4-dihydroxyphenylglycol formed from noradrenaline) in the periphery
- oxidises other monoamines, e.g. dopamine and 5-HT
what is MAO inhibited by?
- various drugs that are used for their effects on the CNS, where three amines have transmitter functions
where is COMT located?
absent from noradrenergic neurons but present in the adrenal medulla and other cells/tissues
what is the action of COMT?
- methylation of one of the catechol hydroxyl groups by COMT to give a methoxy derivative
what is the final product formed by the sequential action of MAO and COMT?
3-methoxy-4-hydroxyphenylglycol (MHPG)
what happens to MHPG?
partly conjugated to sulfate or glucuronide derivatives, which are excreted in the urine
- most converted to vanillylmandelic acid and excreted in the urine in this form
how are noradrenaline, adrenaline and isoprenaline removed from circulation?
- noradrenaline is removed by NET
- adrenaline more dependent on EMT
- isoprenaline is not a substrate for NET, and is removed by a combination of EMT and COMT
why is noradrenaline content of the cytosol usually low?
due to MAO in nerve terminals
what causes transmitter release in noradrenergic transmission?
- normally by Ca2+ mediated exocytosis from varicosites on the terminal network
- non-exocytotic release occurs in response to indirectly acting sympathomimetic drugs (e.g. amphetamine) which displace noradrenaline from vesicles
- noradrenaline escapes via the NET transporter (reverse transport)
what is transmitter action in noradrenergic transmission terminated by?
mainly by reuptake of noradrenaline into nerve terminals via the NET transporter; NET is blocked by tricyclic antidepressent drugs and cocaine
what is noradrenaline release controlled by?
autoinhibitory feedback mediated by alpha2 receptors
what is the action of the drug noradrenaline? what are its uses/functions, unwanted effects and pharmacokinetic aspects?
main action: alpha/beta agonist
uses/function: hypotension in intensive care, transmitter at postganglionic sympathetic neurons
unwanted effects: hypertension, vasoconstriction, tachycarida, ventricular dysrhythmias
pharmacokinetic aspects: poorly absorbed by mouth, rapid removal by tissues, metabolised by MAO and COMT, plasma t1/2: 2 min
what is the action of the drug adrenaline? what are its uses/functions, unwanted effects and pharmacokinetic aspects?
main action: alpha/beta agonist
uses/function: asthma, anaphylactic shock, cardiac arrest, anaesthetic solutions, adrenal medulla
unwanted effects: hypertension, vasoconstriction, tachycarida, ventricular dysrhythmias
pharmacokinetic aspects: poorly absorbed by mouth, rapid removal by tissues, metabolised by MAO and COMT, plasma t1/2: 2 min
what are the stages of a drug development project?
- drug discovery
- preclinical development
- clinical development
what happens overall in drug discovery?
candidate molecules are chosen on the basis of their pharmacological properties
what happens overall in preclinical development?
a wide range of non-human studies (e.g. toxicity testing, pharmacokinetic analysis and formulation) are performed
what happens overall in clinical development?
selected compound is tested for efficacy, side effects and potential dangers in volunteers and patients
what are the stages of drug discovery?
- target selection
- lead finding
- lead optimisation
- pharmacological profiling
what happens in target profiling in drug discovery?
- drug targets are mostly functional proteins
- identification of targets comes from biological intelligence
- 100s to 1000s potential drug targets have yet to be exploited therapeutically
- conventional, biological wisdom, drawing on a rich knowledge of disease mechanisms and chemical signalling pathways and genomic data is the basis on which novel targets are chosen
what is involved in lead finding in drug discovery?
- when the biochemical target has been decided and the feasibility of the project has been assessed, the next step is to find lead compounds
- cloning of the target protein
- assay system developed, to measure the functional activity of the target protein
what are the requirements for assays used in lead finding (drug discovery)?
- assay system measures the functional activity of the target protein
- could be cell-free enzyme assay, membrane-based binding assay or cellular response assay
- must be engineered to run automatically, in a miniaturised multiwell plate format for speed and economy
- robotically controlled assay facilities are often used
what is the process of screening in assays in lead finding (drug discovery)?
- large compound libraries are maintained by large companies, with a growing collection of a million or more synthetic compounds
- these compounds in the library are routinely screened whenever a new assay is set up
- X-ray crystallography etc is used to produce 3D structure of a target protein to generate possible lead structures and reduce the number of compounds to be screened
- aim is to identify appropriate lead compounds with pharmacological activity that are modifiable
what are ‘Hits’ detected in initial screening? what are they used for?
- turn out to be molecules with features undesirable in a drug, e.g. too high a molecular weight, excessive polarity and groups associated with toxicity
- computational prescreening of compound libraries are used to eliminate such compounds
- used as a basis for preparing sets of homologues by combinatorial chemistry to establish critical structural features
what are examples of natural products used as lead compounds?
- penicillin
- streptomycin
- other antibiotics
- vinca alkaloids
- paclitaxel
- ciclosporin
- sirolimus (rapamycin)
how are natural products utilised as lead compounds in drug discovery?
- fungal and plant sources have been used in anti-infective, anticancer and immunosuppressant drugs
- fungi and microorganisms are ubiquitous, highly diverse and easy to collect and grow in the lab
- compounds from plants, animals or marine organisms are troublesome to produce commercially
what is the main disadvantages of natural products as lead compounds in drug discovery?
they are complex molecules that are difficult to synthesise/modify by conventional synthetic chemistry
- lead optimisation may difficult
- commercial production expensive
what is the aim of lead optimisation?
- to increase potency of the compound on its target and to optimise it with respect to other characteristics, e.g. selectivity and pharmacokinetic properties
- to identify one or more drug candidates suitable for further development
what are the tests applied in lead optimisation in drug development?
- broader range of assays on different test systems
- studies to measure the activity and time course of the compounds in vivo
- checking for unwanted effects in animals, evidence of genotoxicity and oral absorption
what is the success rate of projects in drug discovery? how long does it take?
2-5 years
100 projects
only about one project in five succeeds in generating a drug candidate
what are problems affecting lead optimisation in drug discovery?
- lead optimisation is often very difficult
- compounds produce the desired effects on the target molecule and have no other defects, but fail to produce the expected effects in animal models of the disease, implying that the target is not good
what are the stages in preclinical development?
- pharmacokinetics
- short-term toxicology
- formulation
- synthesis scale-up
what is the aim of preclinical development?
to satisfy all requirements that have to be met before a new compound is deemed ready to be tested for the first time in humans
what are the four categories of work involved in preclinical development?
- safety pharmacology: testing to check that the drug doesn’t produce any obviously hazardous acute effects
- preliminary toxicological testing to eliminate genotoxicity and to determine maximum non-toxic dose.
- animals are checked for weight loss/gross changes, and examined post mortem to look for histological and biochemical evidence for tissue damage - ADME studies in animals, to link the pharmacological and toxological effects to plasma concentration and drug exposure
- chemical and pharmaceutical development to assess the feasibility of large-scale synthesis and purification, assess the stability of the compound and to develop a formulation suitable for clinical studies
what is the work of preclinical development done under? what does it cover?
Good Laboratory Practice: covers record keeping procedures, data analysis, instrument calibration and staff training
what is the aim of GLP?
to eliminate human error as far as possible and to ensure reliability of data
- labs are regularly monitored for compliance to GLP standards
what is required for permission to proceed with drug studies in humans?
- ‘inestigator brochure’ submitted alongside specific study protocols to regulatory authorities
- European Medicines Evaluation Agency
- US Food and Drugs Administration
how long does preclinical development take and how many compounds go through it?
1.5 years
20 compounds
what are the phases involved in clinical development?
phase I, II and III
what are the steps involved in phase I of clinical development? how many compounds go through it?
- pharmacokinetics
- tolerability
- side effects in healthy volunteers
10 compounds involved
what are the steps involved in phase II of clinical development? how many compounds go through it?
- small scale trials in patients to assess efficacy and dosage
- long term toxicology studies
5 compounds involved
what are the steps involved in phase III of clinical development? how many compounds go through it?
- large-scale controlled clinical trials
2 compounds involved
what happens in phase I studies? what is assessed?
- performed on a small group (20-80) of volunteers
- to check for signs of dangerous effects, tolerability, pharmacokinetic properties
- pharmacodynamic effects in volunteers (proof-of-concept studies)
what happens in phase II studies? what is assessed?
- performed on groups of patients (100-300)
- determine pharmacodynamic effect in patients, and if confirmed, to establish the dose regimen to be used in phase III
- cover several distinct clinical disorders to identify possible therapeutic indications for the new compound and the dose required
what happens in phase III studies? what is assessed?
- definitive double-blind, randomised trials
- performed as multicentre trials on thousands of patients
- aimed at comparing the new drug with commonly used alternatives
- expensive, difficult to organise and take years to complete
what does good clinical practice cover?
covers every detail of the patient group, data collection methods, recording of information, statistical analysis and documentation
what does pharmacoeconomic analysis assess?
clinical and economic benefits of a new drug
what is involved in regulatory approval after phase III studies?
- drug submitted to relevant regulatory authority for licensing
- required dossier is a massive and detailed compilation of preclinical and clinical data
- takes a year or more
what is the approval rate of phase III drugs?
- 2/3 of submissions gain marketing approval
- only 11.5% of compounds entering phase I are eventually approved
what is involved in phase IV studies?
- obligatory postmarketing surveillance designed to detect any rare or long-term adverse effect resulting from use of the drug in a clinical setting in thousands of patients
- may lead to limiting use of the drug to particular patient groups, or withdrawl of the drug
what are biopharmaceuticals?
therapeutic agents produced by biotechnology rather than conventional synthetic chemistry
what is the proportion of biopharmaceuticals in new products registered each year?
about 30%
what are advantages/disadvantages of biopharmaceuticals?
- have fewer toxicological problems than synthetic drugs
- more problems with production, quality control, immunogenicity and drug delivery
what are the main key points about pharmaceutical projects?
- high-risk business, with only one drug descovery in 50 reaching its goal
- takes 12 years on average
- very expensive
how long does patency usually last?
20 years in most countries
