NEUS 609 - Neuropharmacology

neural action

drugs that target receptors, ion channels, transporters, and enzymes are used to modulate ___

pharmacology

study of effects of chemical agents (drugs) on and in living systems

pharmacokinetics

what the body does to the drug

• absorption, distribution, biotransformation, and excretion

pharmacodynamics

what the drug does to the body

• effects and mechanisms of actions of drugs

toxicology

study of unwanted or undesirable effects of drugs or other chemicals (toxicants) on the body and the mechanisms of those effects

pharmacotherapeutics

treatment or prevention of illness and disease by using drugs

pharmacogenomics

study of how the genetic variation contributes to individual variability in response to drugs, and conversely, how drug exposure affects gene expression

neuropharmacology

study of drugs in the nervous system, and how the nervous system utilizes chemical neurotransmission

• typically, chemical neurotransmission

Basics Principles of pharmacology

1- drugs do NOT create functions but modify existing functions within the body

2- No drug has a single action

3- drug action is determined by how the drug interacts with the body

• chemically alter body fluids (antacids and chelators)

• physically alter cell membranes (anesthetics solvents)

• act through specific proteins mediating intracellular processes (receptors, enzymes, transport proteins, voltage-gated channels)

neuroactive

most ___ drugs act by activating or inhibiting receptors

drug

any small molecule that alters the body’s functions at the molecular level

receptor

component of a cell to which the drug binds and initiates a response

true receptor

protein whose function is to receive an extracellular signal, usually the binding of a hormone, NT, or autocrine/paracrine factor

ligand

any compound that specifically binds to a receptor

agonist

a compound which when bound to a receptor leads to a measurable biological response

• aka activates the receptor

binds to a receptor and produces a response whether excitatory or inhibitory

antagonist

compound when bound to a receptor blocks the effects of an agonist

• binds but does NOT activate the receptor

• intrinsic activity alpha = 0

binds to the receptor but produces no response (blocks effect of agonist or endogenous ligand)

Dose=response curve

aka concentration-effect curve

Graph plotting measured response/effect against known drug concentration

slow

__ dissociation = high affinity (tightly bound to receptor)

association

binding of ligand to receptor, does not vary a lot between ligand

dissociation

separation of ligand from receptor, very variable between ligands 

• used for affinity measurement

affinity

measure of how tightly the drug binds to the receptor

• expressed as Kd aka the concentration of drug at which 50% of receptor are bound which is the reciprocal of __

• low Kd means high ___ and vice versa

efficacy

degree or magnitude of the response that a drug produces when it binds to the receptor

• maximal effect produced by a drug which is related to the degree to which drug activates the receptor when bound

maximal effect

receptor density

number of receptors the cell contains

• aka Rt or Bmax

• number of moles of receptor per cell or per unit of tissue

quantification of receptors

• Blots, rtPCR which measure protein or RNA expression

• Binding assays which measure receptor binding and can be used to deduce receptor density and ligand affinity

  • direct or indirect

  • broken cells, tissue sections, whole animals

  • detection via radiolabeled or fluorescent ligand

radioligand receptor binding

• homogenize tissues (broken cell preparation)

• centrifuge, keep pellet, and resuspend in buffer

• incubate lower than physiological temperature

• add excess UNLABELED drug for NONSPECIFIC binding

• filtration assay

Ligand is either free in solution or bound to receptor forming a complex

Measure complex formation and nonspecific binding

saturation curve

Graph of receptor-ligand complex formation [RL] against varying ligand concentration [L]

• determine [RL] by separating bound from unbound drug (non-specific binding has a linear progression)

• finite number of receptors which is why it is called a ___

specific binding

high affinity & low capacity binding

non-specific binding

low affinity, high capacity binding

equilibrium dissociation constant

Kd = (k1/k-1) = [R][L]/[RL]

fundamental constant of affinity of ligand for receptor

direct binding assay

binding assay determining the affinity and maximal number of binding sites

LANGMUIR equation governs the saturation curves

[RL] = (Bmax[L])/([L]+Kd)

Langmuir equation

[RL] = (Bmax[L])/([L]+Kd)

Criteria for receptor

1 - bind specifically (total-nonspecific binding) and stereo-specifically (active vs inactive isomers require different [L])

2 - specific binding is saturable (finite number of binding sites)

3 - tissue linearity (binding is linear with tissue amount aka more tissue = more receptors available)

4 - specific binding is reversible (ligand comes off receptor at some point) and functional response is antagonist reversible (can reverse/inhibit function using antagonist which is possible because binding is reversible so antagonist binds when agonist comes off)

5 - localization is relevant: receptor is found in tissue where functional response occurs

• regional = receptor located in appropriate organs and cell types

• subcellular = receptor located where it can receive signal (on plasma membrane or intracellular)

6 - RECEPTORS DO NOT METABOLIZE THE LIGAND (aka receptor is NOT a degradative enzyme)

Scatchard/Rosenthal plot

linearize receptor binding data

• curvilinear when there are multiple binding sites leaves a mirrored comma shaped curve

• y-axis = B/F (bound/free)

• x-axis = B (bound)

• slope = -1/Kd

• x-intercept = Bmax

Scatchard equation

With [RL] = B and [L] = F

Can track change in Bmax aka the number of receptors available

(B/F) = (Bmax-B)/Kd

low

__ Kd = high affinity

indirect binding assay

aka competitive binding assay

determines affinity and selectivity

single concentration of labelled drug is displaced by varying concentrations of unlabeled drug

Cheng-Prushoff equation

Cheng-Prushoff equation

Ki = IC50/(1+[L]/Kd)

where Kd = Kd of radioligand & [L] = concentration of radioligand

• try using concentration of radioligand closer to Kd as possible so denominator of correction as close to one as possible

IC50

inhibitory concentration 50

concentration that inhibits 50% of radiolabeled ligand binding

competition curves 

• structure-activity relationship

• displacement (y-axis) against drug log concentration (x-axis)

• application of Cheng-Prushoff equation

hill plot

• linearize binding data (works best with data between 5 and 95%)

• determine cooperativity or multiple sites

• hill coefficient = slope of hill plot & what determine cooperativity type

• y axis = log (B/(Bmax-B))

• x-axis = log [L]

slope = 1

hill coefficient

NO cooperativity, one binding site type, no interaction between sites

slope >1

hill coefficient

positive cooperativity between sites

slope <1

hill coefficient

negative cooperativity (does not really exist) OR multiple binding sites with different affinities for ligand

autoradiography

binding assay

1- cut frozen tissue on cryostat & freeze

2- wash in buffer & incubate with radioligand

3- rinse in buffer then H2)

4- dry and expose to film for months

5- analyze using densitometry

Darker radioactivity = more binding

exact measure of binding and where it is

advantages of autoradiography

• anatomical resolution

• requires less tissue per CNS region

• can be combined with other approaches like immunostaining

advantages of filtration binding assay

• easier to run multiple ligand concentrations

• determine Kd and Bmax

• data available more quickly than autoradiography

• can be combined with other approaches like western blots

receptor activity

MAJOR DISADVANTAGE OF ALL RECETOR BINDING ASSAYS is that they do NOT provide any information on ____

• cannot distinguish between agonists, antagonists, or determine functional effects of receptor occupancy

potency

concentration required to produce an effect

EC50

concentration of drug necessary to produce 50% of maximal effect

greater

lower EC50 = __ potency

Clark’s theory

• recognized relationship between dose and effect is proportional

• proposed that magnitude of effect is proportional to the fraction of total receptors occupied

E = ([L]Emax)/([L]+EC50)

basically, drug affinity = drug potency

explains potency NOT EFFICACY (all drugs also assumed to bind to the same receptor)

Ariens’ theory

introduced the concept of intrinsic activity of a ligand

problems

• assume full occupancy of receptor for full effect

• not accounting for effect of differences in Rt aka Bmax

• not accounting for inverse agonism

E/Emax = a[AR]/Rt

with [AR] is occupied receptor, Rt is total # of receptors (~Bmax), and a is intrinsic activity

potency determined by affinity, and efficacy determined by intrinsic activity

full agonist

intrinsic activity alpha = 1

produce max effect

number

same fractional occupancy can lead to different effect based on the cell receptor __

• cells with less need higher occupancy for same effect

partial agonist

in between no effect and max effect

0< alpha < 1

binds to receptor but produces a less than maximal response, determined by properties of ligand, tissue and functional measure

also partial antagonist

neutral agonist

no effect

inverse agonist

stabilizes inactive form of receptor decreasing baseline activity leading to opposite effect

binds to receptor and decrease basal activity

competitive antagonist

orthosteric, single binding site: apparent EC50 of agonist is increased, but apparent efficacy (maximal effect) is the same

potency calculated as Ke 

Ke = [ANT]/(DR-1)

DR = EC50 with agonist - EC50 alone

DRC SHIFTS RIGHT

non-competitive antagonist

1- allosteric = binds to a site different from agonist binding site & decrease Emax

2- irreversible = binds covalently, show no change in agonist EC50, but decrease Emax

Stephenson’s theory

effect is a function of the stimulus produced 

• does not assume full occupancy for full effect

• DOES NOT ACCOUNT FOR effect of differences in absolute values of Rt on total stimulus produced

S = e[AR]/Rt

where S = stimulus produced by ligand & e is its efficacy

Furchgott theory

• does not assume full occupancy for full effect

• stimulus depends on total number of occupied receptors and intrinsic efficacy

• if Rt is high, then greater [AR] at any [AR]/Rt

S = E[AR]

more receptor = more effect

general pharmacodynamic equation

Effect = E*Rt*([L]/[L]+Kd)

• E = intrinsic ligand efficacy

• Rt = receptor density aka Bmax

• ([L]/[L]+Kd) = fractional occupancy = B/Bmax

ceiling effect

phenomenon where increasing the dose of a drug does not lead to an increase in its therapeutic effect, often due to receptor saturation.

• depends on response being measured

Ki/EC50 ratio

• ratio >1 = receptor reserve aka excess

• ratio = 1 = partial agonist

• Ki is basically Kd

higher ratio = greater affinity

intrinsic efficacy

Er x (Ki/EC50 + 1) x 0.5

• Er = relative efficacy = Er = Emax of ligand/Emax of standard full agonist