Medicinal Chemistry I IInd Module Affinity, efficacy, and potency

Affinity, efficacy, and potency

Once a new drug molecule has been synthesized, one has to verify that it possesses the functional activity and therapeutic efficacy expected. his is done by evaluating the properties of the drug in a logically ordered se!uence of tests, the screening architecture, that starts with simple in vitro tests but later also includes sophisticated in vivo experiments" each result provides a piece of evidence con# firming or infirming the potential interest of the compound. $n idea of the various types of biological tests that may be included in a screening architecture, how the results are expressed and what they imply are schematized at the right.
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Affinity, efficacy, and potency

he affinity of a drug for a receptor is a measure of how strongly that drug binds to the receptor. Efficacy is a measure of the maximum biological effect that a drug can produce as a result of receptor binding. &t is important to appreciate the distinction between affinity and efficacy. $ compound with high affinity does not necessarily have high efficacy. 'or example, an antagonist can bind with high affinity but has no efficacy. he potency of a drug refers to the amount of drug re!uired to achieve a defined biological effect ( the smaller the dose re!uired, the more potent the drug, it is possible for a drug to be potent )i.e. active in small doses* but have a low efficacy. $ffinity can be measured using a process +nown as radioligand labelling. $ +nown antagonist )or ligand* for the target receptor is labelled with radioactivity and is added to cells or tissue such that it can bind to the receptors present. Once an e!uilibrium has been reached, the unbound ligands are removed by washing, filtration, or centrifugation. he extent of binding can then be measured by detecting the
amount of radioactivity present in the cells or tissue, and the amount of radioactivity that was removed.

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Binding
he aim of binding experiments is to determine the affinity )the strength with which a compound binds to a site* of the compound for its biological target and to chec+ its selectivity versus other binding sites or biological off#targets. Binding studies usually represent an initial step in compound charac# terization. -chematically, membranes are prepared from the tissue of interest )heart, bladder, brain ...* or from mammalian cells that express the receptor of interest. he receptors can be native, that is, they are constitutively expressed by the cells or the tissue, or transduced, that is, a c./$ coding for the receptor isolated from any appropriate species has been inserted into the cell. Chinese hamster ovary )C0O* or human epithelial +idney 21% cells )02321%* are generally used. &n the latter, transfection can be stable and cells can proliferate while continuing to express the receptor, or it can be transient and cells rapidly loose their ability to express the receptor. -table transfection in cell lines is often used to perform binding studies with human receptors since compound affinity may differ mar+edly between receptors isolated from animals and man.

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Binding Constant
he e!uilibrium constant for bound versus unbound radioligand is defined as the dissociation binding constant )3d*.

567 and 568l can be found by measuring the radioactivity of unbound ligand and bound ligand respectively, after correction for any bac+ground radiation. 0owever, it is not possible to measure 587, and so we have to carry out some mathematical manipulations to remove 9 87 from the e!uation. he total number of receptors present must e!ual the number of receptors occupied by the ligand )5687* and those that are unoccupied )587*, i.e. 8tot : 587 ; 5687 and 587 : 8tot # 5687 -ubstituting this into the first e!uation and rearranging leads to the -catchard e!uation, where both 5687 and 567 are measurable<

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Scatchard Plots
>e are still faced with the problem that 3d and 8tot cannot be measured directly. 0owever, these terms can be determined by drawing a graph based on a number of experiments where different concentrations of a +nown radioligand are used. 5687 and 567 are measured in each case and a Scatchard plot is drawn which compares the ratio 5687?567 versus 5687. his gives a straight line, and the point where it meets the x#axis represents the total number of receptors available. he slope is a measure of the radioligands affinity for the receptor and allows 3d to be determined.

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Scatchard Plots
>e are now in the position to determine the affinity of a novel drug that is not labelled. his is done by repeating the radioligand experiments in the presence of the unlabelled test compound. he test compound competes with the radioligand for the receptorAs binding sites and is called a displacer. he stronger the affinity of the test compound, the more effectively it will compete for binding sites and the less radioactivity will be measured for 5687. his will result in a different line in the Scatchard plot. &f the test compound competes directly with the radiolabeled ligand for the same binding site on the receptor, then the slope is decreased but the intercept on the x#axis remains the same. &n other words, if the radioligand concentration is much greater than the test compound it will bind to all the receptors available

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Scatchard Plots
Cenerally, it is assumed that, when affinity is high, the compound is less li+ely to interfere with other, possibly unwanted off#target sites. 0owever, this is not always true and selectivity has to be chec+ed by evaluating the affinity of the compound for a large panel of receptors, enzymes and ion channels. &t is obvious that selectivity has limits that depend on the size of the panel that has been investigated, but also on the sci# entific +nowledge available at the time when the studies are performed. 'or these reasons, it is thus always possible that a compound considered to possess a high degree of specificity may nevertheless induce unexpected biological effects.

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Cheng-Prusoff E uation
ypically, displacement experiments give rise to sigmoid curves. he drug concentration that displaces half of the maximum bound radioactive ligand represents the &C4D. >hen membranes are incubated with various concentrations of the radiolabeled ligand, a plot of bound?free against bound ligand )Scatchard plot* generally gives rise to a straight line. &n these saturation experiments, Emax )the maximal number of binding sites per unit of tissue or protein weight* is determined from the intercept of the line with the abscissa and 3d )the dissociation constant* from the negative reciprocal of the slope of the line. >hen such experiments are performed in the presence of various concentrations of the compound under study, they give rise to a family of lines. &f Bma! remains unchanged and the slope of the lines decreases with increasing concentrations of the compound, the dis# placement is competitive. Fnchanged slope and decreased Emax indicate that the displacement is non#competitive. he lower the &C4D or 3d, the higher the affinity. 8esults can also be expressed as a 3i, the inhibitory )or affinity* constant of the displacer compound for the receptor. 3i and &C4D are not independent and are very simply related when the displacement is non#competitive )3 : &C4D*, but the relationship becomes more complicated ) ChengPrusoff e uation* for a competitive displacement 5"i # IC$%&'( ) *+,&"d- where 567 is the concentration of the radioactive ligand7. >hen designing new drugs, high affinity is often sought and may represent a crucial parameter, particularly in cases where the affinity of the endogenous ligand for its binding site is very high.

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Cheng-Prusoff E uation
$gents that bind to the receptor at an allosteric binding site do not compete with the radioligand for the same binding site and so cannot be displaced by high levels of radioligand. 0owever, by binding to an allosteric site they ma+e the normal binding site unrecognizable to the radioligand and so there are less receptors available. his results in a line with an identical slope to line $, but crossing the x#axis at a different point, thus indicating a lower total number of available receptors. he data from these displacement experiments can be used to plot a different graph which compares the percentage of the radioligand that is bound to a receptor versus the concentration of the test compound. his results in a sigmoidal cur.e termed the displacement or inhibition cur.e, which can be used to identify the &C4D value for the test compound. he inhibitory or affinity constant )3i* for the test compound is the same as the &C 4D value if non#competitive interactions are involved. 'or compounds that are in competition with the radioligand for the binding site, the inhibitory constant depends on the level of radioligand present and is defined with the Cheng-Prosoff e uation<

where 3d is the dissociation constant for the radioactive ligand and 567 tot is the concentration of radioactive ligand used in the experiment.
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Schild Plots
Efficacy is determined by measuring the maximum possible effect resulting from receptor#ligand binding. Potency can be determined by measuring the concentration of drug re!uired to produce 4DH of the maximum possible effect )2C4D*. he smaller the value of 2C4D, the more potent the drug. &n practice, p/0 is ta1en as the measure of potency where p.2 : #log2C4D.

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Schild Plots
$ Schild analysis is used to determine the dissociation constant )3d* of competitive antagonists. $n agonist is first used at different concentrations to activate the receptor, and an observable effect is measured at each concentration. he experiment is then repeated several times in the presence of different concentrations of antagonist. Comparing the effect ratio 2observer?2maximum versus the log of the agonist concentraion )log5agonist7* produces a series of sigmoidal curves where the 2C 4D of the agonist increases with increasing antagonist concentration. &n other words, greater con# centrations of agonist are re!uired to compete with the antagonist.

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Schild Plots
$ Schild plot is then constructed, which compares the reciprocal of the dose ratio versus the log of the antagonist concentration. ) he dose ratio is the agonist concentration re!uired to produce a specified level of effect when no antagonist is present, compared to the agonist concentration re!uired to produce the same level in the presence of antagonist.* he line produced from these studies can be extended to the x#axis to find p$2 : #log 3d*, which represents the affinity of the competitive antagonist.

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Schild Plots
Einding experiments are performed in order to characterize the affinity of a compound for a receptor but they do not establish whether a compound behaves as an agonist, an antagonist or an inverse agonist. -uch determinations necessarily involve functional measurements of ligand#receptor interaction#induced changes in an intracellular signal. -uch experiments also represent the initial step in compound characterization as a transporter or enzyme inhibitor, or as a voltage#activated cation channel modulator. &n this latter case, the compound potency in functional experiments is often much higher than that expected from the affinity determined in binding experiments but the reasons for this discrepancy are largely un+nown to date. -ince antagonists bloc+ an existing ligand#activated functional effect, the receptor has to be incubated with a given concentration of an agonist and the effects of various concentrations of the putative antagonist are then studied. he resulting curves are called Schild plot and the results are expressed as an &C4D, the drug concentration that produces half of the maximal response )2max in H* measured in the absence of the antagonist. $lternatively, the effects of the agonist in the presence of different concentrations of the antagonist can be studied )one concentration for each curve* thus giving rise to a family of curves and allowing the calculation of a p$2 )#log molar concentration of antagonist producing a 2#fold shift of the concentration response curve, that is, a 2#fold increase in agonist concentration in order to obtain a similar effect*.
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Schild Plots2 competiti.e and non-competiti.e anatagonists

Competitive antagonists induce a parallel rightward displacement of the curves with increasing concentrations of the antagonist, but, with non#competitive antagonists a rightward displacement of the curves with a decrease in 2max is observed.

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Schild Plots2 potency

$gonists are characterized by incubating the receptor with the compound under study and the functional response is compared to that obtained in the presence of a ligand already identified as a full agonist. &n order to fully characterize the effect of a drug it is necessary to ta+e into account both the efficacy, the 2max, and the potency, the 2C4D, that is, the effective concentration needed to reach 4DH of the maximal effect. he results can also be expressed as a p/0 )p/0 # -log*EC$%,*. &ndeed, two agonists may possess a similar efficacy but one of them may be less potent than the other )rightward displacement of the curve*. Conversely, two agonists may be e!ually potent but the efficacy of one of them can be lower )smaller maximal response*. 6igands with an efficacy that is a fraction of the effects induced by a full agonist are named partial agonists. $ number of CPC8s display a measurable basal activity in the absence of any endogenous or exogenous agonist, either constitutively in the native state or follow# ing transfection with a mutated protein. he effects of full or partial agonists described above are unaffected by basal receptor activity. 0owever, some ligands are able to decrease constitutive receptor activity, a property +nown as inverse agonism. &n the absence of constitutive activity, inverse agonists behave as competitive antagonists but the mechanisms by which inverse agonists and neutral antagonists achieve their effects are different.
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Schild Plots2 Partial Agonists

8eceptor theory assumes that the receptor can exist in at least two separate forms< one inactive form denoted by 8 and one active form denoted by 8I that are in e!uilibrium. $ full agonist has a much higher affinity for the active form of the receptor and will displace the e!uilibrium toward the active form and a full inverse agonist has a much higher affinity for the inactive form of the receptor. /eutral antagonists have a similar affinity for both receptor forms.

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Protean Agonism

$ new functional property has recently been characterized< protean agonism. 6igands belonging to this class of drugs act as partial agonists in !uiescent silent systems and as inverse agonists in systems that show a high level of constitutive activity. he name protean comes from the Cree+ god Proteus who had the ability to change his shape at will. he reversal from agonism to inverse ago# nism would only occur when an agonist produces an active conformation of lower efficacy than a totally active conformation )an other 8J species distinct from 8 and 8I*. herefore, the higher the constitutive activity, the greater chance to see this other conformation.
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Allosteric interaction
he ligand#induced functional effects described above can occur when a drug binds to the site recognized by the endogenous ligand, the orthosteric site, leading to competitive interactions or to a site located extremely close to the orthosteric site inducing non#competitive interactions. 0owever, the entire receptor surface )other than the orthosteric binding domain* can be considered as bearing potential binding sites for a drug. -uch sites, distinct from the orthosteric binding domain, are allosteric sites and drugs that recognize these sites are allosteric modulators. >hen a drug binds to an allosteric site, protein conformation is altered, resulting in changes in the affinity between ligands and the orthosteric site. $lthough allosteric modulators were initially defined as ligands possessing no intrinsic agonist or inverse agonist properties, this assumption has been challenged and some allosteric modulators may give rise to agonist or inverse agonist effects in the absence of the orthosteric ligand. Modulators are able to shift radioligand binding curves, but the allosteric nature of the interaction is revealed as progressively higher concentrations of antagonist fail to cause significant displacements of the radioligand saturation curve, in contrast to what would theoretically be expected with an antagonist.
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Allosteric interaction
Plots of fractional orthosteric ligand#receptor occupancy as a function of log5orthosteric ligand concentration7. Curves shifts induced by a competitive antagonist )a* or a negative allosteric modulator )b*. /ote the limits in curve shifts with an allosteric modulator )ceiling effect*.

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Allosteric interaction
$ common graphical method for assessing the relationship between radioligand saturation binding and antagonist concentration involves the determination of the affinity shift, that is, the ratio of radioligand affinity in the presence )3$pp* to that obtained in the absence )3$* of each concentration of antagonist. $ plot of log )affinity shift#G* versus log 5antagonist7 should yield a straight line with a slope of G for a competitive interaction, but a curvilinear plot for an allosteric interaction. Eut an allosteric modulator can also alter the lin+ between the orthosteric site and the functional response and therefore modify the efficacy of the orthos# teric ligand. his parameter can sometimes be appreciated by the shift between the 2C4Ds for functional concentration#response curves obtained in the absence or in the presence of the allosteric potentiator. &n general, the overall effect of an allosteric ligand results from the balance between the modulation of affinity and efficacy and it is usually necessary to also measure cooperativity factors and dissociation rates.

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Allosteric interaction
he use of allosteric ligands offers certain distinct advantages over orthosteric ligands. he first is a saturability of effect that is retained irrespective of the dose that is administered therapeutically. $ second advantage of positive allosteric modulators relates to the fact that they do not replace the endogenous ligand to produce full receptor activation, but selectively KtuneK tissue responses in those organs where the endogenous agonist exerts its physiologi# cal effects. 'inally, a modulator may display the same affin# ity for each subtype of a receptor but still exert a selective effect by having different degrees of cooperativity at each subtype. $bsolute subtype selectivity may therefore be obtained when a modulator remains neutrally cooperative at all receptor subtypes except the one targeted for thera# peutic purposes. 0owever, since the structure of allosteric sites is in most cases un+nown, selectivity versus other receptors has to be carefully chec+ed and this might not be such an easy tas+ due to the probe dependence of allos# teric phenomena and the difficulty in validating allosteric effects. Compilation of useful structure#activity relationship data for allosteric ligands is thus not simple.
Medicinal Chemistry

he shift )2C4Dwo?2C4Dw* of functional concentration response curves obtained in the absence or in the presence of the allosteric potentiator is a measure of the efficacy of the modulator
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Cellular and tissular functional responses

6igand#receptor or drug#enzyme interaction is expected to alter cellular function but in intact cells, a number of functional events may interfere with the initial intracellular signaling and modify the final response. 'or example, receptor function may be under control of other, possibly ill#defined, regulatory mechanisms. he compound under evaluation may also interfere with other receptor subtypes that are un+nown at the time the study is performed. 'inally, since the targeted change in tissue function is generally the conse!uence of a cascade of intracellular events, many of the biochemical steps involved in this se!uence may be subLected to tight regulatory mechanisms.

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Cellular and tissular functional responses

&t is thus necessary to confirm the existence of modified cellular function following ligand#receptor or drug# enzyme interaction. -uch in vitro experiments, performed on isolated cells, either native or transfected with the protein of interest, can be underta+en for mechanistic and?or therapeutic purposes. he data depicted in 'igure illustrate the fact that, in 02321% cells in culture, inhibi# tion of the enzyme glycogen synthetase +inase % )C-3#%* decreases tau protein hyperphosphorylation, one of the anatomopathological hallmar+s of $lzheimerAs disease, and may thus represent a potential therapeutic approach for this disease.
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Cellular and tissular functional responses

$lternatively, this experimental set#up can also be used for mechanistic purposes to characterize the efficacy of compounds as C-3#% inhibitors. >hen compared to simple in vitro experiments in which the activity of the purified enzyme is measured in the presence of a drug, results similar to
those shown in 'igure provide at least three important pieces of information. G* 2* %* he drug penetrates into the cell, a property that is rather difficult to assess directly. &nhibition effectively ta+es place with the enzyme in situ. he inhibitor does not possess any overt toxicity )cells remain viable*.

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Cellular and tissular functional responses

-tudies performed with isolated tissues, that have been ta+en from a living animal, represent a more complicated situation in terms of the number and diversity of biochemical steps that lin+ receptor stimulation and the final functional tissular response since it will integrate ligand#receptor interaction#induced changes in single cells, and the resulting interac# tions between many cells of the same type and cells of different types. &n most cases, drug effects are expressed, as in binding experiments, as 2C4D, p.2, &C4D or p$2 . Eut in some rather sophisticated experiments the effects of only a very small number of drug concentrations will be evaluated and the final result will be expressed as a minimal acti.e concentration 'MAC-. $lthough this value represents the first concentration that induces a statistically significant change when compared to control cells or tissue, no general definition can be given since it may also include other re!uirements specific to the experimental set#up, such as the fact that drug effect should be greater than 4DH. 2xpressing results this way may appear unsatisfactory but this paucity of experimental data is generally dictated by practical reasons such as difficulties in obtaining the tissue or technical difficulties.

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E3 4I45 E3PE6IME78S
2x vivo experiments generally represent the next step in the characterization of drug effects although they cannot be underta+en with all biological targets. 2x vivo means that the drug has been administered by different routes to a living animal or to humans and that the evaluation of drug effects are performed in vitro with tissue samples or fluid ali!uots of the organism under study. $n example of an ex vivo study is the inhibition of platelet aggregation. Putative inhibitors of platelet aggregation are administered systemically to the animal, drug effects ta+e place inside the body of the animal, blood is sampled after a pre#determined period of time, platelets are isolated and aggregation is induced in vitro following addition of $.P. &n ex vivo studies, the drug concentration in the test tube is un+nown )but can eventually be determined* and drug effects will basically be expressed as a function of the dose administered or time, depending on the aim of the study.

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E3 4I45 E3PE6IME78S
-ince the only drug#related parameter +nown is the initial dose that has been administered, results can be expressed as a minimal acti.e dose 'MA/-, that is, the lower administered dose that induces a statistically significant effect when compared to animals treated with the vehicle. &f it has been possible to study a relatively large number of experimental groups treated with different drug doses, depending on the experimental set#up drug potency will be expressed as an &.4D )inhibitory dose 4DH*, that is, the dose that reduces by 4DH the effect measured in control animals or an 2.4D )efficacy dose 4DH* the dose that induces an effect which is half the maximal effect that can be obtained. 2.4D and &.4D are expressed in mg?+g, that is, the amount of drug )generally of the free base if the compound is a salt* per unit of body weight, and the route of administration is also specified )see below K&n vivoK part*. &n the ex vivo binding experiment shown in 'igure, data have been reported as H of receptor occupancy for each administered dose, which represents the fraction of 0% receptors occupied by the antagonist versus the total number of 0% receptors in the absence of the drug. &n fact, what is actually measured is the number of receptors that remain free in each experimental condition and are thus able to bind the radioactive ligand.

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E3 4I45 E3PE6IME78S
8eceptor occupancy depends on the pharmacodynamic )affinity of the drug for the receptor* and pharmaco+inetic )drug tissue concentrations* characteristics of the drug. his latter parameter is a crucial determinant of drug potency ex vivo and in vivo< it has been, for example, suggested that dopaminergic .2#receptor occupancy by antipsychotics should lie in an optimal therapeutic window between M=4H and MBDH in order to gain a clinical response. $lternatively, the drug may be administered at a pre#determined efficacious dose and drug effects are then stud# ied as a function of time. &f a functionally meaningful parameter is chosen, then the duration of action of the drug can be determined, that is, the time beyond which the drug will no longer be efficacious.

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E3 4I45 E3PE6IME78S
2x vivo experiments are an important step in compound characterization as they investigate compound activity following systemic drug administration to a living animal. hey provide a lot of important information concerning the fate of the drug following its administration. &f the drug has been administered orally, drug activity implies that< N N he drug has been absorbed< &nsufficient, or lac+ of, absorption )the fact that the drug passes from the gastro#intestinal tract into the blood* is often a problem when designing new drugs. he drug has not been subLected to extensive metabolism< 'ollowing metabolism the drug may loose its pharmacological properties or may no longer be able to penetrate into the tissue. Eut even if the drug is extensively metabolized, the expected functional change can sometimes ta+e place due to the formation of an active metabolite. he drug has reached, and penetrated into, the targeted tissue or cell and it has recognized the biological target )e.g. receptor or enzyme* of interest< $chieving good tissue penetration may also be a problem, particularly when the brain is concerned since this organ is very efficiently protected from drug entry by the blood#brain barrier.

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I7 4I45 E3PE6IME78S
he aim of in vivo experiments is to confirm that the compound has the therapeutic efficacy expected, that is, that it will interfere with a pathological mechanism involved in an illness and induce beneficial effects. &n preclinical in vivo studies, the compound under study is administered to an animal and drug effects are !uantified by measuring either the behavior of the intact animal placed in a pathological situation, a physiological parameter or drug#induced changes in an insult#related tissue alteration by biochemical or histological methods on tissue samples ta+en from the animal. Clearly, it is !uite impossible to give an idea of a standard protocol, due to the very large number of experimental models that can be set up. 2ach global research field )oncology, cardiovascular research ...* deals with field#related pathologies )e.g. anxiety or depression in psychiatric research* that re!uire specialized experimental models that are aimed to mimic the pathology. 0owever, before being tested in highly sophisticated models, compound evaluation is generally done in rela# tively simple ones in a first instance. hese models are most often performed on small laboratory rodents )mice, rats* and for those that are acute and technically simple, size and number of experimental groups should be large enough to express drug effects as an 2.4D or &.4D. -ometimes results are expressed as a M$. and the potency of the compound is then compared to that of a given reference drug, if avail# able, that may, or may not, have been included in the study, and drug effects may be expressed as simply as better, e!ual to or less interesting than the reference.

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I7 4I45 E3PE6IME78S
'urthermore, again due to the severity of the model, maxi# mal drug effects are not expected to exceed M4DH and the 2.4D would represent the dose that induces an effect of O24H that is li+ely not to be statistically significant. he results of the study shown in 'igure will be presented as the effects at the maximally effective dose )#,BH at G4mg?+g, p.o.* together with experimental details that will influence drug efficacy )number of administrations, delay between artery occlusion and first drug administration, duration of artery occlusion*. &mportant additional information arises from the shape of the dose#effect curves. -ome drugs display inverted F#shaped curves, that is, drug efficacy increases with increas# ing doses up to a dose beyond which it decreases. his progressive loss of efficacy is often indicative of a drug#induced deleterious mechanism )toxicity*, generally unrelated to the main effect of the drug. he dose that induces the greater pharmacological effects is very important for clinical development since, if a biological mar+er is available )for purposes of comparison between the animal and man*, it may help the clinician to determine the dose that can be administered to humans that will display maximal efficacy and minimal drug#related ris+s.

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I7 4I45 E3PE6IME78S
he route of administration is an important aspect of an in vivo experiment. .rugs may be administered in many ways but the most widely used are orally postoperati.e )p.o.*, intraperitoneally )i.p., in the abdomen*, intra.enously )i.v.*, subcutaneously )s.c.* and intracerebro.entricularly )i.c.v., directly in the cerebrospinal fluid into the brain* although other routes )intra#thecally, trans#dermally* may also be used. &n early in vivo experiments the drug is generally administered i.p. since this route is easy to use in rodents, bypasses possible gastric absorption problems and is successful even for compounds with poor solubility. &n more complex models, the choice of a route of administration depends on the targeted pathology, the physicochemical properties of the drug and aim of the study. 'or treating acute, life# threatening insults )heart or brain infarcts* the drug has to reach its site of action as !uic+ly as possible and drugs will be inLected i.v. his can be performed in the awa+e mouse but generally re!uires anesthesia or arterial catheterization in other species and the maLor issue is drug solubility. &n most other pathologies, particularly those that re!uire long#term treatment )e.g. depression or hypertension* the oral route will be selected, the drug being administered by oral gavage, gastric tubing or inclusion in the food. here are of course exceptions for drugs with a proven therapeutic utility and that are poorly absorbed and?or !uic+ly metabolized )insulin for diabetes* and?or that display high systemic toxicity )anticancer drugs* in which case the s.c. or i.v. route will be selected. he i.c.v. route is devoted to proof#of#concept experiments for drugs acting on a cerebral target, that is, to ascertain that the drug has the mechanistic or therapeutic effects expected in the absence of any other interfering parameter )crossing of blood#brain barrier, absorption, metabolism*.

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