Pharmacology and Physiology,

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Pharmacology and Physiology, Pharmacology
Lectures BIOL243 / BMSC 213
Dr Paul Teesdale-Spittle School of Biological
Sciences KK713 Phone 6094
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Introduction What is pharmacology The study of
the interaction between exogenous chemicals (or
xenobiotics) and living organisms. 1. How an
organism affects a xenobiotic. Transport,
distribution and metabolism. 2. How a xenobiotic
affects an organism. Molecular targets, mode of
action and toxicology. Pharmacology is not just
descriptive, but also quantitative.
Mathematical quantification and physical
chemistry. The purpose of the pharmacology
lectures is to introduce the principles of the
subject and show how it can be used to understand
modulation of physiological function through the
action of drugs.
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• Pharmacology is important for
• Chemists
• designing new drugs.
• Clinicians
• administering drugs.
• Toxicologists
• explaining toxicity.
• Biochemists, Physiologists, Psychologists
• using drugs to modify the normal functioning of
  cells or organisms.

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Definitions Xenobiotic A chemical that is not
endogenous to an organism. Endogenous Made
within. Drug A chemical taken that is intended
to modulate the current physiological status
quo. Ligand A compound that binds to another
molecule, such as a receptor protein. Bioavailabil
ity The amount or proportion of drug that
becomes available to the body following its
administration. Pharmacokinetics What the body
does to a drug. Pharmacodynamics What a drug
does to the body .
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History Ancient knowledge of the materials that
could relieve pain, alter moods and perceptions,
aid against infection, poison etc. The first
written treatises were generated by the Chinese
e.g. Pen Tsao was written 2700 B.C., describing
uses and classifications of medicinal plants.
The ancient Egyptians by 1550 B.C. had written
prescriptions using a range of pharmaceutically
active ingredients and vehicles for their
delivery. At about the same time, similar
medical advances were being made in Babylonia and
India. Between about 400-300 B.C. the Greeks
made enormous advances in the knowledge of
anatomy and physiology.
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• Philippus Theophrastus Bombastus von Hohenheim
  (1493-1541), also known as Aureolus Paracelcus,
  took up the pharmacological baton.
• He is often referred to as the grandfather of
  pharmacology (and also the grandfather of
  toxicology) because of his impact on the
  understanding between dose and response All
  things are poisons, for there is nothing without
  poisonous qualities. It is only the dose which
  makes a thing a poison.
• No real further advances until the sciences of
  chemistry and physiology had developed
• To provide pure compounds.
• Allow careful monitoring of their physiological
  effects.
• This combination of circumstances arose in the
  early 19th Century.

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• Thus, the history of pharmacology has shown the
  importance of the subjects of
• Biochemistry
• Chemistry and
• Physiology.
• The main aims of the subject are to
• Evaluate the mode and site of action of drugs
• Their distribution, metabolism and elimination
  and
• The molecular interactions by which they
  function.

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• Drug action
• A drug is a compound that can modify the response
  of a tissue to its environment.
• A drug will exert its activity through
  interactions at one or more molecular targets.
• The macromolecular species that control the
  functions of cells.
• May be surface-bound proteins like receptors and
  ion channels or
• Species internal to cells, such as enzymes or
  nucleic acids.

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• Receptors
• Receptors are the sites at which biomolecules
  such as hormones, neurotransmitters and the
  molecules responsible for taste and odour are
  recognised.
• A drug that binds to a receptor can either
• Trigger the same events as the native ligand -
  an agonist.
• Or
• Stop the binding of the native agent without
  eliciting a response - an antagonist.
• There are four superfamilies of receptors.

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Type 1. These have 4 or 5 membrane-spanning
helical subunits. Their N- and C-terminii are
found in the extracellular fluid. This family
includes ion channels. Type 2. These have 7
helical transmembrane regions. Their N- terminal
is extracellular and the C-terminal in
intracellular. This family is coupled to the
action of G-proteins They are known as the
G-protein coupled receptors. Type 3. These are
tyrosine kinase-linked receptors with a single
transmembrane helix. The insulin and growth
factor receptors fall within this family. Type 4.
These receptors are found in the cell nucleus
and are transcription factors. They have looped
regions held together by a group of four cysteine
residues coordinating to a zinc ion. These
motifs are called zinc fingers. The receptor
ligands include steroids and thyroid hormones.
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• Read the sections on ion channels G-protein
  coupled receptors
• Then answer the following
• Name two therapeutic uses for ion channel
  blockers.
• Are channel blockers agonists or antagonists?
• How many transmembrane helices are there in
  GPCRs?
• Where does GTP bind?
• Why GTP and not ATP?
• How do receptors amplify the signal of a single
  ligand?

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• Enzymes
• They are proteins that catalyse the reactions
  required for cellular function.
• Generally specific for a particular substrate, or
  closely related family of substrates.
• Molecules that restrict the action of the enzyme
  on its substrate are called inhibitors.
• Inhibitors may be irreversible or reversible.
• Reversible inhibitors may be
• Competitive.
• Non-competitive.
• Enzyme inhibitors might be seen to allow very
  fine control of cellular processes.

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• Nucleic acids
• Potentially the most exciting and valuable of the
  available drug targets.
• BUT designing compounds that can distinguish
  target nucleic acid sequences is not yet
  achievable.
• There are compounds with planar aromatic regions
  that bind in-between the base pairs of DNA or to
  the DNA grooves.
• These generally inhibit the processes of DNA
  manipulation required for protein synthesis and
  cell division.
• Suitable as drugs for applications where cell
  death is the goal of therapy - such as in the
  case of the treatment of cancer.
• Name another use where cell death is desirable.

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• Mechanisms and Specificity of Drug Binding
• The majority of binding and recognition occurs
  through non-covalent interactions.
• These govern
• The folding of proteins and DNA.
• The association of membranes.
• Molecular recognition (e.g. interaction between
  an enzyme and its substrate or the binding of an
  antibody).
• They are generally weak and operate only over
  short distances.
• As a result large numbers of these interactions
  are necessary for stability, requiring a high
  degree of complementarity between binding groups
  and molecules.

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• Covalent bonds
• The sharing of a pair of electrons between two
  atoms.
• These electrons largely occupy the space between
  the nuclei of the two atoms.
• A very stable interaction
• Requires hundreds of kilojoules to disrupt.
• Compounds that inhibit enzymes through formation
  of covalent interactions are called suicide
  inhibitors.
• Not all covalent bond formation is irreversible
• Hydrolysis.
• Action of repairing proteins.

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• Non-covalent interactions
• The forces involved are
• Hydrogen bonds
• van der Waals forces
• Ionic / electrostatic interactions
• Hydrophobic interactions.
• Generally, such interactions are weak
• vary from 4-30 kJ/mol.

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• van der Waals
• Weak, but significant over many atoms.
• Attractive over short distances
• A strong repulsive force at very short
  distances.
• From temporary dipole - induced dipoles
• Every atom
• No directionality
• Less entropically unfavourable

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• Hydrogen bonds
• Strong
• Longer distance
• Directional
• Most common is between the CO and NH groups on
  the peptide backbone.

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• Electrostatic
• Two common classes of electrostatic interactions
• Ionic and dipolar.
• Ionic interactions arise between basic and
  acidic functionalities, typically amines and
  carboxylic acids.
• Can be spread over more than one atom.
• Salt bridges.
• These are the strongest non-covalent
  interaction.
• Dipolar interactions are also extremely
  important.
• Interaction of partially charged regions of
  molecules or as a result of aromatic ?-systems.
• Dipolar interactions are much weaker than ionic
  interactions.

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• Hydrophobic interaction
• Water hating - oil / water principle
• Why?
• DG DH TDS
• Either
• Water H-bonding disrupted (DH ve but DS -ve)Or
  
• Water forms an ordered clathrate cage around
  solute (DH -ve but DS ve)
• In both cases DG ve.
• At low temps, formation of clathrate cages least
  unfavourable

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• Combinbation of 2 clathrate cages gives a smaller
  overall surface area.
• Leads to smaller amount of ordered H-bonding
  surface
• and therefore less unfavourable DG.

• Hydrophobic moieties tend to combine

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• Recognition
• Forces of interactions are weak
• Need many co-operative forces as binding
  entropically unfavourable.
• i.e. lots of small -ve DHs to make DG -ve.
• So need good complementarity between binding
  groupsand molecules
• lock key
• Only a small range of conditions under which most
  molecularassemblies will operate
• e.g. Effected by temp, pH, metal concentration
  etc.

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• Conformation effects
• Binding also locks a mobile, flexible molecule
  into a restricted conformation.
• These losses of motion are entropically
  unfavourable (?S negative).
• Since ?G ?H -T?S, then the entropic energy loss
  must be compensated for by the enthalpic
  contribution.
• Configuration effects
• Differences in configuration (e.g.
  stereochemistry) can lead to startling
  differences in the biological effect.
• e.g. The L enantiomer of penicillamine is highly
  toxic and only the (S) enantiomer of indomethacin
  acts as an anti-inflammatory agent.
• The wrong configuration will lack required
  interactions or add undesired ones

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Protein Surface
 
X-Ray Diffraction Structure Of Hiv-1 Protease
Complexed With SB203238
(Drawn from Brookhaven database file
1hbv.pdb. K.A.Newlander, J.F.Callahan, M.L.Moore,
T.A.Tomaszek, W.F.Huffman A Novel Constrained
Reduced-Amide Inhibitor Of HIV-1 Protease Derived
From The Sequential Incorporation Of Gamma-Turn
Mimetics Into A Model Substrate J.Med.Chem. 1993,
36, 2321.)
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Selectivity, toxicity and therapeutic index Drugs
may bind to both their desired target and to
other molecules in an organism. If interactions
with other targets are negligible then a drug is
said to be specific. In most cases drugs will
show a non-exclusive preference for their target
- selective. The interaction with both their
intended target and other molecules can lead to
undesirable effects (side effects). Establish
the concentrations at which the drug exerts its
beneficial effect and where the level of side
effects becomes unacceptable. Commonly used
values are ED50 and LD50. For obvious reasons
LD50 tests are not carried out on human
volunteers!
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• One measure of the margin of safety is the
  therapeutic index. Therapeutic index LD50 /
  ED50
• Drugs with low therapeutic indices are only used
  in life or death type situations.
• Exercise it can be argued that the ratio LD1 /
  ED99 might be a more realistic estimate of
  safety. Why?
• There are other side effects of drugs that are
  undesirable.
• e.g. Drowsiness, nausea, impairment of immune
  functions and so on.
• The protective index is defined as the ratio of
  ED50s of the desired and undesired effects.
• Should be gtgt1

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• Agonists antagonists
• Activity of a drug is the result of two
  independent factors
• Affinity is the ability of a drug to bind to its
  receptor.
• Efficacy describes the ability of the bound drug
  to elicit a response.
• The two state model. Receptors can at rest or
  activated.
• An agonist stabilises the active state
  preferentially.
• An antagonist shows no preference or it
  stabilises the resting state.
• The efficacy of a compound in the two state model
  is the degree of selectivity for stabilising the
  active or resting state of the receptor.

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• The degree of selectivity can be expressed in
  terms of the ratio of the equilibrium binding
  constant, K for each receptor state.
• Kactive / Kresting gt 1, then the compound is an
  agonist. The higher the ratio, the higher will
  be the efficacy.
• Kactive / Kresting ? 1, then the compound is an
  antagonist. The smaller the ratio, the higher
  will be the efficacy.
•  
• There is not a direct proportionality between
  receptor occupancy and response.
• The maximum possible cellular response may occur
  at levels of lower than 100 receptor occupancy
  with a strong agonist.
• Due to the amplification inherent in receptor
  response

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• There are 2 classes of agonist
• Full agonists which elicit the maximum
  possible response at some concentration
• Partial agonists which never elicit the
  maximum possible response from the receptor.
• There are also 2 classes of antagonist
• Competitive antagonists which compete for the
  agonist binding site, and require higher agonist
  concentration to elicit a given response.
• Non-competitive agonists these bind at a site
  other than the agonist binding site, or even to a
  completely different molecular target. The
  result is the lowering of the maximum possible
  response in addition to the usual antagonist
  effect of displacing agonist activity to higher
  concentration.

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Concn vs response curves The amount of drug could
be expressed in terms of 1. Amount of drug
administered 2. Dose per unit bodyweight of the
subject 3. Concentration of drug in plasma or
serum Usually expressed in terms of 2.
(clinically useful) or 3. (useful in research).
  The monitored effect might be Quantised (e.g.
dead/alive, cured/not cured) Continuous (e.g.
Days of remission, percentage reduction in
swelling)
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• In either case, data is often normalised
• Responses given as a fraction (or percentage) of
  the group as a whole for quantised data or of the
  maximum response for an individual subject for
  continuous data.
• It is common to use a logarithmic scale for
  response curves (i.e. plotting log(dose) or
  plotting dose on a logarithmic scale).

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Quantised data Here there is a specific response
being measured as an effect of concentration.
Each subject will demonstrate that response at
some concentration. The data can be represented
by a graph of the cumulative fraction of animals
displaying the response at a given concentration.

Requires large numbers of test subjects and
repeated experiments to be statistically valuable.
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Continuous data A continuum of level of effect as
concentration changes. Drug administered until
the effect becomes saturated with a single
individual. The data is plotted normalised to the
maximum effect, to give a concentration vs
fraction of maximum effect curve. Different test
subjects usually give responses that are shifted
along the concentration axis relative to each
other. Use large test sets. ED50 can be
calculated as a mean from this data, along with a
measure of distribution, such as standard
deviation.
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• Exercise Try constructing dose-response curves
  for the following systems
• 3 different full agonists of differing activity.
• A full agonist and a partial agonist.
• An agonist in the presence of no antagonist and
  2 increasing doses of a competitive antagonist
• An agonist in the presence of no antagonist and
  2 competitive antagonists of differing activity
• An agonist in the presence of no antagonist and
  2 increasing doses of a non-competitive
  antagonist

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Some Physical Chemistry It is possible to explain
these response curves based on the equilibrium
associations involved. Response and occupancy are
not always directly proportional, however this is
assumed for the sake of simplicity. Agonist
binding
RateF k1A.R RateR k-1AR
The equilibrium constant K is given by the ratio
K k1/k-1
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AR/R K.A
Rtot R AR
Consider situation if A 0, small, big, ?
As K increases, then the dependence of the
response on K decreases. Focc becomes
progressively a function of concentration. When K
is very high, 1/K becomes effectively zero, so
Focc becomes 1.
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See Excell graphs
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Competitive Antagonists
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AR/R K.A NR/R KN.N
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Define the ratio, r, of new (A) to old (A)
concentrations as
Setting Focc equal for situation with and without
antagonist
r 1 KN.N log(r-1) logN log KN
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Example Problem An agonist, A, provides 50 of
its maximum response at a concentration of 30
?M. Calculate the required concentration to
reproduce this response in the presence of an
antagonist, N, whose equilibrium binding
constant, KN, is 6x104 M when the concentration
of N is 20 ?M.
Solution The required increase in A is given
by r 1 KN.N In this case, KN 6x104 M
and N 2x10-5 M (notice the conversion back to
molarity, the equation would not work
otherwise). So r 1 (6 x 104).(2 x
10-5) 1 12 x10-1 2.2 So the required
concentration of A is 2.2 times the concentration
required to produce the same effect without the
antagonist, i.e. 66 ?M.
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See Excell graphs
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• Summary
• A ligand for a receptor may be a full agonist, a
  partial agonist, a competitive antagonist or a
  non-competitive agonist.
• An agonist stabilises the active state of the
  receptor.
• An antagonist stabilises its resting state.
• The degree of stabilisation reflects the
  efficacy of a ligand.
• Ligand-response curves generally demonstrate a
  saturating response with increasing agonist
  concentration.
• A competitive antagonist shifts the curve to
  higher agonist concentration.
• A non-competitive agonist lowers the maximum
  possible response.
• These effects can be quantified in simple models
  using physical chemistry.