03.affinity Efficacy Potency 5r3053

Medicinal Chemistry I IInd Module Affinity, efficacy, and potency

Affinity, efficacy, and potency Once a new drug molecule has been synthesized, one has to that it possesses the functional activity and therapeutic efficacy expected. This is done by evaluating the properties of the drug in a logically ordered sequence 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. An 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. Medicinal Chemistry

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Affinity, efficacy, and potency The 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. It is important to appreciate the distinction between affinity and efficacy. A compound with high affinity does not necessarily have high efficacy. For example, an antagonist can bind with high affinity but has no efficacy. The potency of a drug refers to the amount of drug required to achieve a defined biological effect — the smaller the dose required, 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. Affinity can be measured using a process known as radioligand labelling. A known 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 equilibrium has been reached, the unbound ligands are removed by washing, filtration, or centrifugation. The 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.

Medicinal Chemistry

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Binding The 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 check its selectivity versus other binding sites or biological off­targets. Binding studies usually represent an initial step in compound charac­ terization. Schematically, membranes are prepared from the tissue of interest (heart, bladder, brain ...) or from mammalian cells that express the receptor of interest. The receptors can be native, that is, they are constitutively expressed by the cells or the tissue, or transduced, that is, a cDNA coding for the receptor isolated from any appropriate species has been inserted into the cell. Chinese hamster ovary (CHO) or human epithelial kidney 293 cells (HEK293) are generally used. In 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. Stable transfection in cell lines is often used to perform binding studies with human receptors since compound affinity may differ markedly between receptors isolated from animals and man.

Medicinal Chemistry

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Binding Constant The equilibrium constant for bound versus unbound radioligand is defined as the dissociation binding constant (Kd).

[L] and [LRl can be found by measuring the radioactivity of unbound ligand and bound ligand respectively, after correction for any background radiation. However, it is not possible to measure [R], and so we have to carry out some mathematical manipulations to remove | R] from the equation. The total number of receptors present must equal the number of receptors occupied by the ligand ([LR]) and those that are unoccupied ([R]), i.e. Rtot = [R] + [LR]

and

[R] = Rtot ­ [LR]

Substituting this into the first equation and rearranging leads to the Scatchard equation, where both [LR] and [L] are measurable:

Medicinal Chemistry

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Scatchard Plots We are still faced with the problem that Kd and Rtot cannot be measured directly. However, these can be determined by drawing a graph based on a number of experiments where different concentrations of a known radioligand are used. [LR] and [L] are measured in each case and a Scatchard plot is drawn which compares the ratio [LR]/[L] versus [LR]. This gives a straight line, and the point where it meets the x­axis represents the total number of receptors available. The slope is a measure of the radioligands affinity for the receptor and allows Kd to be determined.

Medicinal Chemistry

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Scatchard Plots We are now in the position to determine the affinity of a novel drug that is not labelled. This is done by repeating the radioligand experiments in the presence of the unlabelled test compound. The test compound competes with the radioligand for the receptor's binding sites and is called a displacer. The stronger the affinity of the test compound, the more effectively it will compete for binding sites and the less radioactivity will be measured for [LR]. This will result in a different line in the Scatchard plot. If 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 x­axis remains the same. In other words, if the radioligand concentration is much greater than the test compound it will bind to all the receptors available

Medicinal Chemistry

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Scatchard Plots Generally, it is assumed that, when affinity is high, the compound is less likely to interfere with other, possibly unwanted off­target sites. However, this is not always true and selectivity has to be checked by evaluating the affinity of the compound for a large of receptors, enzymes and ion channels. It is obvious that selectivity has limits that depend on the size of the that has been investigated, but also on the sci­ entific knowledge available at the time when the studies are performed. For 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 Equation Typically, displacement experiments give rise to sigmoid curves. The drug concentration that displaces half of the maximum bound radioactive ligand represents the IC50. When 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. In these saturation experiments, Bmax (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 Kd (the dissociation constant) from the negative reciprocal of the slope of the line. When such experiments are performed in the presence of various concentrations of the compound under study, they give rise to a family of lines. If Bmax remains unchanged and the slope of the lines decreases with increasing concentrations of the compound, the dis­ placement is competitive. Unchanged slope and decreased Bmax indicate that the displacement is non­competitive. The lower the IC50 or Kd, the higher the affinity. Results can also be expressed as a Ki, the inhibitory (or affinity) constant of the displacer compound for the receptor. Ki and IC50 are not independent and are very simply related when the displacement is non­competitive (K = IC50), but the relationship becomes more complicated (ChengPrusoff equation) for a competitive displacement [Ki = IC50/(1 + [L]/Kd) where [L] is the concentration of the radioactive ligand]. When deg 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.

Medicinal Chemistry

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Cheng-Prusoff Equation Agents 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. However, by binding to an allosteric site they make the normal binding site unrecognizable to the radioligand and so there are less receptors available. This results in a line with an identical slope to line A, but crossing the x­axis at a different point, thus indicating a lower total number of available receptors. The 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. This results in a sigmoidal curve termed the displacement or inhibition curve, which can be used to identify the IC50 value for the test compound. The inhibitory or affinity constant (Ki) for the test compound is the same as the IC 50 value if non­competitive interactions are involved. For 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 equation:

where Kd is the dissociation constant for the radioactive ligand and [L] tot is the concentration of radioactive ligand used in the experiment. Medicinal Chemistry

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Schild Plots Efficacy is determined by measuring the maximum possible effect resulting from receptor­ligand binding. Potency can be determined by measuring the concentration of drug required to produce 50% of the maximum possible effect (EC50). The smaller the value of EC50, the more potent the drug. In practice, pD2 is taken as the measure of potency where pD2 = ­logEC50.

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Schild Plots A Schild analysis is used to determine the dissociation constant (Kd) of competitive antagonists. An agonist is first used at different concentrations to activate the receptor, and an observable effect is measured at each concentration. The experiment is then repeated several times in the presence of different concentrations of antagonist. Comparing the effect ratio Eobserver/Emaximum versus the log of the agonist concentraion (log[agonist]) produces a series of sigmoidal curves where the EC 50 of the agonist increases with increasing antagonist concentration. In other words, greater con­ centrations of agonist are required to compete with the antagonist.

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Schild Plots A Schild plot is then constructed, which compares the reciprocal of the dose ratio versus the log of the antagonist concentration. (The dose ratio is the agonist concentration required to produce a specified level of effect when no antagonist is present, compared to the agonist concentration required to produce the same level in the presence of antagonist.) The line produced from these studies can be extended to the x­axis to find pA2 = ­log Kd), which represents the affinity of the competitive antagonist.

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Schild Plots Binding 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. Such determinations necessarily involve functional measurements of ligand­receptor interaction­induced changes in an intracellular signal. Such experiments also represent the initial step in compound characterization as a transporter or enzyme inhibitor, or as a voltage­activated cation channel modulator. In 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 unknown to date. Since antagonists block an existing ligand­activated 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. The resulting curves are called Schild plot and the results are expressed as an IC50, the drug concentration that produces half of the maximal response (Emax in %) measured in the absence of the antagonist. Alternatively, 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 pA2 (­log molar concentration of antagonist producing a 2­fold shift of the concentration response curve, that is, a 2­fold increase in agonist concentration in order to obtain a similar effect). Medicinal Chemistry

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Schild Plots: competitive and non-competitive anatagonists Competitive antagonists induce a parallel rightward displacement of the curves with increasing concentrations of the antagonist, but, with non­competitive antagonists a rightward displacement of the curves with a decrease in Emax is observed.

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Schild Plots: potency Agonists 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. In order to fully characterize the effect of a drug it is necessary to take into both the efficacy, the Emax, and the potency, the EC50, that is, the effective concentration needed to reach 50% of the maximal effect. The results can also be expressed as a pD2 (pD2 = -log[EC50]). Indeed, 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 equally potent but the efficacy of one of them can be lower (smaller maximal response). Ligands with an efficacy that is a fraction of the effects induced by a full agonist are named partial agonists. A number of GPCRs 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. The effects of full or partial agonists described above are unaffected by basal receptor activity. However, some ligands are able to decrease constitutive receptor activity, a property known as inverse agonism. In 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. Medicinal Chemistry

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Schild Plots: Partial Agonists Receptor theory assumes that the receptor can exist in at least two separate forms: one inactive form denoted by R and one active form denoted by R* that are in equilibrium. A full agonist has a much higher affinity for the active form of the receptor and will displace the equilibrium toward the active form and a full inverse agonist has a much higher affinity for the inactive form of the receptor. Neutral antagonists have a similar affinity for both receptor forms.

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Protean Agonism

A new functional property has recently been characterized: protean agonism. Ligands belonging to this class of drugs act as partial agonists in quiescent silent systems and as inverse agonists in systems that show a high level of constitutive activity. The name protean comes from the Greek god Proteus who had the ability to change his shape at will. The 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 R@ species distinct from R and R*). Therefore, the higher the constitutive activity, the greater chance to see this other conformation. Medicinal Chemistry

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Allosteric interaction The ligand­induced 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 non­competitive interactions. However, the entire receptor surface (other than the orthosteric binding domain) can be considered as bearing potential binding sites for a drug. Such sites, distinct from the orthosteric binding domain, are allosteric sites and drugs that recognize these sites are allosteric modulators. When a drug binds to an allosteric site, protein conformation is altered, resulting in changes in the affinity between ligands and the orthosteric site. Although 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. Medicinal Chemistry

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Allosteric interaction Plots of fractional orthosteric ligand­receptor occupancy as a function of log[orthosteric ligand concentration]. Curves shifts induced by a competitive antagonist (a) or a negative allosteric modulator (b). Note the limits in curve shifts with an allosteric modulator (ceiling effect).

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Allosteric interaction A 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 (KApp) to that obtained in the absence (KA) of each concentration of antagonist. A plot of log (affinity shift­1) versus log [antagonist] should yield a straight line with a slope of 1 for a competitive interaction, but a curvilinear plot for an allosteric interaction. But an allosteric modulator can also alter the link between the orthosteric site and the functional response and therefore modify the efficacy of the orthos­ teric ligand. This parameter can sometimes be appreciated by the shift between the EC50s for functional concentration­response curves obtained in the absence or in the presence of the allosteric potentiator. In 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 The use of allosteric ligands offers certain distinct advantages over orthosteric ligands. The first is a saturability of effect that is retained irrespective of the dose that is istered therapeutically. A 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 "tune" tissue responses in those organs where the endogenous agonist exerts its physiologi­ cal effects. Finally, 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. Absolute subtype selectivity may therefore be obtained when a modulator remains neutrally cooperative at all receptor subtypes except the one targeted for thera­ peutic purposes. However, since the structure of allosteric sites is in most cases unknown, selectivity versus other receptors has to be carefully checked and this might not be such an easy task due to the probe dependence of allos­ teric phenomena and the difficulty in validating allosteric effects. Compilation of useful structure­activity relationship data for allosteric ligands is thus not simple. Medicinal Chemistry

The shift (EC50wo/EC50w) 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 Page 22

Cellular and tissular functional responses Ligand­receptor or drug­enzyme 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. For example, receptor function may be under control of other, possibly ill­defined, regulatory mechanisms. The compound under evaluation may also interfere with other receptor subtypes that are unknown at the time the study is performed. Finally, since the targeted change in tissue function is generally the consequence of a cascade of intracellular events, many of the biochemical steps involved in this sequence may be subjected to tight regulatory mechanisms.

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Cellular and tissular functional responses It is thus necessary to confirm the existence of modified cellular function following ligand­receptor or drug­ enzyme interaction. Such in vitro experiments, performed on isolated cells, either native or transfected with the protein of interest, can be undertaken for mechanistic and/or therapeutic purposes. The data depicted in Figure illustrate the fact that, in HEK293 cells in culture, inhibi­ tion of the enzyme glycogen synthetase kinase 3β (GSK­3β) decreases tau protein hyperphosphorylation, one of the anatomopathological hallmarks of Alzheimer's disease, and may thus represent a potential therapeutic approach for this disease. Medicinal Chemistry

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Cellular and tissular functional responses Alternatively, this experimental set­up can also be used for mechanistic purposes to characterize the efficacy of compounds as GSK­3β inhibitors. When 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 Figure provide at least three important pieces of information. 1)

The drug penetrates into the cell, a property that is rather difficult to assess directly.

2)

Inhibition effectively takes place with the enzyme in situ.

3)

The inhibitor does not possess any overt toxicity (cells remain viable).

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Cellular and tissular functional responses Studies performed with isolated tissues, that have been taken from a living animal, represent a more complicated situation in of the number and diversity of biochemical steps that link receptor stimulation and the final functional tissular response since it will integrate ligand­receptor interaction­induced changes in single cells, and the resulting interac­ tions between many cells of the same type and cells of different types. In most cases, drug effects are expressed, as in binding experiments, as EC50, pD2, IC50 or pA2 . But 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 active concentration (MAC). Although 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 requirements specific to the experimental set­up, such as the fact that drug effect should be greater than 50%. Expressing 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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EX VIVO EXPERIMENTS Ex vivo experiments generally represent the next step in the characterization of drug effects although they cannot be undertaken with all biological targets. Ex vivo means that the drug has been istered 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 aliquots of the organism under study. An example of an ex vivo study is the inhibition of platelet aggregation. Putative inhibitors of platelet aggregation are istered systemically to the animal, drug effects take place inside the body of the animal, blood is sampled after a pre­determined period of time, platelets are isolated and aggregation is induced in vitro following addition of ADP. In ex vivo studies, the drug concentration in the test tube is unknown (but can eventually be determined) and drug effects will basically be expressed as a function of the dose istered or time, depending on the aim of the study.

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EX VIVO EXPERIMENTS Since the only drug­related parameter known is the initial dose that has been istered, results can be expressed as a minimal active dose (MAD), that is, the lower istered dose that induces a statistically significant effect when compared to animals treated with the vehicle. If it has been possible to study a relatively large number of experimental groups treated with different drug doses, depending on the experimental set­up drug potency will be expressed as an ID50 (inhibitory dose 50%), that is, the dose that reduces by 50% the effect measured in control animals or an ED50 (efficacy dose 50%) the dose that induces an effect which is half the maximal effect that can be obtained. ED50 and ID50 are expressed in mg/kg, 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 istration is also specified (see below "In vivo" part). In the ex vivo binding experiment shown in Figure, data have been reported as % of receptor occupancy for each istered dose, which represents the fraction of H3 receptors occupied by the antagonist versus the total number of H3 receptors in the absence of the drug. In 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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EX VIVO EXPERIMENTS Receptor occupancy depends on the pharmacodynamic (affinity of the drug for the receptor) and pharmacokinetic (drug tissue concentrations) characteristics of the drug. This latter parameter is a crucial determinant of drug potency ex vivo and in vivo: it has been, for example, suggested that dopaminergic D2­receptor occupancy by antipsychotics should lie in an optimal therapeutic window between ~65% and ~80% in order to gain a clinical response. Alternatively, the drug may be istered at a pre­determined efficacious dose and drug effects are then stud­ ied as a function of time. If 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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EX VIVO EXPERIMENTS Ex vivo experiments are an important step in compound characterization as they investigate compound activity following systemic drug istration to a living animal. They provide a lot of important information concerning the fate of the drug following its istration. If the drug has been istered orally, drug activity implies that: •

The drug has been absorbed: Insufficient, or lack of, absorption (the fact that the drug es from the gastro­intestinal tract into the blood) is often a problem when deg new drugs.

•

The drug has not been subjected to extensive metabolism: Following metabolism the drug may loose its pharmacological properties or may no longer be able to penetrate into the tissue. But even if the drug is extensively metabolized, the expected functional change can sometimes take place due to the formation of an active metabolite.

•

The 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: Achieving 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 blood­brain barrier.

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IN VIVO EXPERIMENTS The 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. In preclinical in vivo studies, the compound under study is istered to an animal and drug effects are quantified by measuring either the behavior of the intact animal placed in a pathological situation, a physiological parameter or drug­induced changes in an insult­related tissue alteration by biochemical or histological methods on tissue samples taken from the animal. Clearly, it is quite impossible to give an idea of a standard protocol, due to the very large number of experimental models that can be set up. Each global research field (oncology, cardiovascular research ...) deals with field­related pathologies (e.g. anxiety or depression in psychiatric research) that require specialized experimental models that are aimed to mimic the pathology. However, before being tested in highly sophisticated models, compound evaluation is generally done in rela­ tively simple ones in a first instance. These 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 ED50 or ID50. Sometimes results are expressed as a MAD 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, equal to or less interesting than the reference.

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IN VIVO EXPERIMENTS Furthermore, again due to the severity of the model, maxi­ mal drug effects are not expected to exceed ~50% and the ED50 would represent the dose that induces an effect of <25% that is likely not to be statistically significant. The results of the study shown in Figure will be presented as the effects at the maximally effective dose (­48% at 15mg/kg, p.o.) together with experimental details that will influence drug efficacy (number of istrations, delay between artery occlusion and first drug istration, duration of artery occlusion). Important additional information arises from the shape of the dose­effect curves. Some drugs display inverted U­shaped curves, that is, drug efficacy increases with increas­ ing doses up to a dose beyond which it decreases. This progressive loss of efficacy is often indicative of a drug­induced deleterious mechanism (toxicity), generally unrelated to the main effect of the drug. The dose that induces the greater pharmacological effects is very important for clinical development since, if a biological marker is available (for purposes of comparison between the animal and man), it may help the clinician to determine the dose that can be istered to humans that will display maximal efficacy and minimal drug­related risks.

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IN VIVO EXPERIMENTS The route of istration is an important aspect of an in vivo experiment. Drugs may be istered in many ways but the most widely used are orally postoperative (p.o.), intraperitoneally (i.p., in the abdomen), intravenously (i.v.), subcutaneously (s.c.) and intracerebroventricularly (i.c.v., directly in the cerebrospinal fluid into the brain) although other routes (intra­thecally, trans­dermally) may also be used. In early in vivo experiments the drug is generally istered i.p. since this route is easy to use in rodents, byes possible gastric absorption problems and is successful even for compounds with poor solubility. In more complex models, the choice of a route of istration depends on the targeted pathology, the physicochemical properties of the drug and aim of the study. For treating acute, life­ threatening insults (heart or brain infarcts) the drug has to reach its site of action as quickly as possible and drugs will be injected i.v. This can be performed in the awake mouse but generally requires anesthesia or arterial catheterization in other species and the major issue is drug solubility. In most other pathologies, particularly those that require long­term treatment (e.g. depression or hypertension) the oral route will be selected, the drug being istered by oral gavage, gastric tubing or inclusion in the food. There are of course exceptions for drugs with a proven therapeutic utility and that are poorly absorbed and/or quickly 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. The i.c.v. route is devoted to proof­of­concept 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 blood­brain barrier, absorption, metabolism).

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