Drug Safety 5 (Suppl. I): 4-23, 1990 0114-5916/90/0001-0004/$10.0/0 © ADIS Press Limited All rights reserved. DSSUP1790

Safety by Design David Jack Formerly of Glaxo Holdings pic, London, United Kingdom

Summary

Safety by design for any kind of drug requires it to act selectively on the cells which mediate the desired effect, and affect other cellular functions as little as possible. To illustrate this, the treatment of bronchial asthma was much improved during the past 20 years by the development of inhalation treatment based on drugs such as salbutamol (albuterol), a selective i32-adrenoceptor stimulant, and beclomethasone dipropionate, a topical anti-inflammatory steroid, from their parent physiological mediators epinephrine (adrenaline) and hydrocortisone (cortisol). Their development and that of HI- and H2antagonists from histamine are described. From these and other sources the general conditions for safe, selective drug action and the range of drug effects that may be attained by modifying physiological mediators are deduced. This involves the identification and definition of type I and type 2 agonism. This analysis led to the discovery of salmaterol (salmeterol), a new uniquely long acting i32-adrenergic bronchodilator, by modification of salbutamol. The development, by modification of serotonin (5-hydroxytryptamine), of ondansetron, a new antiemetic for use in cancer chemotherapy, and sumatriptan, a new type of drug for treating migraine, are also described. All these new drugs are more efficacious and safer than their predecessors.

In drug research, safety by design simply means using what we know to conceive and to create better medicines of a new kind or to improve existing medicines. Most often the objective is a new drug with a desired therapeutic effect and no uselimiting side effects. The key to success is selective drug activity at an appropriate cellular target. This is true for all kinds of drugs whether for the chemotherapy of infections or the achievement of a particular change in mammalian physiological functioning. Appropriate selectivity of action is the absolute requirement for both efficacy and safety in any medicine. Safety by design is such a huge topic that it is impossible to deal with it adequately in a short paper of this kind. Therefore, one of the major ap-

proaches to drug research is used to demonstrate some general principles that are important in drug research. The selected approach is the manipulation of physiological mediators and their receptor proteins. This article is essentially a personal view of some of the work done in Glaxo and elsewhere over a 25-year period. The main subjects are adrenergic bronchodilators and steroids in bronchial asthma, drugs based on histamine, and drugs based on serotonin (5-hydroxytryptamine).

1. Adrenergic Bronchodilators and Steroids in the Treatment 0/ Bronchial Asthma Our approach to drug research in bronchial asthma is based on a belief that there are 2 quite different causes of the increased airway resistance

Safety by Design

5

Table I. Summary of Ahlquist's evidence for the classification of adrenoceptors into a and {J types Type receptor

Biological responses

Order of potency of catecholamines

Effect of dibenamine

a-Adrenoceptor

Contraction of smooth muscle in the arteries of the skin and viscera, and in the uterus, ureter iris and nictitating membrane Relaxation of smooth muscle in the gut

Epinephrine > norepinephrine > a-methyl norepinephrine > a-methylepinephrine > isoprenaline (isoproterenol)

Blockade

{J-Adrenoceptor

Relaxation of smooth muscle in the bronchi, uterus and in the arteries supplying the heart and skeletal muscles Stimulation of the heart

Isoprenaline> epinephrine > a-methylepinephrine > a-methylnorepinephrine > norepinephrine

No blockade

that characterises the disease. The first is bronchospasm caused by contraction of bronchial smooth muscle and the second is progressive blockage of the airways because of damage to the bronchial mucosa and accumulation of tenacious secretions in the bronchi. Bronchospasm is the dominant cause of airway obstruction in less severe asthma and is easily controlled in most patients by bronchodilators, the most effective of which are the highly selective {j2-adrenoceptor stimulants given by inhalation. Salbutamol (albuterol), the first drug of this kind, is discussed in section 1.2. The occlusion of the bronchi which occurs as asthma worsens is the main life-threatening process in the disease. It is not controlled by bronchodilators and is reliably controlled only by glucocorticoid steroids. Systemic steroids such as prednisone are effective but have to be used cautiously because serious side effects develop with long term use. How this problem has been partly overcome by inhaled anti-inflammatory steroids is explained in section 1.3.

1.1 Ahlquist's Classification of Adrenoceptors and its Consequences Ahlquist (1948) divided adrenoceptors into ex and {j types on the basis of the relative activities of a few close analogues of epinephrine (adrenaline) in a range of pharmacological tests. The re-

suits of this brilliantly simple work are summarised in table I. The profound significance of Ahlquist's contribution to drug research was not recognised at the time. Indeed, even today it is still not fully realised that, apart from clarifying adrenergic physiology and pharmacology, his use of close analogues of a physiological mediator as biological probes provided the first general method for detecting chemical differences between the receptor proteins for particular mediators that are the essential basis for selective drug activity. All of this is discussed more fully in a late section on the biochemical basis for adrenergic activity (section 1.5). The tree of innovation that grew directly from Ahlquist's work is shown in figure I. The forest that is growing by application of his method to other mediators will soon require an aerial photograph to illustrate it.

1.2 Salbutamol Epinephrine used to be commonly given by injection and occasionally by inhalation to relieve acute bronchospasm. However, when we considered its many pharmacological actions only 2 seemed to be relevant in asthma and some of the others were potentially harmful. The relaxant effect of epinephrine on bronchial muscle was obviously desirable but its ability to inhibit antigeninduced release of histamine from sensitised guinea-

Drug Safety 5 (Suppl. 1) 1990

6

{:I-Blocker branch

{:I-Stimulant branch

/

Brittain et al. (1988)

I

Brittain et al. (1968)

{:I,-

~

o,.st;m""",

Propranolol

I

Salbutamol

r

,q-Blocker branch

Atenolol

Salmaterol

Prazosin

Clonidine

Pronethalol Black & Stephenson (1962)

J

and {:I2-Adrenoceptors

''1-

Lands et al. (1967)

and "2-Adrenoceptors Starke (1972)

\

(t-

J

I

and {:I-Adrenoceptors Ahlquist (1948)

Fig. 1. The tree of adrenergic innovation (after Ahlquist 1948).

pig lung (Mongar & Schild 1957) hinted that an additional, possibly more fundamental, prophylactic effect might also be achievable with the right compound. The ultimate objective of our work was, therefore, an epinephrine analogue with these desirable actions and as few as possible of the others. After Ahlquist's work, isoprenaline (isoproterenol), because of its selective action at {j-receptors, was used by inhalation in preference to epinephrine as a bronchodilator but its intense stimulant action on the heart precluded its parenteral use. About 1960, Lands and his colleagues at Winthrop extended Ahlquist's work to epinephrine analogues with larger N-substituents and found them to be significantly more active at some {j-receptors than others. The result was their proposal that {j-receptors be subdivided into {jl and {j2 subtypes (Lands et al. 1967a,b). Their conclusions are summarised in table II. The important feature of this classification for asthma research is that the desirable relaxant action on the bronchi and the undesirable stimulant actions on the heart are mediated by different kinds of receptors.

At that time we were working on noncatechol analogues of epinephrine in the hope of finding a longer acting bronchodilator that would be active by mouth or inhalation. That and more was achieved when we found the saligenin analogues of catecholamines to be very highly selectively active at {j2-receptors. The relative activities of selected catecholamines and their saligenin analogues on bronchial Table II. Lands' classification of i3-adrenoceptors in mammalian

tissues (after Lands et al. 1967) Responses mediated by i3,-adrenoceptors

Responses mediated by i32-adrenoceptors

Increased force and rate of contraction of cardiac muscle

Relaxation of smooth muscle in bronchi, uterus and arteries supplying skeletal muscles

Relaxation of smooth muscle Decreased twitch tension in in the alimentary tract skeletal muscle Lipolysis

Glycolysis Glycogenolysis

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Table III. Relative activities and affinities of selected fj-adrenoceptor stimulants at preparations containing fj1- or fj2-receptors Relative activity Equipotent concentration; (-)isoprenaline=1

Compound

Relative affinity (-)-lsoprenaline=1

guinea-pig tracheal

guinea-pig left atria

rat lung membrane

muscle

(fj1-adrenoceptors)

(fj2-adrenoceptors)

(fj2-adrenoceptors) Catechols

HO

rat left atrial membrane (fj1-adrenoceptors)

OH

HO%~HCH2NHR

(-) Norepinephrine

E.

56

10

80

14

1-) Epinephrine

CH3-

5

12

6

10

(-) Isoprenaline

IC H3)2 CH -

1 (pECSO = 7.7)

1 (pECSO = 7.9)

1 (pKi = 6.71)

1 (pKi = 6.52)

ICH3)3-C-

0.4

3.6

CH3

0.1

0.5

H

580

10000

I ±) AH4053

CH3-

430

3500

I±) AH3021

ICH 3)2CH -

6

>

3.7

> 2000a

10

26

1.3

> 2000a

0.5

> 2000a

0.07

0.5

H

(±) t-Bulylnorepinephrine

(±) p·Hydroxyphenylisopropyl·

Saligenins HOCH2 I±) AH3364

OH

HO%~HCH2NHR

ICH313CCH3 Ho-@-bHCH2-

( ±) Salbutamol

I±) AH4553

( ±) Salmaterol

a

I

HO-@- CHCH2-

norepinephnne

@-ICH2)4 0 ICH2)6 -

1000a

Partial agonists.

Abbreviations: pECso = negative logarithm of concentration producing half-maximal response; pKi = affinity constant.

muscle and heart muscle are summarised in table III. The most selective catechol is about 10 times more active on bronchial than on heart muscle but with salbutamol and some other saligenins the ratio of these activities is more than 1000 times. Salbutamol, the first highly selective i32-adrenoceptor stimulant, was marketed in 1969 and ultimately became the adrenergic bronchodilator of choice throughout the world. Salbutamol is at its best by inhalation because its intrinsic i32-selectivity is reinforced by local application within the airways. Near-maximal

bronchodilatation is achieved within a few minutes with 200~g doses without significant systemic side effects. Its effective duration of action in chronic asthma is about 4 hours, which is a weakness because this is not long enough to control nocturnal asthma or for convenient maintenance treatment of the disease. The bronchodilating effect obtained with standard 4mg oral doses of salbutamol is less than with the inhaled drug and the duration of action is no longer. Systemic use of salbutamol is also complicated by side effects, most noticeably tremor of skeletal muscles and tachy-

Drug Safety 5 (Suppl. 1) 1990

8

cardia in sensitive individuals, which are caused by generalised activation of ~2-receptors in the body. Fortunately, the systemic effects of salbutamol, though troublesome for some, are not intrinsically dangerous. As shown in table IV the cardiovascular effects of salbutamol are markedly different from those of isoprenaline. Both drugs cause tachycardia in patients with mitral valve disease but the reasons are different. The increase in heart rate with isoprenaline is caused by a direct chronotropic action of the drug whereas that with salbutamol is mainly of reflex origin and secondary to dilatation of the arteries in the skeletal muscle beds. An even more important difference is that salbutamollacks the intense inotropic action of isoprenaline which is so biochemically wasteful and, therefore, dangerous in hypoxic heart muscle. Even in overdosage, the cardiovascular effects of salbutamol are surprisingly mild. The effects of 80mg of oral salbutamol in 3 young male volunteers are illustrated in figure 2 in which the relatively small effect on diastolic blood pressure is noteworthy. ~2-Selectivity contributes to the safety

as well as to the efficacy of salbutamol and similar drugs. 1.3 Selective Glucocorticoid Steroid Treatment of Bronchial Asthma The introduction of hydrocortisone (cortisol) and related systemic glucocorticoid steroids from 1950 onwards was a real godsend for severe asthmatics because they were the first drugs that could reliably contain and reverse the inflammatory process in their lungs. Only the anti-inflammatory effects of these drugs is beneficial; their other glucocorticoid actions give rise to very serious side effects with prolonged use. Unfortunately, this problem has not been solved. Despite much effort, separation of the anti-inflammatory effect from other glucocorticoid actions in a systemic steroid has not been achieved. An important advance in steroid drugs, however, was made at the end of the 1950s when triamcinolone acetonide was found to be a potent antiinflammatory agent when applied to the skin. Topical anti-inflammatory steroids of this kind trans-

Table IV. Effects of intravenous isoprenaline and salbutamol in patients with mitral valve disease (from Gibson & Coltart 1971)

Response measured

Heart rate (beats/min) Cardiac output (L/min) Aortic pressure (mm Hg) Mean ejection rate (mlfsec) Oxygen uptake (ml/min)

Isoprenaline !ltg/kg) control

0.05

0.1

70.0 2.8 98.0 135.0 170.0

+0.8*· +0.7·· -9.5·

+10.6** +1.1** -10.0 +62.0*· +47.5··

+42.0** +26.7··

Salbutamol !ltg/kg)

Heart rate (beats/min) Cardiac output (L/min) Aortic pressure (mm Hg) Mean ejection rate (ml/sec) Oxygen uptake (ml/min)

• = p < 0.05; •• = P < 0.01.

control

0.4

78.0 3.0 102.0 160.0 185.0

+5.7* +0.2 +3.0 +1.0 0

2

+17.5·· +0.6·· -6.7* +4.0 +11.0

+25.5·· +1.1·· -16.0· +8.0 +21.0·

9

Safety by Design

200

~

::>

'"'" III a.

"0

8

iii 100

..,j.___:....

Ii=" -~ ""

Diastolic

0

'2

150

:§.

'" iii e ~ III

100

t:

'" Q)

J:

50

r: ~ -1

o

to test the idea because of its high topical activity and relat\vely modest systemic glucocorticoid activity. The essential differences between BDP and systemic steroids are summarised in table V. The systemic glucocorticoid activity of BDP in dogs is greater than in humans and they were obviously 'Cushingoid' after 6 months of treatment with inhaled and oral doses of the drug. Nevertheless, at the end of the experiment the lungs of the animals were found to be normal, and infection had not been a problem. We were encouraged by the results of toxicity tests in the dog to proceed to human trials. Fortunately, the same was true in patients and the drug proved to be an important advance for treating asthma. A substantial anti-inflammatory effect is achieved in the airways with doses which do not cause significant systemic side effects. The maximal attainable effect, however, is equivalent to about Smg of prednisone daily, so systemic glucocorticoid treatment is still needed in severe asthma. Beclomethasone dipropionate inhaler was marketed in 1972 and has proved to be very safe in therapeutic use.

I

2

Hours Salbutamol (80mg) Fig. 2. The effects of a large single dose of salbutamol on the blood pressure and heart rate of 3 healthy young male volunteers.

formed the treatment of inflammatory skin diseases, the cause of so much distress. The proposal to use a topical anti-inflammatory steroid by inhalation for asthma, first made by Wilfrid Simpson and Eric Snell in Glaxo, immediately posed several questions. Would the bronchial mucosa resemble the skin in being sensitive to their anti-inflammatory action? Would the function of lung fibroblasts be inhibited like those in the skin? Would treatment favour the spread of infections in the lung? Beclomethasone dipropionate (BDP) was chosen

1.4 Current Treatment and Research in Asthma The therapeutic consequences of this, largely empirical, work for the treatment of asthma are summarised in figure 3. Today, patients treated intelligently with concurrent inhaled Ih-selective bronchodilators and topical steroids, supplemented with systemic steroids when necessary, are better controlled and much less likely to die during exacerbations of their disease than ever before. With continuing research, knowledge of the cells and chemical mediators involved in asthma has greatly increased. A very complicated position is summarised in table VI. This new knowledge has not yet led to improved drug treatments, perhaps for want of understanding of how the actions of the various cells and mediators are integrated. Nevertheless, many research groups are trying to inhibit the formation or to antagonise the actions of particular mediators in the hope that one or a

10

Drug Safety 5 (Suppi. 1) 1990

Table V. Relative systemic and topical activities of selected steroids in humans Biological activity

Cortisol

Betamethasone

Beclomethasone dipropionate

Fluticasone propionate

Systemic glucocorticoid

Oral 1

Oral 25

Systemic mineralocorticoid Topical anti-inflammatory (McKenzie skin test)

Oral 1 0.1 (Cortisol acetate

0 0.6

Intravenous 20 Oral 4 0 500

Intravenous active (not quantified) Oral "'0 0 950

= 1)

Chemical structure

few of them play key roles in the inflammatory process. The leukotrienes appear to be good candidates for such research and we await with interest the results of trials now under way with 5lipoxygenase inhibitors. The data in table VI explain why the concurrent use of i32-selective bronchodilators and steroids is unusually effective in asthma. Between them they directly inhibit the activation of all the proinflammatory cells except the platelets. As already indicated, however, neither salbutamol nor BDP is ideal for its intended purpose and so we tried to improve them. Fluticasone propionate, whose properties are summarised in table V, is now in clinical trial as a possible advance on BDP because it has greater topical activity and less systemic glucocorticoid activity. Our hope is for a more intense anti-inflammatory action within the lungs than is achievable with BDP. The search for a better salbutamol was particularly satisfying because it started with a theoretical analysis of selective i3-stimulant activity (Brittain et al. 1976; Jack 1977). I have outlined my current thinking in the next section in the hope that it will be useful to other drug researchers and in the belief that it is of general application to the manipulation of mediators similar to epinephrine.

1.5 The Biochemical Basis of i3-Adrenergic Activity and of Selective i32-Agonism My interpretation of the voluminous literature on the mechanism of action of epinephrine at 13receptors is illustrated in figure 4. Epinephrine is considered to be an allosteric activator of its receptor protein which is an enzyme which forms iXsGTP units from Gs-protein molecules in the cell membrane. In order to emphasise this concept the receptor proteins for epinephrine and similar mediators are referred to as receptor enzymes in the rest of this article. The function of the iXs-GTP units is to activate adenylate cyclase, the effector enzyme which converts ATP into cAMP. This second messenger continues the cellular response by activating specific kinases inside the cell. The coupling of a receptor enzyme and an effector enzyme by an intermediate G-protein is a common mechanism whereby the action of extracellular mediators at their specific receptors on the external surfaces of their receptor enzymes lead to the formation of second messengers inside the cell or in the cell membrane (Neer & Clapham 1988). The effector enzymes whose actions are well established are adenylate cyclase, which generates cAMP, and phospholipase C which converts phosphatidylinositoI4,5-diphosphate into 1,2-diacylgly-

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High

1 Normal

"'::~

Blockage due to mucosal damage and hypersecretion

Bronchospasm _ _ _ _ _ _ _ _ _ _ _ _ _-' Mild

Moderate

Severe

Status

Seventy of asthma

Treatment

Injected steroid Oxygen

Fig. 3. The causes of airway obstruction and the treatment of bronchial asthma.

cerol and inositol triphosphate. Like cAMP these mediators catalyse the succeeding step in cellular responses, the former by activating protein kinase C in the cell membrane, and the latter by releasing calcium ions from intracellular storage sites. Activation of phospholipase C is also coupled with the opening of calcium channels in the cell wall and this provides more calcium ions to initiate and maintain the cellular response. Selected extracellular mediators and their associated G-proteins, effector enzymes and second messengers are listed in table VII to illustrate the versatility of this important process. ,B-Agonists that are analogues of epinephrine activate the ,B-receptor enzyme in the same way as epinephrine but, because the agonists vary in struc-

ture, each agonist-receptor enzyme complex is unique. The active centre of each is therefore more or less perfectly formed and consequently may process Gs-proteins at different rates. It is this rate which determines the efficacy of each ,B-agonist. The concept of efficacy as the intrinsic stimulating capacity of an agonist was first introduced by Stephenson (1 95U). The efficacy of the i3-agonist, which may have any value from zero to a large number, is the first determinant of a ,B-adrenergic stimulus. The second determinant of the stimulus is the average life ofthe ,B-agonist-receptor enzyme complex (Jack 1977, 1989). This is determined by the average duration of receptor occupancy by the {3agonist and this, in tum, by its chemical affinity for its receptors. I consider these receptors to be small assemblies of chemical groupings on the outer surfaces of the receptor enzymes. Affinity may be expressed as .:lG, the free energy change associated with the formation ofthe agonist-receptor enzyme complex or a function of it. Increasing .:lG values increases the stimulus. In summary, the stimulus delivered by a {3agonist to a cell under steady-state conditions is determined by its concentration, and by the product of its efficacy and its affinity for its receptors because these determine the number of active enzyme complexes in each affected cell, their qualitative nature and their persistence. Partial agonists are drugs whose efficacy is so low that the stimulus delivered to a cell is insufficient to provoke a full response even when they occupy all their receptors. Pure ,B-blockers disorder their receptor enzymes and have zero efficacy; their potency is determined only by their affinities for their receptors. These generalisations were derived from data such as those presented in table III. The potencies of nonselective catecholamines and of i32-selective agonists clearly increase as their affinities for their receptors increase. Affinity increases with the size of the N-substituents because the larger molecules undergo additional interactions with the receptor enzyme each of which increases .:lG. The more stable the agonist-receptor enzyme complex the more efficiently it processes G-proteins. It is also clear that the larger molecules tend to

12

Drug Safety 5 (Suppi. 1) 1990

Table VI. Cells and mediators involved in the pathology of bronchial asthma Type of cell

Mediators released

Stimulated by

Inhibited by

Acetylcholine, (vagal reflex), histamine, 1'12-Adrenoceptor stimulants leukotrienes, thromboxane A2,

Bronchial smooth muscle

prostaglandin Fa, prostaglandin 02 Mast cells

Histamine, serotonin

Allergen

1'12-Adrenoceptor stimulants

Anti-inflammatory steroids

(S-hydroxytryptamine), leukotrienes, prostaglandin 02, thromboxane A2 eosinophil chemotactic factor, neutrophil chemotactic factor, platelet-activating factor Basophil

Histamine, prostaglandins, platelet activating factor

Allergen

Eosinophil

Leukotriene B4, thromboxane A2, prostaglandins, platelet-activating factor, major basic protein, proteolytic enzymes, peroxidase

Leukotriene B4, lipopolysaccharides, Anti-inflammatory steroids immune complexes, complement factors

Neutrophil

Leukotriene B4, prostaglandins, thromboxane A2, major basic protein, proteolytic enzymes, peroxidase

Leukotriene B4, lipopolysaccharides, Anti-inflammatory steroids immune complexes, complement factors

Alveolar

Leukotriene B4, leukotriene C4, prostaglandin 02, thromboxane A2, platelet-activating factor, proteolytic enzymes, complement factors

Allergen immune complexes

Anti-inflammatory steroids

Monocyte

Leukotriene B4, leukotriene C4, cytokines

Platelet-activating factor

Anti-inflammatory steroids

Platelet

Thromboxane A2, prostaglandins, serotonin

Platelet-activating factor

Thromboxane A2 antagonist?

macrophage

be selectively active at ,82-receptors. This effect does not result simply from relatively greater chemical affinities for ,82-receptors because with salbutamol, for example, the ,82 :,81 affinity ratio is only about 2 whereas its ,82 :,81 potency ratio is at least 500. The efficacy of salbutamol must therefore be much greater at ,82- than at ,81-receptors and this means that its ,81- and ,82-receptor-enzyme complexes and, therefore, the ,81- and ,82-receptor enzymes themselves are chemically different.

Ahlquist's use of simple methyl epinephrine analogues to distinguish a- and ,8-receptor enzymes for epinephrine is identical in principle to the above. His small molecules were sufficiently powerful probes to detect the relatively large differences between these proteins but bigger and more

complicated analogues of a mediator are usually needed to detect smaller differences between its receptor enzymes in different kinds of cells. I find it helpful to regard the physiological mediator as a skeleton key, that fits all the members of a family of more or less related receptor enzymes, from which more complex keys that fit only one or some of them can be made. The consequences of the application of this process to serotonin (5-hydroxytryptamine) and its receptors are described later. 1.6 Salmaterol Intrinsic to the proposed mechanism for ,8agonism is the supposition that ,8-agonist-receptor enzyme complexes process G protein molecules for

Safety by Design

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Epinephrine

Activated receptor enzyme

+GTP -GDP

Activated adenyl ate cyclase

ATP

cAMP

GDP Fig. 4. Diagram showing how epinephrine (adrenaline) activates its receptor enzyme to convert a trimeric Gs-protein into ll's-GTP units, which, in turn, activate adenylate cyclase, the effector enzyme. The effect of epinephrine terminates when it disengages from its receptors and that of ll's-GTP by similar disengagement and by hydrolysis to ll's-GDP by virtue of its intrinsic GTPase activity. While they exist, each epinephrine receptor enzyme complex and each ll's-GTP effector enzyme complex processes several of its substrate molecules. A very low concentration of the extracellular mediator, therefore, gives rise to a much greater concentration of cAMP within the cell. The Gs-protein is represented by ~

as long as they exist. Accordingly, in 1981 we decided to seek a selective {j2-stimulant that formed a stable complex with the receptor enzyme because it should be efficacious for a long time and, given by inhalation to avoid systemic {j2-stimulation, might remedy the only major flaw in salbutamol. The outcome of the resultant project in the form of salmaterol has exceeded our initial hopes. How salmaterol was derived from salbutamol is illustrated in figure 5. The chemists at Glaxo replaced its squat N-t-methyl substituent by flexible nonpolar groups of varying lengths in the hope of finding one that would bind firmly to a nonpolar

area of the {j2-receptor enzyme accessible to the epinephrine receptor and thereby hold the active phenylethanolamine head of the new drug in close proximity to that receptor. Such a drug would, of course, have unusually high affinity for its receptor. In the event, the optimum N-substituent proved to be the phenylbutoxyhexyl group which is present in salmaterol. Its oxygen atom is critical for prolonged activity possibly because it has a directing influence on the binding of the rest of the N-substituent. As shown in table III, salmaterol has the predicted high affinity for its {j2-receptors and is se-

Drug Safety 5 (Suppl. J) 1990

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Table VII. Selected extracellular physiological mediators and their corresponding effector enzymes and intracellular mediators Membrane mediator

Extracellular mediator Epinephrine (iJ-effects) Histamine (H2-effects) Serotonin (S4 effects ?) Adrenocorticotrophic hormone (ACTH) Luteinising hormone (LH) Luteinising hormone releasing hormone (LHRH) Follicle stimulating hormone (FSH)

O';-GTP

Epinephrine (a2-effects) Somatostatin Epinephrine (a,-effects) Acetylcholine (muscarinic effects) Histamine (H,-effects) Serotonin (SHT2-effects)

lectively active at them. It has a uniquely persistent action on pharmacological preparations that contain Ih-receptors. For example, its relaxant effect on electrically stimulated guinea-pig trachea, which is illustrated in figure 6, shows no sign of waning even after many hours of washing with drug-free Kreb's solution. Under these conditions, this preparation recovers from the effects of isoprenaline or salbutamol within a few minutes. HOCH2

HO

Intracellular mediator

Adenylate cyclase

Cyclic AMP

Adenylate cyclase (inhibition)

None

Phospholipase C

Diacylglycerol (in cell membrane) Inositol triphosphate Calcium ions

The slow onset of action of salmaterol on the trachea is attributable to the long time such a large flexible molecule needs to fit at its ultimate site in the receptor enzyme. When it does succeed, however, a profound conformational change has occurred and the resultant enzyme complex is stable. This is thought to be the cause of its persistent actions (Brittain et al. 1988). Salmaterol also has a persistent inhibitory effect

OH

-@-~HCH'_NH;- ------------------~j ~-----------------_/

a

"

HOCH2

HO b

Effector enzyme

OH

~lHCH'NH ~H,cH,cH,cH,cH,cH,OCH,CH,cH,cH, -



Salmaterol

Fig. 5. Rationale for and structure of salmaterol. (8) Postulated structural requirements for a long acting Ih-adrenoceptor stimulant; I = active phenylethanolamine head to engage the physiological tl2-receptor; II = large nonpolar grouping to ensure tl2-selectivity and to anchor the whole molecule by interaction with an accessible nonpolar region of the receptor enzyme. (b) Structure of salmaterol.

Safety by Design

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7h ........AJ........A"A.,A.,.II,.,A..A..J........AJ........A..A.,A.,.II,.,A..A..J........AJ...A.A..A.A..J........AJ • •••••

....u

U

1 ~ J

I

I 10 min

Salmaterot 10 nmol/L

] lIillLdl II I1111 IIJ c:::J

Isoprenaline 30 nmot/L

Salbutamot 100 nmot/L

F'1g. 6. The effects of salmaterol, isoprenaline and salbutamol on electrically stimulated guinea-pig isolated tracheal muscle.

on the release of histamine and other proinflammatory mediators from allergen-challenged sensitised human lung. For the first time, therefore, this drug offers the possibility of a therapeutic benefit resulting from continuous inhibition of mast cell function in the lungs of asthmatic patients in addition to prolonged bronchodilatation. In patients, single doses of 50J,Lg of salmaterol by inhalation induce intense bronchodilatation for more than 12 hours and do not cause significant systemic side effects (Ullman & Svedmyr 1988). More importantly, 50J,Lg taken night and morning gave much better control of asthma and especially nocturnal attacks than 200J,Lg of salbutamol taken 4 times daily in double-blind studies involving hundreds of patients and many months of treatment. No evidence of pharmacological tolerance to the drug was found. Salmaterol is an exciting new drug which may make a substantial change in the treatment of bronchial asthma. It is also a good example of greater efficacy and possibly of safety by design, even after the contribution of serendipity is gratefully acknowledged.

2. Drugs Based on Histamine Histamine resembles epinephrine in having many pharmacological actions and in the broad correspondence of its HI- and H2-receptors with al- and tt-adrenoceptors. In each case, the effector enzyme for the first is a phospholipase C and for the second, adenylate cyclase. The experimental basis for the histamine H I/H2-receptor classification is summarised in table VIII and it is noteworthy that Black et al. (1972) used simple methylhistamines in their first differentiation of the receptors. Histamine differs from epinephrine in that few of its pharmacological actions correspond with established physiological roles. That it is an important mediator in allergic disorders was confirmed by the clinical effectiveness of mepyramine and other early HI-antagonists which were developed by Bovet and others in the 1940s. Similarly, after more than 50 years of argument, the role of histamine as a physiological mediator of acid secretion by parietal cells was finally established by Black and colleagues in 1972 and the critical evidence

Drug Safety 5 (Suppl. 1) 1990

16

Table VIII. Pharmacological classification of histamine receptors (after Black et al. 1972) Histamine H,-receptors

Histamine H2-receptors

Selective agonist

2-Methylhistamine

4-Methylhistamine

Selective antagonist

Mepyramine

Burimamide

Biological responses

Contraction of smooth muscle in the lungs and gut

Relaxation of uterine muscle Stimulation of heart muscle Stimulation of gastric acid secretion

\

¥

~

Relaxation of smooth muscle in small blood vessels

was that burimamide, the first selective H2-antagonist, inhibited acid secretion induced by food as well as that induced by histamine or pentagastrin. Physiological roles for histamine at other peripheral sites remain unproven because, apart perhaps from sedation with HI-antagonists, none of the side effects of HI- or H2-antagonists is attributable to blockade of histamine receptors. It is too early to say whether the localised distribution of histamine in the brain reflects physiological function. 2.1 Histamine Antagonists Specific histamine HI- and H2-antagonists, pioneered by Bovet and Black, respectively, were both milestones in medical practice. The former transformed the treatment of allergic disorders and the latter, with the introduction of cimetidine in 1976, provided the first reliable means for healing peptic ulcers. How the selectivity of action and the safety of the HI- and H2-antagonists has been improved is shown in tables IX and X, respectively. In each case, real improvement was possible because side effects were caused by blockade of cellular receptors other than the target receptors. Terfenadine (Sorkin & Heel 1985) and astemizole (Richards et al. 1984) are rapidly becoming market leaders because they do not pass the blood-brain barrier and are safer because they are non sedative. Similarly, it was the cleaner, safer action of ranitidine which enabled it to displace cimetidine as the world's bestselling medicine. In particular, unlike cimetidine, it does not inhibit the hepatic cytochrome P450

mixed function oxygenase enzyme system and does not, therefore, potentiate the toxicity of drugs such as theophylline, phenytoin and warfarin which are normally inactivated by that enzyme. 2.2 Possible Improvement of Ranitidine Recommended therapeutic doses of ranitidine and similar competitive H2-antagonists are designed to inhibit acid secretion during the night and to allow essentially normal acid secretion during much of the day in order to avoid bacterial growth and the formation of mutagenic N-nitroso compounds in the stomach. These doses heal about 80% of ulcers after 4 weeks and more than 90% after 8 weeks. Maintenance dosage, which is usually half of the therapeutic dose, reduces the annual relapse rate to about 20% compared with about 75% with placebo treatment. Ordinary antiulcer dosage, however, is inadequate to provide the 24-hour control of acid secretion needed to relieve pain and heal oesophageal lesions in severe reflux oesophagitis. Accordingly, we decided to look for an H2-antagonist with a more intense longer action to treat this disease and the even more difficult Zollinger-Ellison syndrome. Loxtidine was ultimately chosen for development because it has a highly specific H2-blocking action, is about 10 times more active than ranitidine, but differs from it in being an insurmountable antagonist and very long acting (Brittain & Jack 1983). The first results in human volunteers

Safety by Design

17

Table IX. From mepyramine to modern histamine Hl-antagonists Comments

Antagonist Mepyramine

Chlorpheniramine

Imperfect selectivity of Hl-blockade, side effects include anti muscarinic activity, sedative, short acting competitive blocker

Better selectivity of Hl-blockade, sedative, medium duration competitive blocker

Selective Hl-blockade, non-sedative, long duration unsurmountable blockers

Table X. From burimamide to ranitidine Antagonist

Comments Weakly active, poorly absorbed from the gastrointestinal tract

Active by mouth, clinically effective in peptic ulcer disease, caused agranulocytosis

N

:9J

l.

N H

N-eN

II

CH,-S-eH,CH,-NH-e-NHCH, CH,

Cimetidine

Active by mouth, clinically effective, well tolerated apart from inhibition of hepatic cytochrome P450 and antiandrogenic action

Active by mouth, clinically effective, well tolerated

(table XI) were encouraging because the drug was long acting and seemed to have the potential to inhibit acid secretion to any desired level. Unfortunately, not every pharmacological advance enhances safety, and work on loxtidine was abandoned when it was found to cause malignant

carcinoid tumours in the fundus of the stomach in the mouse and rat after 2 years' dosage. The malignancy is almost certainly secondary to unremitting achlorhydria induced by the large doses used in the toxicity tests and there is considerable evidence that hypergastrinaemia facilitates tumour development

Drug Safety 5 (Suppl. 1) 1990

18

Table XI. Inhibitory effects in 4 human volunteers of 7 daily doses of loxtidine given at 9am on acid secretion stimulated by intermittent l-hour infusions of pentagastrin (Glaxo Group Research data) Percentage inhibition of acid secretion

Loxtidine dose (mg)

20

40

period after dosing on day 1

period after dosing on day 7

3 to 4 hours

11 to 12 hours

23 to 24 hours

Mean (range)

98 (94-100)

54 (39-73)

26 (19-29)

Mean

100

80 (70-90)

64 (46-83)

(range)

because gastrin is a known trophic factor for enterochromaffin-like cells. The development of malignancy, regardless of its cause, was sufficient reason for us to abandon loxtidine because we thought it wrong to use a potential carcinogen to treat reflux oesophagitis, an essentially benign condition, which could be controlled by increasing the dosage of known safe H2-antagonists. The only proper indications for a drug such as loxtidine appeared to be the ZollingerEllison syndrome, which is itself a malignant condition, and other serious conditions such as Barrett's oesophagus. None of the H2-antagonists reported in recent years is an obvious improvement on ranitidine or loxtidine. However, omeprazole is clearly different in that it inhibits acid secretion in animals and humans by irreversibly inhibiting parietal cell H+/K+ ATPase. Achlorhydria, an inevitable consequence of this mechanism of action occurs with therapeutic doses of the drug (Sharma et al. 1984). This is why it is so effective in reflux oesophagitis and the Zollinger-Ellison syndrome. Unfortunately, since it increases the levels of nitroso-compounds in the stomach of treated patients (Wormsley 1988) and induces carcinoid tumours in the rat stomach after prolonged administration, it is unlikely to be safer than loxtidine. Only time will tell if drugs of this kind will be safe in ordinary use. Other safe means of inhibiting gastric acid are still being sought and gastrin antagonism is an attractive mechanism. Carcinoid formation might be avoided and concurrent inhibition of pepsinogen

23 to 24 hours

54 (42-62)

and acid secretion might provide better treatment for all acid-aggravated diseases.

3. Drugs Based on Serotonin Serotonin, like epinephrine, is a physiological mediator at many sites but this role does not involve its general distribution in the body. It is a nerve transmitter in the brain and the gastrointestinal tract and it is released from aggregating platelets and promotes further aggregation of them. Serotonin released from platelets has also been implicated as a trigger for the migraine syndrome, and an ensuing relative deficiency of the amine in the blood is a suggested cause of the dilatation of cerebral vessels that occurs during the attack (Lance 1973).

A systematic investigation of serotonin and its pharmacological receptors was started in 1972 because of the possibility that drugs which mimicked or antagonised some of its actions might be therapeutically useful. The possible involvement of serotonin in migraine also encouraged us to proceed. Apart from a possible aetiological role, infusion of serotonin itself had been reported to relieve the acute pain of the disease but also to cause intolerable side effects (Kimball et al. 1960); the useful but toxic ergot alkaloids were known to antagonise the vasoconstricting effect of serotonin. By 1976 Patrick Humphrey and his colleagues had identified 4 distinct pharmacological receptors for serotonin which they called S I, S2, S3 and S4. This classification and the recent internationally

Safety by Design

19

agreed classification of serotonin receptors (Bradley et al. 1986) are shown in table XII together with the key test preparations, agonists and antagonists. Further subdivision of functional serotonin receptors will almost certainly be required but, in my view, no attention should be paid to 'receptors' identified solely by ligand binding studies because they only complicate the situation. The Glaxo project has already identified 2 new kinds of drugs and more are likely. Ondansetron (GR 38032) is a highly selective 5HT3 (serotonin S3) antagonist, and sumatriptan (GR 43175) is a 5HTI-like agonist (serotonin S2-type) [see table XII]. The chemical structures of these 5HT analogues are shown in figure 7. 3.1 Ondansetron Michael Tyers and his team (1989) have found ondansetron to have a number of pharmacological effects in animals which indicate possible therapeutic uses in man. It is presently being tested in anxiety states because it relieves induced anxiety in the rat and primates, and in schizophrenia because it prevents the peculiar hyperactivity induced by infusion of dopamine into the midbrain of the rat. In the latter test, haloperidol and other neuroleptic agents are also effective but only ondansetron does not impair the normal activity of

the animals. A further use, at least as important, may be to control withdrawal of drugs of abuse such as alcohol and benzodiazepines (Costall et al. in press). The only use of ondansetron that is so far fully established in man is as an antiemetic in cancer patients being treated with cytotoxic agents or irradiation. The beneficial effect results from blockade of 5HT3 receptors in sensory vagal nerve endings in the gut and in nerve cells in the area postrema (Higgins et al. 1989; Stables et al. 1987). The latter has been shown by in vivo specific ligand binding studies to contain many 5HT3 receptors (Kilpatrick et al. 1987). Recently, metoclopramide, the drug currently most used to control vomiting during cancer chemotherapy, has also been shown to act by 5HT3 receptor blockade. Its blocking action at these receptors is, however, at least 40 times less than that at dopamine DA2 receptors and so effective doses of the drug cause severe Parkinsonian side effects, especially in young patients. Ondansetron is a better antiemetic with fewer adverse effects (Marty 1989; Priestman & Priestman 1989; Schmoll 1989). 3.2 Sumatriptan The discovery and the general properties of sumatriptan have already been described (Humphrey et al. 1989). For the present it is enough to say that,

Table XII. Pharmacological classification of serotonin (5-hydroxytryptamine) receptors (after Humphrey, personal communication; Bradley et al. 1986) Subtype of serotonin receptor

Test preparation

Selective agonist

Selective antagonist

Dog saphenous vein (contraction)

GR 43175 5-Carboxamidotryptamine

Methiotepin (poorly selective)

Cat saphenous vein (relaxation)

5-Carboxamidotryptamine

Methiotepin (poorly selective)

S,

Rabbit aorta (contraction) Platelets (aggregation)

a-Methyl-5HT

Ketanserin

S3

Rat vagus nerve (depolarisation)

2-Methyl-5HT

GR 38032

Original Glaxo nomenclature

5HT,

5HT3

20

Drug Safety 5 (Suppl. I) 1990

o

~:::lOJ ~~~ I

CH3

a 200 160

N

120

Ondansetron

80 40 0 -40 b

200

-;R e....

160

1

Q)

u

HO ~CH2CH2NH2

~y)

Serotonin

H

c:

~ II)

'iii ~ ~

given by intravenous or subcutaneous injection or by mouth, the new drug has a unique ability to relieve established migraine syndrome or abort attacks (Doenicke et al. 1988). The basis for its therapeutic action appears to be selective constriction of cerebral vessels and, in particular, of the arteriovenous anastomoses in the cerebral circulation (Feniuk et al. 1989). The selective action of the drug on vascular resistance in the carotid bed in the anaesthetised dog is illustrated in figure 8. The therapeutic effect of ergot alkaloids in migraine may also depend on constriction of cerebral vessels but, unlike sumatriptan, they cause intense constriction of peripheral blood vessels which, if maintained, can cause gangrene. Sumatriptan, the result of pharmacological manipulation by Humphrey and colleagues, is a much better and very much safer drug than any ergot derivative and this was one of the starting objectives (Humphrey et al. 1989).

80

:; u

II)

os

> .!:::

Fig. 7. Chemical structures of serotonin, ondansetron and sumatriptan.

120

Q)

Ol

c: os

40 0

~

u

-40

c

200

I

1

I

10

I

100

1000

160 120 80 40 0 -40 I

1

I

10

I

100

1000

Sumatriptan (ltg/kg IV) Fig. 8. The effects of intravenous sumatriptan on vascular resistance in [a] carotid artery bed (0) and vertebral artery bed (e), [b] coronary artery bed (e) and carotid artery bed (0), and [e] total peripheral resistance (e) and carotid artery bed (0) in the anaesthetised dog.

Safety by Design

4. Generalisations and Conclusions Variation in the cellular receptor proteins, similar to that already demonstrated for epinephrine, histamine and serotonin, is a general phenomenon for non peptide physiological mediators. The receptor proteins for some peptide mediators such as endorphins are similarly varied because selectively acting agonists and antagonists have been found. However, no such selectivity has been found in analogues of somatostatin and some other peptide hormones; their receptor proteins may be uniform. It is the exact nature and distribution of the receptor proteins that determines whether selective drug activity is possible. The purpose of this concluding section is to identify the factors which determine the range of cellular responses that are attainable because they are the ultimate determinant of both efficacy and safety. 4.1 General Conditions for Safe Selective Drug Activity in Analogues of Physiological Mediators 1. The receptor proteins for the chosen mediator must vary in different kinds of cells. 2. The receptor proteins which mediate the desired effects must be located in different cells from those which mediate unwanted effects. 3. For clinically significant selective agonism an analogue of the mediator must be found which delivers a stimulus at the target cells at least 100 times greater than that at other cells. It must, therefore, have greater efficacy and, preferably, greater affinity for the target receptor enzyme. 4. For clinically useful, selective antagonism the affinity of the required analogue should be at least 100 times greater for its target receptors than for others.

4.2 The Range of Cellular Responses to Pharmacological Agonists In my view, cells respond to pharmacological stimulation in one of two general ways, called type I and type 2 agonism. Their distinguishing features are summarised below.

21

4.2.1 Type 1 Agonism The essential characteristics of type I agonism are: I. The affected cell is capable of responding continuously to a continuing stimulus. 2. Pharmacological tolerance does not occur with stimuli of physiological dimensions or, as a rule, with tolerated doses in man. 3. Increasing efficacy and increasing receptor affinity enhances the potency of agonists. 4. Prolonged agonism is achievable by formation of stable agonist-receptor protein complexes. S. Antagonists form inactive receptor protein complexes and their potency is determined solely by their affinities for their receptors. Type I agonism is believed to be the commonest mechanism whereby physiological mediators affect cells. If, for example, we consider the mediators dealt with in this paper it applies for all the responses to epinephrine, histamine and steroids and to the agonists derived from them, and to serotonin responses mediated by SHT,-like and SHT2receptors. It is also the rule for mediators such as prostanoids and leukotrienes. 4.2.2 Type 2 Agonism The essential characteristics of type 2 agonism are: I. The affected cell must recover after an effective stimulus before it can respond to another. 2. For a continuing cellular response, unnatural agonists must mimic the physiological activation process. 3. High efficacy and receptor affinity enhance the initial stimulus but the target cell is desensitised if the agonist persists at its receptors for longer than the physiological recovery time for the cell. 4. The recovery process involves the generation, by one means or another, of a sufficient number of free receptors to react with the agonist and create an effective stimulus. S. Two kinds of receptor blockers exist. The first is the same as a type I antagonist and has zero efficacy and relatively high affinity for its receptors. The second is an agonist with high affinity

22

Drug Safety 5 (Suppl. 1) 1990

which first stimulates and then desensitises as in the third characteristic above. The nicotinic actions of acetylcholine at the motor end-plate in skeletal muscle and in autonomic ganglia are the best known examples of type 2 agonism. The whole sequence of events takes only milliseconds. Other mediators which cause similar rapid localised depolarisation and firing of their target cells are also likely to be type 2 agonists. The action of serotonin at 5HT 3-receptors in vagal sensory nerve cells and the excitatory effects of amino acid transmitters in the central nervous system are obvious examples of this possibility. Type 2 agonism is not restricted to very short events because the intermittent stimulant action of lutein ising hormone releasing hormone (LHRH) on the anterior pituitary cells which secrete luteinising hormone (LH) is a clear example of it. LHRH is released from hypothalamic cells for about 5 to 10 minutes in a series of short pulses every 90 minutes or so. The cell responds by secreting LH, also over a short period, and then has to recover before it can respond again 90 minutes later. To be effective, hormone replacement treatment with LH in man has to mimic the physiological process. The first kind of antagonist in type 2 agonism simply forms an inactive complex with its receptor enzyme. d-Tubocurarine and other curari sing drugs are examples, and ondansetron may be another. The paralysing action of the depolarising blockers decamethonium and suxamethonium is the archetype for type 2 antagonism. Another recent important exam pie is the use of a parenteral sustained release formulation of goserelin acetate, a potent analogue of LHRH which is relatively stable in the body, first to stimulate and then, by the continuing presence of the drug, to inhibit secretion ofLH from the pituitary. The consequence is an equally effective but more acceptable alternative to castration for controlling prostatic cancer. 4.3 Conclusion

Because of the great increase in understanding during the past 25 years of how physiological mediators affect cells it is now possible to define the

limiting conditions for selectivity of action in analogues of physiological mediators and the range of drug effects attainable with them. The corresponding growth in the understanding of enzymes and their substrates has also led to many important new medicines and to general ways to manipulate false substrates and/or' inhibitors of particular mammalian or parasite-derived enzymes. These are, of course, the two great rational approaches to drug discovery and, with continuing research, they will undoubtedly yield better, safer medicines for the many ailments that are poorly controlled today. Other narrower but significant approaches, such as drug targeting by means of monoclonal antibodies, will also contribute to the safer use of drugs and so it is easy to be optimistic for safety by design in the future.

Acknowledgement The author thanks his colleagues, past and present, in Glaxo Research Limited for their help and friendship through the years and for their forbearance of his using their work, yet again, to illustrate his notions about pharmacological agonism and antagonism.

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Safety by Design

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Neer EJ, Clapham DE. Roles of G-protein subunits in transmembrane signalling. Nature 333: 129-134, 1988 Priestman TJ, Priestman SG. Studies with ondansetron in the management of radiation-induced emesis. Ondansetron Symposium (Glaxo), Queen Elizabeth" Conference Centre, london, June 29-30, pp. 34-37, 1989 Richards DM, Brodgen RN, Heel RC, Speight TM, Avery GS. Astemizole. A review of its pharmacodynamic properties and therapeutic efficacy. Drugs 28: 31-61, 1984 Schmoll HJ. The role of ondansetron in the treatment of emesis induced by non-cisplatin-containing chemotherapy regimens. Ondansetron Symposium (Glaxo), Queen Elizabeth" Conference Centre, London, June 29-30, pp. 39-47, 1989 Sharma BK, Walt RP, Pounder RE, Gomes MDA, Wood EC, Logan LH. Optimal dose of oral omeprazole for maximal 24 hour decrease of intragastric acidity. Gut 25: 957-964, 1984 Sorkin EM, Heel RC. Terfenadine. A review of its pharmacodynamic properties and therapeutic efficacy. Drugs 29: 34-56, 1985 Stables R, Andrews PLR, Baily HE, Costall B, Gunning SJ, et al. Antiemetic properties of the 5HT3-receptor antagonist, GR 38032F. Cancer Treatment Reviews 14: 333-336, 1987 Starke K. Alpha-sympathomimetic inhibition of adrenergic and cholinergic transmission in the rabbit heart. Naunyn-Schmiedeberg's Archives of Pharmacology 274: 18-45, 1972 Stephenson RP. A modification of receptor therapy. British Journal of Pharmacology II: 379-393, 1956 Tyers MB. The pharmacological and anti-emetic properties of ondansetron. Ondansetron Symposium (Glaxo), Queen Elizabeth " Conference Centre, London, June 29-30, pp. 18-23, 1989 Ullman A, Svedmyr N. Salmeterol, a new long acting inhaled beta2-adrenoceptor agonist: comparison with salbutamol in adult asthmatic patients. Thorax 43: 674-678, 1988 Wormsley KG. Risks of therapeutic achlorhydria. Scandinavian Journal of Gastroenterology 23 (Suppl. 153): 35-51, 1988

Author's address: D. Jack, 6 The Slype, Gustard Wood, Wheathampstead, Hertfordshire AL4 8RY, United Kingdom.