Pharmacodynamics

The study of neurochemistry and behavior involves a considerable use of pharmacological information. Of course, one reason is that the use of drugs is one of the most practical means of manipulating neurochemical systems. There is also, however, a deeper reason. It is now implicitly accepted that most psychoactive drugs have the effects they do because they modify the function of neurochemical systems. In this context the study of drug effects and the study of the neurochemical basis of brain function converge. At some level we therefore expect that the effects of drugs of known neurochemical actions tell us something about  the role of these neurochemical systems in brain function. However drugs have many effects, not all of which are explained by their actions on the brain, or on particular neurochemical systems - these we call side effects, but it must be remembered that they are just as genuine effects as the one we are primarily interested in.

The use of drugs to study neurochemical substrates of behavior necessarily involves finding solutions for two sets of problems which I call 'pharmacological specificity' and 'behavioral specificity`. The problem of pharmacological specificity is to determine whether a given drug effect is mediated by the particular neurochemical system under study, and whether it depends on the appropriate pharmacological mechanism. For example, let us say we are interested in the role of dopamine systems in motivation and we decide to examine the effects of amphetamine on feeding because we know that amphetamine is a dopamine releaser. We find that amphetamine produces anorexia - i.e. blocks feeding. How are we to interpret this fact? Amphatamine has a number of side effects it releases noradremaline and serotonin as well as dopamine. It also blocks reuptake of these amines into the presynaptic terminal. Pharmacological specificity depends on being able to show that amphetamine's effect on feeding is due to its effect on dopamine release rather than some other effect.

The question of 'behavioral specificity' is whether our observed effect of amphetamine on feeding represents a change in motivation for food. We might like to argue that the animal or person does not eat because he is not hungry. However, there are other possibilities -- perhaps the drug makes the animal feel sick -- we do know that dopamine receptors in the region of the area postrema are involved in vomiting. Perhaps the drug affects the motor system to interfere with feeding reflexes so that the animal is 'hungry' but can't eat. we know that high doses of drugs like amphetamine cause involuntary movements.

You can see that to come to a clear understanding of the role of dopamine in feeding we need to get satisfactory answers to both sets of questions. In general, pharmacologists tend to be most interested in the question of pharmacological specificity while psychologists tend to be more concerned with behavioral specificity. Nevertheless both problems have to be solved if we are to understand either the effects of psychoactive drugs, or the role of neurochemistry in brain function. In this course we will therefore be quite concerned with pharmacological issues and an understanding of basic pharmacology is important.

Elements of pharmacology

Pharmacology is the study of drug effects and drug action. A drug effect is a drug-induced change in the activity of a biological process or system. The term drug action refers to the site and mechanism whereby the drug produces its effects. In order to produce its effect a drug must reach the site of action, bind to the receptors to indicate the biological action, and be excreted or metabolized to terminate the effect. How quickly a drug reaches its site of action depends on where it is administered (stomach or vein) and on its physio-chemical characteristics that determine how it will be absorbed by different tissues. The size and duration of the effect depends on how the drug interacts with the receptors but also on the characteristics of absorption and excretion of the drug.

Time course of drug action

If we administer a drug we find that the effects have a timne course which is characteristic for the particular drug. At some time after administration we will see the biginnings of an effect which will then rise to some maximum and gradually decline. This is the time course of the drug. The rising and falling phases of the effect are generated by several processes, the most important of which are the processes of drug absorption and elimination. The rates of these processes are governed by a number of factors.

FMQ Fig1.1 The pathways of drug disposition.

1. Route of administration

2. Characteristics of distribution

3.Charcteristics of binding

4. Route of inactivation

5. Route of elimination

Route of administration.

The choice of a route of administration depends to some extent on the chemical characteristics of the drug. Drugs that are insoluble in water cannot usually be given intravenously, while drugs that are broken down by digestive enzymes cannot be given by mouth.

The most rapid absorption occurs from an intravenous injection which places the full dose directly into the blood stream , no first pass effect. ( Restrictions - Water soluble, not damage vasc. in high conc. not affect other organs in high conc.)

Risks transient very high concentrations, infection.

The next most rapid is intraperitoneal injection which places the substance near the large blood vessels of the intestine. Absorption is rapid but the blood passed through the liver on its way to the general circulation. Since the liver is one of the major sites of drug metabolism many drugs are partially inactivated by liver enzymes before they reach the target tissue. Thus IP injection may be less efficient than IV injection. IP injection is often used in animal experiments but rarely with humans.

Intramuscular injection produces slower absorption than IV or IP injection but like IV injection the drug does not pass through the liver before going into the circulation. The drug can be injected as a suspension but in general IM administration cannot be used with irritating substances.

Subcutaneous administration is often used with small animals. Absorption is slow and even and the method can be used with non-acqueous solutions, eg. oil. It is also possible to implant pellets of drug which will dissolve out slowly -- sometimes over days.

Oral - easy, popular with humans but may be stressful to animals. Drug must be resistant to acid and enzymes and may be variable onset of effect becasue of changes in gut movement. Sublingual also used for rapid absoption of lipid sol drugs.

Inhalation Used for anesthtics and a few gassifiable or atomizeable drugs. Very rapid because of the huge surface area of lung and rich blood supply.

Topical A few very lipid soluble drugs can penetrate the skin. e.g. nerve gas, estrogen. More rapid absorption through mucous membrane (cocaine sniffing) Central distinguished from systemic. Finally, it is also possible to inject minute quantities of drugs directly into the brain or spinal cord either through a canula implanted into the brain (intracranial) or into the cerebrospinal fluid in the ventricles (intraventricular) or spinal subdural space (intrathecal). This method is used to localize receptors or to administer drugs that do not readily cross the blood- brain barrier.

The concentrations of drugs attained from a given dose vary with the method of administration. IV administration results is a rapid rise of the concentration of drug in the blood followed by a rapid fall as the drug moves into other compartments and is excreted. On the other hand subcutaneous injection leads to a much slower rise and fall in blood concentration because the drug moves into other compartments as it is being absorbed. Furthermore the peak concentration is not as high as that obtained after IV injection.

Kinetics

(draw IV and subcut time course of plasma level) The half-life Fig 1.3 FMQ steady state at 5 half lives Redistribution - fluid/fat. Influence of age, bodyweight and sex. Larger implies lower plasma concentrations and less drug delivered to tissues like the brain
Fatter means lipid soluble drugs sequester in fat, reducing plasma concentration and maybe extending duration of action.

Females have more fat per kg body weight so will have smaller fluid volume. this implies higher concentration of drugs in the plasma but more redistribution to the fat.

Chemical Characteristics and Absorption

In order to reach the target sites a drug usually has to cross several cell layers and the membraines of the cells that comprise them. Membranes have a high lipid content and drugs that will not dissolve in liquid will not cross membranes very readily. This fact is of particular importance in understanding the passage of drugs into the brain. In most capilliaries there are small gaps between adjacent cells through which small molecules can diffuse. (see Fig. 5 F & Q) In addition, there are transport vesicles which envelope and transport large molecules through the cappilliary wall. In brain capilliaries the adjacent cell membranes form 'tight
junctions' that block the passage of even small molecules. Pinocytotic vesicles are are and there are numerous mitochondria. Finally the brain capilliaries are surrounded by astrocytic processes (glial feet) which cover about 85% of the basement membrane surrounding the capilliary cells.

FIG.1.6 FMQ

Thus the movement of a drug into the brain is heavily dependent on its lipid solubility. Lipid solubility is in turn dependent on the degree of ionization of the drug in solution. The prediction of penetration can be made on the basis of the partition coefficient.

Drugs that readily ionize in aqueous solution, do not move readily from an aqueous solution to a lipid because of the electrostatic attraction between the drug and H+ and OH ions in the water. A classic example is the contrast between morphine and heroin. The two drugs have similar action at receptors indeed heroin is believed to be converted to morphine before binding to receptors. However, heroin is much faster acting and more potent when given systemically. Heroin is more lipid soluble than morphine and thus readily crosses the blood-brain barrier while morphine is absorbed slowly. Windows are area postrema caudal IV ventricle, median eminence, attachment of chorionic plexus, pineal gland.

Pharmacodynamics

The dose-response relation.

Let us now consider what happens when we administer a series of doses of a drug to a subject and measure some response that we expect to be altered by the drug.

As the dose is increased from some low level their is initially no effect but at some point the effect becomes apparent and then, as the dose is further increased, the effect increases to a maximum. These data can be represented graphically as a dose-response curve. It is usual to plot the effect as a percentage of the maximal effect and the dose as log dose.

dose effect curves

A drug's effectiveness varies from subject to subject. Sometimes this variability is itself significant and we can obtain useful information but examining the proportion of subjects showing the effect at each dose. In this case our "effect" goes from 0% to 100%. From such cureves it is possible to estimate some parameters and their confidence intervals - in particular the ED50 and LD50.

Two other parameters are important - the slope and the position of the curve on the abscissa which is an indicator of drug potency. Potency is not itself a particularly important pharmacological property since it is determined by many unrelated aspects of drug action (e.g. kinetics). However, comparisons of s drugs potency in producing different effects are of interest since this given an index of the selectivity of a drug e.g LD50/ED50, ratio of analgesic to cataleptic effects. Another important parameter is efficacy - the height of the asymptote of the curve which tells us how much of the maximum possible effect a drug can produce. The information contained in dose-response curves is used to explore drug action - the effect of a drug on its receptors. The initial intereaction of drug and receptor is termed the drug action and the succeeding events - depolarisation, increase in heart rate, induction of adenylate cyclase etc represent drug effects.

An agonist binds to a receptor to produce a given effect while an antagonist binds to a receptor or effector mechanism to inhibit the effect of an agonist. Pure antagonists produce no effect themselves. As we shall see there are also drugs that are intermediate in their characteristics - the partial agonists, and some novel agents that have the reverse effect to a traditional agonist - the inverse agonists.

The pattern of interaction of agonists and antagonists as revealed by dose- response curves, provides information about the processes involved. A competitive antagonist shifts the dose-response curve to the right. That is the antagonism can be overcome by increasing the dose of the agonist. In contrast a non-competitive antagonist reduces the maximum effect - i.e. it removes a proportion of the tissues capacity to produce the effect. It could be because the antagonists irreversibly binds some of the receptors or because it blocks the effector mechanism.

Binding

One of the deficiencies of a purely pharmacological analysis of effects elicited by systemic drugs is that the results may identify the class of receptors involved but give no clues as to the localization of the receptors. A powerful technique derived from pharmacological principles that provides evidence of localization is the ligand receptor binding study. The idea is that drugs with high affinity for particular receptors can be used to locate the receptors if the drug molecules can be labelled.

Since many drugs are complex hydrocarbons it is often possible to radioactively label one of the carbons or hydrogens (14C or 3H). The procedure is to incubate the tissue with the labelled ligand and then wash off the excess ligand. The tissue is then dissolved to liberate the bound ligand an the radioactivity measures with a scintillation counter. Alternatively the tissue may be apposed to a photographic plate which is eventually developed to show a photographic map of the intensity of radioactivity. It is quantitated by measuring the density of the developed silver grains. As most ligands bind nonspecifically to tissue as well as to the tube it is essential to determine the amount of nonspecific binding. This is done by comparing the binding of the ligand alone with another sample in which the ligand is accompanied by another drug known to bind to the same receptor. This second drug presumably acts as a competitive antagonist of the ligand. The nonspecific binding is therefore the difference between the radioactivity of the ligand alone and the ligand plus the antagonist.

If the measurements are made for a range of concentrations of the ligand, the amount of bound ligand will increase as the concentration of ligand increases until saturation occurs. It is conventional to represent the results of such experiments
in a Scatchard plot in which the ratio of total bound ligand to total free ligand is plotted against the total bound ligand.

bound/free

bound

When there is only a single population of binding sites the points make a straight line which intercepts the X axis at a point which represents the maximum binding or Bmax. The slope of the line is proportional to the affinity of the ligand for the receptor slope=-1/K where

K is the equilibrium constant. This allows us to estimate K. You will often see K quotes as a measure of the affinity for the receptor. Drugs with a very low K are likely to be very potent.

As we shall see, much can be learned from these experiments though they must be interpreted with reservations - the most important one being that binding does not establish a receptor. There are binding sites which are not associated with any effector mechanism such as in depot binding. For example the blood contains albumin - a protein which has many binding sites for amino acids. many drugs will bind to albumin so that a proportion of the drug is not available at effectors and cannot be metaboilised by liver enzymes. Depot binding may thus slow down metabolism.

Proof of receptor identification demands evidence that the binding site is connected to an ion channel, second messenger system or some effect. An important inference is the expectation that binding affinity should be correlated with the potency of a drug when appropriate allowances are made for drug distributional factors.

Behavioural specificity

The problem of behavioural specificity is really the psychology part of psychopharmacology. The issue is what does the drug actually do. Take an example like - memory. imagine that we have a drug that is supposed to promote memory. the evidence is that subjects seem to do better in recall tests when taking the drug. This might suggest that the drug is somehow improving the memory storage process. However when we look at the drugs effects we find that it is a mild stimulant, it reduces sleep time and increases locomotor activity. Now we know that efficiency of learning is influenced by the level of alertness and attention. perhaps the drug has no effect on memory processes but improves alertness or attention (e.g. amphetamine-like drugs do this very well) and the improved memory is really just the result of better studying. The drug could still be useful but brain processes underlying its effects are different in the two cases. To study it in animals we have to find tests that will measure memory, and find tests for attention and alertness.

Many such tests have been developed and some of them are described in Chapter 2 of FMQ. If you are not familiar with basic research in several areas of psychology you should read this chapter to familiarize yourself with the kinds of tests that are used.