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NEUROPHARMACOLOGY:504

ABERCROMBIE

LECTURE 1: INTRODUCTION TO GENERAL PHARMACOLOGY – Pharmacokinetics and Pharmacodynamics

How drugs work and how their effects are measured. Drugs can act to increase or decrease the normal function of tissues or organs, but they do not confer any new functions on them; effects of drugs are quantitative, never qualitative. A particular effect measured as a change in a definite physiologic process may be brought about in several ways.

Nomenclature

Pharmacology - the study of the effects of drugs on the body. However, given the broad nature of this subject area, pharmacology includes a number of subdisciplines:

Neuropharmacology - the subdiscipline devoted to the study of the effects of drugs on cells of the nervous system.

Psychopharmacology - the subdiscipline devoted to the study of the effects of drugs on psychological processes and behavior.

Clinical Psychopharmacology - the subdiscipline of psychopharmacology concerned mainly with the use of drugs to treat abnormal human behaviour.

Clinical Neuropsychopharmacology - you guessed it, a combination of all three subdisciplines of pharmacology, i.e. the subdiscipline concerned with the study of the effects of drugs on the nervous system, their effects on psychological and behavioural processes, and their use in the treatment of abnormal human behaviour.

PHARMACOKINETICS AND PHARMACODYNAMICS

Nomenclature

Pharmacokinetics - the study of the absorption, distribution, and elimination of drugs, that is, what the body does to the drug.

Pharmacodynamics - the study of the action of drugs at cellular receptors, that is, what the drug does to the body.

Drug - any chemical agent that affects living processes via actions at receptor sites and include exogenous chemicals that are ingested deliberately or inadvertently, and also endogenous chemicals such as neurotransmitters and hormones.

Drug absorption - the process by which a drug gets from the site of administration to the blood plasma (the fluid of the blood minus the red and white blood cells; blood serum is the plasma minus the blood protein, fibrinogen).

Drug distribution - degree to which a drug reaches its receptor targets or a biological fluid (e.g., blood plasma). Once in blood plasma, it is distributed throughout the body via circulation. How much drug will be available to the CNS depends on how well the drug crosses the Blood brain barrier- a term used to describe the low permeability of capillaries that supply the CNS with blood and the amount of drug that binds to blood proteins. Some drugs cannot be used clinically because of low blood brain barrier permeability, while others bind to proteins.

Drug elimination - two processes by which drugs are eliminated from the body: (a) metabolism, performed mainly by the liver; and (b) excretion, performed mainly by the kidneys.

Drug clearance - ability of the body to eliminate a drug. Elimination half-life is time required for drug concentration to decay 50% of initial peak concentration. Drugs usually stay in body for ~4-5 half-lives.

PHARMACOKINETICS AND PASSAGE ACROSS BIOLOGICAL BARRIERS (relevant reading: Chapter 1 M & Q)

Drugs must move from where they are administered to the tissues or cells where they will act. Selectivity of migration of drug is consequence of physicochemical properties and structural configuration of barrier and of molecule. Transport defined as translocation of a solute from one phase to another. Passive diffusion, Facilitated diffusion, Active transport. PASSIVE DIFFUSION: directed movement of solute through a biologic barrier from the phase of higher concentration to the phase of lower concentration, the process requiring no direct expenditure of energy by system. Concentration gradient & permeability.

DRUG ABSORPTION - drug must be absorbed and be transported to site of action, target site; transport often over very large distances. Bloodstream as carrier. How and where drug is administered will determine how quickly drug is absorbed and how completely it gets into bloodstream (definition of absorption). Disparities in movement of materials across different biological barriers must lie in anatomical arrangement of barriers themselves.

E.G., drugs entering the body by way of the GI tract, skin, or lungs must first traverse an epithelial barrier before entering the interstitium. Drugs given S.C. or I.M. bypass the epithelial barrier. Drugs given by any route except I.V. must traverse the capillary wall in order to enter circulation. Vascularity at site of absorption thus an important variable. ENTERAL ADMINISTRATION (via GI tract)

1) Sublingual - thin epithelium, high vascularity, and slightly acid pH make oral mucosa very conducive to absorption. Compound must dissolve rapidly and be effective in small amounts. Advantage that drug gains access to general circulation without first traversing liver (also true of lower rectum).

2) Oral administration -Stomach fluid is very acidic, thus absorption of weak acids (like aspirin) is promoted, weak bases (e.g. nicotine) not absorbed to any significant degree. Major factor is rate of stomach emptying into small intestine. Small intestine exquisitely suited for absorption because of enormous surface area, true whether substance is lipid soluble or is a weak electrolyte. Enormous quantitative difference in area available for absorption. Along length of intestine, pH changes from slightly acidic to barely alkaline. PARENTERAL ADMINISTRATION (bypass GI tract) 1) Transdermal application - very slow. Sustained release preparations, blood level achieved never very high. 2) Inhalation - Very large surface area and high degree of vascularization of lungs. Lungs receive half of blood pumped from heart. Most effective absorptive area of body. Gases are almost always small molecules of high lipid solubility and are almost instantaneously absorbed when inhaled. Aerosol preparations.

3) Subcutaneous injection - Bypass epithelial layer and must pass endothelial layer of capillaries to enter bloodstream. Rate of capillary blood flow/vascularization main determinant. Some drugs too irritating.

4) Intramuscular injection - Again, barrier is capillary wall. Muscle somewhat more blood flow, especially when active. Larger volumes, more irritating substances can be administered. 5) Intravenous injection - most rapid and precise since barriers are eliminated and therefore exact quantity of drug administered is amount absorbed. Can be used for drugs otherwise too irritating. Least safe, large variations in blood level.

DIFFUSION ACROSS MEMBRANES DEPENDS ON RELATIVE LIPID SOLUBILITY (lipid/water partition coefficient)

The rate of passive diffusion is dependent on the degree of lipid solubility. Compounds that are highly soluble in lipids diffuse rapidly, whereas those that are relatively lipid insoluble diffuse more slowly. Size also a factor. See NEURONAL MEMBRANE (below).

MOST AGENTS OF PHARMACOLOGICAL INTEREST ARE WEAK ELECTROLYTES. They ionize, but only partially such that they are present in aqueous solution as both undissociated and dissociated entities.

Weak acids: substance that is a proton donor, net negative charge (aspirin)

Weak bases: substance that ionizes by accepting a proton, net positive charge.

Ammonium ion type coordinate bond formation important for many biologically active molecules (ionization of amines e.g.) Majority of commonly used drugs are weak bases. Tendency to ionize for a given weak electrolyte = ionization constant.

HA = H+ + A- HA is the undissociated acid

B + H+ = BH+ B is the unionized base

Fractions are in equilibrium.

DRIVE THESE REACTIONS BY CHANGING HYDROGEN ION CONCENTRATION, pH.

pH is expression of reciprocal of H+ ion concentration.

1) degree of ionization of a weak electrolyte is dependent on its ionization constant and on the pH of the aqueous medium in which it is dissolved.

2) degree of ionization of a weak acid tends to be greater at higher pH and lower at lower pH.

3) degree of ionization of a weak base tends to be greater at lower pH and lower at higher pH.

THE RATE OF PASSIVE DIFFUSION OF WEAK ELECTROLYTES IS DEPENDENT ON THEIR DEGREE OF IONIZATION: THE GREATER THE FRACTION THAT IS NONIONIZED, THE GREATER THE RATE OF DIFFUSION, SINCE THE RATE OF DIFFUSION IS MAINLY DETERMINED BY THAT OF THE UNDISSOCIATED PORTION.

ASPIRIN EX.,

Stomach: pH~1.0 Small intestine: pH=5.0-6.6 Blood: pH~7.4 Urine: pH=4.5-7.0

Aspirin is a weak acid. If we put aspirin into the stomach (increase H+, low pH of stomach) we drive reaction to left, or increase non-ionized form of drug. This increases the amount of aspirin that can be absorbed through stomach lining into blood, where it is carried to target site to reduce pain. Absorption is relatively lessened in small intestine, where reaction is driven to the right and more aspirin is in the ionized form. pK is pH at which 50% of compound is ionized (e.g. catecholamines are weak bases with pK of 9.0; caffeine is weak base with pK of 0.5).

TRANSPORT: Specialized transport processes give membrane flexibility and selectivity to control movement of specific substances.

FACILITATED DIFFUSION- movement of lipid insoluble drugs down a concentration gradient via a carrier, thus has a limiting or maximal value (# carriers in membrane). No energy is required. GLUCOSE. Temporary binding to transporters (proteins) involves same kind of bond formation as drug-receptor interactions.

ACTIVE TRANSPORT- movement of lipid insoluble compounds against a concentration gradient via a carrier thus requiring energy from the cell to perform the work.

DRUG DISTRIBUTION -High endothelial surface area (capillary wall) conducive to penetration and compounds will distribute throughout body. Penetration of drugs into CNS is a special case.

BLOOD-BRAIN-BARRIER: Rate of penetration of water-soluble and ionized compounds into CNS relatively slow compared with rate of distribution into peripheral tissue. Lipid soluble drugs distribute relatively fast since brain receives one-sixth total blood leaving heart. Endothelial cells of brain capillaries appear to be more firmly joined to one another than periphery. Also, surrounding brain capillaries are astrocytic processes, or glial feet, that form an additional membrane barrier.

DRUG INACTIVATION - absorption and distribution determine the speed of onset of drug effect. Processes of excretion and biotransformation terminate the actions of drugs.

EXCRETION: process whereby materials are removed from body to external environment. Drugs eliminated unchanged or as metabolites.

The two major organs of excretion of water-soluble drugs are the kidney and the liver. KIDNEYS are pair of organs that filter out products of metabolism and maintain appropriate blood levels of various ions and other substances. Functional unit of kidney is nephron. 1 to 2 million nephrons per kidney, each consists of a knot of capillaries through which blood flows from renal artery to renal vein. Single layer of epithelial cells lines lumen of nephron throughout entire length. The capillary endothelium contains large pores that discriminate against the passage of blood constituents on the basis of molecular size; hydrostatic pressure of blood supplying kidney is high. Fluid filters out of the capillaries into Bowman’s capsule -ultrafiltrate, filtrate of plasma lacking only the plasma proteins. 99% of original filtrate is reabsorbed and returned to body via a second capillary bed. Driving force for reabsorption is either passive diffusion down a concentration gradient or active transport across tubular epithelium. Nutrients such as glucose, amino acids and some vitamins are reclaimed by active transport. Active removal of Na+ in exchange for H+ (acidification of urine). Passive diffusion of water down osmotic gradient established by active transport of Na+.

EXCRETION OF DRUGS - Because of concentration gradient, kidney itself is insufficient for eliminating drugs from body - reabsorption into bloodstream must be prevented. Drugs will passively diffuse back into the circulation in accordance with their lipid/water partition coefficients, degree of ionization and molecular size. Distal segments, urine is more acidic - reabsorption of weak acids and excretion of weak bases.

BIOTRANSFORMATION: metabolism, process by which chemical reactions carried out by body convert a drug into a different compound. Generally yields products more readily excreted than parent compound. Excretion and biotransformation determine the duration of action of a drug.

LIVER allows metabolism into substance that is less lipid soluble and less capable of being reabsorbed. Conversion of fat-soluble drugs into water-soluble metabolites that can be excreted by kidney -biotransformation. Biotransformation usually, but not always, decreases pharmacological activity of the drug. Drug biotransformations are catalyzed reactions - carried out by special system of enzymes in liver cells. These microsomal enzymes are interesting in that they can only catalyze reactions of compounds which are lipid soluble. Many drugs can increase the rate at which the microsomal enzyme system metabolizes drug. Induce an increase in both enzyme activity and total amount of enzyme - one mechanism for producing pharmacological tolerance.

TOLERANCE: Diminished response to drug administration after repeated exposure to that drug. Increasingly larger doses must be administered to obtain the same magnitude of pharmacological effect observed with the original dose. Tolerance is reversible.

Drug Disposition Tolerance - reduction in concentration of drug at receptor with repeated administration (metabolic tolerance). Increase in rate of drug metabolism, e.g. by induction of liver microsomal enzymes. Effect of this type of tolerance on blood drug level depends on route of administration.

Pharmacodynamic (cellular) Tolerance - Cannot be explained on the basis of altered metabolism or altered concentration of drug reaching brain. Some change in receptor: Change in number of receptors or in their sensitivity (affinity).

Cross-Tolerance - development of tolerance to one drug administered over a period of time can diminish the pharmacological effectiveness of a second drug. Ex., alcohol and barbiturates.

Individual Variation in Drug Effect and Change in Drug Effect Over Time

Contributing factors :

• Drugs do not affect everyone in the same way, even given exactly the same dose by the same route of administration.

• Differences in the way individuals respond to a drug may be due to pharmacokinetic or pharmacodynamic factors, or both.

• Pharmacokinetic explanations for variation in drug effect between individuals include differences in the absorption, distribution, and metabolism of a drug.

• Differences in drug metabolism are particularly important because the capacity to metabolise a specific drug can vary with genetic history, age, disease, pregnancy, and environmental factors that alter liver enzyme induction.

• Differences in drug metabolism are important in geriatric populations in whom liver function may be reduced, resulting in substantially longer half-lives for some drugs.

• The distribution of a drug can be affected if other drugs are being taken concurrently.

• If different drugs compete with one another in binding to blood plasma proteins, the proportion of one drug that is bound to protein may decrease, resulting in higher concentrations of the other drug in the CNS.

• Differences in the effect of a drug may also be due to pharmacodynamic factors such as variation in the state of neurotransmitter receptors due to different drug histories and environmental experiences.

• The CNS effects of a drug may be radically altered if other drugs are being taken concurrently, due to interactions between the drugs at the receptors (e.g., alcohol potentiates the sedative effects of benzodiazepines).

• The effect of a drug may change with repeated administration over time (e.g., drug tolerance due to increased metabolism of the drug by the liver (metabolic or pharmacokinetic tolerance) or to a change in the way that CNS receptors respond to the drug (functional or pharmacodynamic tolerance, or both).

NEURONAL MEMBRANE – PHOSPHOLIPID BILAYER WITH SPECIALIZED PROTEINS (RECEPTORS, CHANNELS, TRANSPORTERS, PUMPS…_

(Chapter 2 Mol. Neuropharm.)

I. THE NEURON-diverse morphology (polymorphic) & function, elementary units of nervous system. Stereotypic neuron is stellate cell body with broad dendrites and axon from one pole.

A. CELL BODY - contains the cellular elements found in all cells of the body such as nucleus, mitochondria (oxidative phosphorylation), endoplasmic reticulum, Golgi apparatus, lysosomes. Nissl substance formed by rough endoplasmic reticulum (studded with ribosomes, protein synthesis).

B. AXON - message carrying part of neuron. Emerges from cell body (axon hillock) and frequently does not branch until near target. Frequently myelinated, increasing efficiency as a conducting unit (oligodendrocytes, Node of Ranvier). Axon hillock as extension of soma. Initial segment, region for generation of action potential. Axon proper, concentration of Na+ channels (at nodal axon of myelinated fibers-rapid, saltatory conduction). At termination, axons have enlargements called terminal boutons where neurotransmitter release in response to arriving electrical signals occurs.

C. DENDRITES - dendritic trees as primary receptive fields of neurons, branched exentsions from soma. Synaptic regions occuring either along dendritic branches or at protuberances known as spines.

D. SYNAPSE - discuss next week

II. NERVE CELL MEMBRANE – REMEMBER BLOOD-BRAIN BARRIER!

Cytoplasmic membrane surrounds all parts of neuron. Important in all cells, in neurons confers special ability to transmit information rapidly to other cells.

MEMBRANE COMPOSITION

LIPIDS. Phospholipids particularly important for structure of cytoplasmic membrane. Phospholipids consist of two fatty acid chains attached through glycerol and a phosphate group to a hydrophilic "characherizing" group. Fatty acid chain is hydrophobic. Thus, phospholipid membranes of cells will line up naturally in a bilayer between two aqueous solutions. At room temperatures the individual molecules are in constant lateral motion and continually exchange places with one another (organic fluid). Length and saturation of fatty acid chains determines membrane fluidity.

Polar = charge, attracted to charged nature of watery environment.

PROTEINS. Polymers built of strings of the 20 different amino acids, chains of amino acid residues linked by peptide bonds (sequence is primary structure). The amino acids mostly share a common structure differing only in their side chains. Side chains are important for determining characteristic conformation of a protein, e.g. charged groups will determine hydrophilic and hydrophobic regions, sites for bond formation on some side chains, side chain group components as sites for protein phosphorylation. Secondary structure of protein determined by hydrogen bonds (transmembrane domains of proteins often have secondary structure of alpha-helices formed by runs of hydrophobic residues - hydrogen bonding of amino acids in same chain with side chains facing outward). A number of amphipathic helices may cluster together to form a hydrophilic pore in a membrane. Tertiary structure is 3 dimensional conformation - binding sites of receptors etc. Tertiary structure is rather fragile so various situations will result in change which alters function, e.g. voltage changes, phosphorylation, second messengers....

Fluid-mosaic model of cytoplasmic membrane: Associated-bind to other components of membrane (cytoplasmic or extracellular protein attached through covalently bonded lipids inserted into bilayer; cytoplasmic protein anchored by hydrophobic peptide segment.) Integral-inserted through the lipid bilayer by presence of regions of outwardly oriented nonpolar amino acid residues. Ion channels, transporters, receptor-effector complexes are integral proteins.

Membrane protein interaction with cytoskeleton, endo- and exocytotic processes such as neurotransmitter release. Ca++ dependent associations with cell membranes-regulation of hydrophobicity of protein-binding surfaces.

SPECIALIZATION OF NERVE CELL MEMBRANE FOR DETECTION, INTEGRATION, AND TRANSMISSION OF SIGNALS.

Constituents of nerve cell membrane plays principle role in conferring the ability of generating and conducting nerve impulses. Key properties for electrical excitability are selective permeability to various ions and ability to selectively transport substances through membrane (ion channels, transporter proteins, respectively). The transmembrane ion gradients produced by the presence of these membrane proteins lead to the development and maintenance of a steady state voltage difference between inside and outside of cell known as RESTING POTENTIAL.

RESTING POTENTIAL - excitable cells at rest display an electrical potential gradient across membrane such that the inside is about 60 to 70 mV more negative relative to the outside.

ORIGIN OF RESTING POTENTIAL. Passive "leak" channels (pores, not gated) with preferential permeability to K+ (Cl-): Hypothetical K+ X- ionic solution. K+ net flux down CONCENTRATION GRADIENT until this drive is offset by electrical potential difference produced by net loss of positive charge (ELECTRICAL ATTRACTION-opposite charges attract one another). When chemical and electrical force on K+ are equal - EQUILIBRIUM POTENTIAL. Hypothetical case, equilibrium potential is EK (no net driving force). Value is described by Nernst equation (electrophysiology course).

POST-SYNAPTIC POTENTIALS SHIFT THE MEMBRANE FROM RESTING POTENTIAL.

DEPOLARIZE-excitatory signals that reduce this difference and make cytoplasm more positive. HYPERPOLARIZE-inhibitory signals that increase this difference and make cytoplasm more negative. Such signalling between neurons involves the chemically mediated alteration of the resting potential, occur by the passive diffusion of ions such as Na+, K+, Ca++, and Cl- through highly selective molecular pores in cell surface membrane called ionic channels. GATING OF ION CHANNELS (Ligand gated - receptors). If sum of signals results in depolarization over firing threshold of neuron the initiation of an action potential at the axon hillock is initiated and a.p. travels down axon to provide means for signalling over distances. State of different ionic channels in membrane (open/close) will determine membrane potential at any given moment. Integration of synaptic inputs at dendrites/soma.

ION FLUXES ALSO EXPLAIN THE NATURE OF THE ACTION POTENTIAL

Transmembrane potential not unique to nerve cells. Self-limiting, all-or-none process of action potential is unique and allows signals to be transmitted over long distances. Hodgkin and Huxley identified currents from two types of ion channels, Na+ and K+, that account almost entirely for all the current in axon membranes. These axonal channels are VOLTAGE GATED - opening and closing conformational changes of voltage gated Na+ and K+ chanel proteins are controlled by the membrane potential and therefore derive energy from work done by the changing membrane electric field on charged domains of the channel protein. Major ionic movements generating nerve impulse are rapid influx of Na+ (depolarization) followed by efflux of K+ (repolarization).

SUMMARY:

1) Small background permeability, primarily to K+, which sets the resting potential (voltage independent, non-gated membrane channels, pores)

2) Brief, dramatic openings of Na+ and K+ channels in sequence to shape action potentials (voltage gated membrane channels)

3) Followed by return to steady state resting potential, restoration of ionic gradients preceeding a.p. (transporter - pump).

VOLTAGE GATED ION CHANNELS

Ion channels form aqueous pores in the lipid membrane. Size of channel pore thought to determine ionic selectivity. Charged domains of the protein are sensitive to shifts in electric field in membrane and drive conformational changes to open or close pore. MOLECULAR BASIS OF GATING, important to relate functional properties to structure of channel proteins - most is known about voltage-sensitive Na+ channels.

Labelled and purified from tissue containing many Na+ channels. In brain, 3 protein subunits, alpha subunit spans membrane and forms pore, other two subunits appear to be associated on external surface of membrane (functional role not clear, maybe regulatory). Alpha subunit forms four domains each with six membrane spanning helices, probably connected by hydrophilic regions protruding from the membrane-these transmembrane domains may form the ion channel pore at their center. This subunit alone seems sufficient to carry out basic function of channel.

Gating of channel by membrane depolarization may involve sequence of positively charged residues on one of the transmembrane segments (channel wall?).

MEMBRANE TRANSPORT

Movement of ions through ion channels limited to discrimination by size and electrical charge, conductance is proportional to electrochemical gradient. Transmembrane ion gradients constitute major store of potential energy drawn on for many purposes (resting to action potential). How are ion gradients maintained, for example, after an action potential has occurred? Transporters (pumps) - integral membrane proteins. Interaction of these proteins with their substrates initiate a cycle of conformational changes of the protein that release the substrate on the opposite face of the membrane. Conformational transitions may be coupled to a chemical reaction that supplies metabolic energy required to generate a concentration gradient (active transport). The principal primary active transport system in neurons is a pump that extrudes Na+ and simultaneously accumulates K+. Antiporter mechanism.

THE ATP-DEPENDENT Na+, K+-PUMP.

We know that at steady state (resting potential) there exists potential energy to generate action potential due to electrochemical gradients formed by transmembrane ion gradients. In particular, Na+ ions are not at equilibrium (∆G not equal to 0) and thus potential energy is available to do work (decrease in free energy of a system is exactly balanced by work done). Nernst equation. When work is carried out by opening of voltage gated Na+ channels and then subsequently by opening of voltage gated K+ channels for repolarization, how is the potential energy (pre-a.p. transmembrane ion gradients) of the membrane restored? Work must be performed by some other process to restore and maintain a steady state.

A Na+/K+ transporter/pump (protein) exists in membrane that is able to transfer 3 Na+ : 2 K+ : 1 ATP. Depends on hydrolysis of ATP (high energy phosphate bond of ATP yields ~12 kcal/mol) to ADP and Pi. Thus, this transporter is termed ATP-dependent Na+/K+ pump. Work done by pump recharges membrane after action potential. Drug ouabain inhibits this pump - used to depolarize cells. Since Na+ binding is not saturated but K+ binding appears to be, activity most related to changes in intracellular Na+ concentration.

Pump is termed electrogenic since there is a net outward flux of positive charge hence a tendency to hyperpolarize membrane. Under most conditions, proportion of membrane potential produced by the pump is relatively small, a few millivolts. Permeability to K+ and Cl- explain most of resting potential.

Much cerebral energy is related to Na+ extrusion by this pump. Energy demands related to: electrical activity, myelination. During a.p. cation flux is 2-3 orders of magnitude greater than at rest. Squid giant axon-Na+ entry and K+ efflux during single a.p.=3x10 -12 mol cm-2. Resting flux (activity of pump) is 12x10 -12 mol cm-2 sec-1. 2.5 cycles per second to maintain steady state if cell fires 10 spikes per sec (activity actually increases so energy requirement is even higher). Post-synaptic potentials (not a.p.) involve less flux but may last much longer. Total ion flux per unit length during a.p. is lower for myelinated vs. unmyelinated axons of equal radius.

SUMMARY: Energy utilized to maintain cation gradients will depend on distribution of differing a.p. frequencies, p.s.p.’s, and resting potentials, and on extent of myelination. Estimated that depending on activity 25-50% of brain energy utilization is related to Na, K-ATPase activity. Cation transport single reaction accounting for largest share of energy flux.

Appears to consist of two identical units, each in turn, consisting of two subunits. Large alpha subunit is catalytic subunit, beta subunit function unclear. Major part of catalytic unit in cytoplasmic domain and contains ATP binding and hydrolysis sites. Thought that initial phosphorylation of protein by ATP occurs after 3 Na+ have bound to cytoplasmic domain. (Dephosphorylation of ATP with transference of cleaved phosphate to site on subunit). Energy released by the phosphorylation initiates an extensive conformation change in molecule which impels 3 sodiums out of cell. K+ attachment leads to reverse conformational change which frees the phosphate from its binding site and impels two K+ ions out of cell. Now returned to original conformation ready to repeat cycle. Details not well worked out.

ALL OF THESE ASPECTS ARE POTENTIAL DRUG TARGETS!

PHARMACODYNAMICS

(Chapter 1 Mol. Neuropharm. & M.&Q.)

DRUG-RECEPTOR INTERACTIONS - Overwhelming majority of drugs show high selectivity and specificity in their actions. Relation between chemical structure and biological effect of drugs suggest that physicochemical aspects of interaction important. Receptors - tissue elements (proteins) with which drugs interact to produce their biologic effect. Drug acts by binding with receptor. Because of its particular shape and charge distribution, can bind reversibly to a specific receptor and, in doing so, change the physiological reactivity of the receptor. CHEMICAL BONDS are the forces that underlie all interactions between drugs and tissue elements.

II. TYPES OF BONDS The atom: Nucleus with positive charge, surrounded by electrons such that total negative charge is equal to positive charge of nucleus. Electrically neutral (also neutrons in nucleus). Configuration of extranuclear electrons determines chemical reactivity.

Electrons moving about nucleus in shells, each of which has a definite number of electrons that can be situated in it. First shell - 2 electrons (hydrogen, helium). Structure of eight electrons in outermost shell is most stable (inert gases). Atoms try to reach chemical stability by attaining configuration of inert gas - do this by giving up or taking on electrons. Ex., Sodium - 11 external electrons, one lone electron in outermost shell. Gives up this electron and has net positive charge. Chlorine - 17 electrons, seven in outermost shell (valence shell). Accepts electron and has negative charge. CHARGED PARTICLES ARE CALLED IONS. THE IONIC BOND: Positively charged Na+ cation and negatively charged Cl- anion. Each is stable and retains this configuration independently of one another. Held together to form a molecule by the electrostatic attraction between them. DEF: electrostatic attraction between oppositely charged ions. Strength depends on distance and diminishes as the square of this distance. THE COVALENT BOND: What about formation of molecules where no ion can be detected? Not only can a bond arise from outright transfer of electrons, stability also arises from sharing of a pair of electrons. Pair of electrons held jointly by 2 atoms, completing stable configuration for each. 20 times stronger than ionic bond.

COORDINATE COVALENT BOND - special case of covalent bond resulting from sharing of electrons supplied by one atom only. Donor atom usually nitrogen, oxygen or sulfur. Ex., When a hydrogen ion approaches the ammonia, the nitrogen allows the proton to share with it the free pair of electrons. Positive charge of proton is retained since there has been no net gain or loss of electrons. Coordinate covalent bond formation is important in ionization of drugs and interactions with receptors. THE HYDROGEN BOND: Hydrogen proton strongly electropositive. When hydrogen is bound by an ionic or covalent bond to a strongly electronegative atom, the hydrogen may further coordinate two more electrons donated by another strongly electronegative atom (O,N,F). Second bond is hydrogen bond, forming bridge between electronegative groups. Ionic in character, but strength is less than true ionic bond. Many hydrogen bonds in a structure can significantly stabilize.

VAN DER WAALS FORCES: Very weak attractive forces between regions of slight charge on neutral atoms, operate only at very close range.

DRUG BINDING - Drug/receptor interaction as selective/specific. Must also be of sufficient stability to permit initiation of action-effect sequence. Usually, not so stable as to be irreversible, however. SELECTIVITY, SPECIFICITY, REVERSIBILITY: require synchronous operation of bond types to be achieved. 1) First force must be one that overcomes random thermal agitation of drug molecule & draw it to receptor. Need rapid bond of sufficient strength that can exert influence while drug is still somewhat distant from receptor. IONIC BOND best suited for this purpose. (covalent bonds essentially irreversible at body temperature). Protein of receptor contains charged groups available for interaction with charged groups of drug. Probably electrostatic attraction that first ties drug to receptor. 2) One or two ionic bonds must be reinforced by other bond formation in order to overcome the energy of thermal agitation, which is great enough at body temp to break a single ionic bond. Additional attractions of hydrogen bonds and van der Waals forces give drug-receptor combination stability essential for drug action. Forces requiring very good fit between drug and receptor since operate only at very small distances. Van der Waals forces as major in conferring specificity.

MODEL FOR COMBINATION OF ACETYLCHOLINE WITH RECEPTOR

Nitrogen group acquires strong positive charge by donating its unshared pair of electrons to carbon to form a coordinate covalent bond. (Quaternary ammonium compound). Thought to be ionically bonded to negative group on receptor. Sufficient to draw ACh to receptor but stability is increased by Van der Waals forces produced by close fit of two methyl groups. Chain of carbons pictured as fitting a flat part of receptor surface. Formation of a hydrogen bond with oxygen may draw other end close to receptor & further increase stability and specificity. Of course, this is actually a complex 3-dimensional relationship. STRUCTURE-ACTIVITY RELATIONSHIPS

THE DOSE-RESPONSE RELATIONSHIP

The qualitative principles discussed so far must find expression in quantitative terms. “Degree of effect pruced by a drug is a function of the quantity of drug administered.” DOSE: amount of drug needed at a given time to produce a particular biologic response. Dose-Response Curve plotted as biological response against dose. Log scale for dose yields S-shaped log dose-response curve. Maximum response corresponds to point at which receptor occupancy is approaching 100%.

Advantages of log curve:

1. Allows expansion of x-axis scale used to depict small doses while still permitting representation of a wide dose range.

2. Typical log dose-response curve has a center of symmetry. Midpoint represents dose at which 50% of the maximum response is elicited (ED50).

3. Middle segment of curve is almost linear, thus making it easier to quantify and compare data.

4. Series of different drugs that produce same response by interacting with common receptor mechanism will give series of parallel log dose-response curves. Curve for drug that interacts most strongly will appear to left of others, since lower doses will be needed to produce biological response. POTENCY of drugs compared in this way. Potency of a drug not related to its safety.

Assessment of agonist and antagonist interactions. Agonists produce direct measureable response by interacting with receptors. Antagonists block the responses elicited by agonists.

COMPETITIVE ANTAGONIST competes with agonist drug for binding to receptor sites. Competitive antagonism can be overcome if dose of agonist is increased. Actions thus show up as shift to right in log dose-response curve of agonist drug.

NONCOMPETITIVE ANTAGONIST blocks receptors in a way that cannot be overcome by increasing agonist dose. Maximum response is decreased.

KEY CONCEPTS/DEFINITIONS

Some principles of drug action:

• Effects drugs elicit depends on extent to which they act on target receptors

• Drugs are distributed to many parts of the body and therefore have the potential to act on receptors other than intended target receptors

• Adverse effects are often result of drug acting on unintended receptors sites

• Molecular biology (cloning of receptors) leading to selectivity in drugs

• Receptor is a complex protein, usually on the cell membrane (fixed vs mobile), which interacts with drug to activate intracellular biochemical events Drug is said to bind to a receptor, and the specific site on the receptor to which the drug binds is referred to as a binding site

• Any drug that binds to a receptor binding site is called a ligand

Receptors have a number of properties that distinguish them from other cellular sites that bind a drug :

1 saturability: there are a finite number of receptors. Therefore, as the drug concentration increases, the occupation of the receptors by drug molecules increases until saturation is reached and there are no more receptors available to interact with the drug.

2 selectivity: the receptor has a high degree of selectivity for the drug (determined by the size, shape, and electrical charge of the drug molecule), rather than binding anything that is molecularly similar to it.

3 reversibility: the interaction between the receptor and the drug molecules is usually reversible.

Four main ways in which drugs act on receptors to affect cell function:

1 a drug may bind to an extracellular receptor that directly regulates ion channel opening (e.g., benzodiazepine anxiolytics effects on Cl-GABAA channel).

2 a drug may bind to an extracellular receptor linked to an intracellular enzyme via an intermediary protein (e.g., cannabis and G-protein/adenylate cyclase)

3 a drug may bind to an extracellular receptor on a transmembrane protein and activate an intracellular enzyme (e.g. growth factor receptors)

4 a drug may cross the lipid cell membrane and act directly on receptors in the cell nucleus (e.g., steroids actions on mobile receptors)

More principles of drug action: Drug effects on a particular type of receptor are determined by:

1 the number and nature of the receptors,

2 the concentration of the drug at the receptors as determined by pharmacokinetic principles, and

3 whether the drug acts as an agonist or antagonist at the receptors.

Affinity and Efficacy - properties of drugs and biological receptors

• Affinity is how avid a particular drug will bind to a particular receptor. If a receptor has a high affinity for a specific drug, then a lower concentration of that drug will be necessary to achieve full occupation of the receptors. Note that: the rate at which drug molecules bind to a receptor (given by the association constant, Kd) is used as an index of the affinity of a receptor for a specific drug. In drug binding studies, Kd is the drug concentration at which half the maximal number of receptors are occupied; the maximal number of binding sites is referred to as Bmax .

Note further: that the formation of a drug-receptor complex is an interaction determined by the nature of the receptor and drug, thus the term affinity is equally applicable to avidity of a receptor for a drug, as well as, avidity of a drug for a receptor.

• Efficacy refers to the capacity of the receptor to elicit an intracellular effect once it has interacted with a particular drug and formed a drug-receptor complex. Note that: Given equal drug concentrations and affinities for a particular receptor, some drug-receptor interactions have a larger effect on a cell than others and therefore have greater efficacy. Note further: Although efficacy strictly refers to the effects of a specific drug-receptor complex, it is often used in reference to specific drugs (e.g., a high efficacy drug is one that has large cellular effects when it interacts with a particular receptor).

Drug-Receptor Complex Nomenclature

• Agonist - a drug that activates a receptor upon forming a drug-receptor complex

Agonists for a particular receptor can differ in both affinity and efficacy for the receptor.

A high efficacy agonist is a full agonist because it elicits the maximal effect from receptors given sufficient concentration, whereas...

A low efficacy agonist is a partial agonist because it cannot elicit the a maximal effect at receptors even at high concentrations (false transmitters).

Direct agonists act on receptors, while indirect agonists facillitate the actions of the endogenous agonist (the neurotransmitter, itself)

• Antagonist - a drug that does not activate the receptor upon forming a drug-receptor complex.

Antagonists also prevent the activation of the receptor by an agonist, thus antagonists are essentially zero efficacy drugs.

A competitive antagonist is one that binds to the same binding site as the agonist and therefore competes with the agonist for that binding site.

A noncompetitive antagonist is one that has a different binding site to the agonist and therefore does not compete with the agonist. Some noncompetitive antagonists have a binding site within the ion channel associated with the receptor complex.

Note that: ionotropic receptors are directly linked to ion channels in the cell membrane that allow the passage of particular ions into or out of the cell when the receptor is activated. Some receptors have multiple allosteric binding sites, in addition to the binding site for the endogenous agonist, that regulate the function of the receptor complex. Drug binding to an allosteric binding site can alter the affinity of the primary binding site for an endogenous agonist (e.g., the actions of benzodiazepine anxiolytic drugs on GABA receptors).

• Effect that a drug has on a biological function (e.g. heart rate) or psychological characteristic (e.g. anxiety or psychosis) can be described using a dose-response or concentration-effect curve.

• Effect of a drug can change markedly with increasing concentration at the receptors.

• Drugs typically lose selectivity for specific receptor types with increasing concentration.

•

Some principles:

Drug Dose-Response Relationships

• Dose-response curves can vary, typically they are sigmoidal, linear, or U or inverted U-shaped

Some characteristics Threshold dose - minimally effective dose that produces a detectable change in response

• Maximum response - the greatest degree of a response that can be achieved with a drug

• Maximum efficacy - (ED100) the dose of drug that produces the greatest response

• Medium effective dose - (ED50) the dose of drug that produces a desired, therapeutic effect in 50% of the population tested

• Medium lethal dose - (LD50) the dose of drug that causes death in 50% of the population tested

• Therapeutic index - (TI) specified in terms of a drug’s lethal dose relative to it’s effective dose (LD50)/(ED50)

• Therapeutic window - (TW) specified in terms of the dose of drug that produces optimal therapeutic results

• Potency - the ability of a drug, relative to others, to induce a given effect; not synonymous with lethality or toxicity.

• Hyper-reactors - individuals who over-respond to the ED50 of a drug

• Hypo-reactors - individuals who under-respond to the ED50 of a drug

• Idiosyncratic-responders - individuals who show an unexpected response or unusual effect to the drug

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