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How animals discover drugs

Sir John Vane FRS

Group R&D Director, The Wellcome foundation Ltd

 

I am particularly pleased to have been asked to give this year’s Stephen Paget memorial lecture. It comes at a time when the opponents of animal experimentation are literally beating a path to the front doors of experimentalists to make their views known.

 

I doubt that any of my predecessors have opened their lectures with a domestic snapshot, but I think what I have just said is best illustrated by my garage door. Figure 1 shows how it appeared one morning after a recent visit by some people who neglected to leave their names and addresses.

 

The remainder of my illustrations will, I hope convey more scientific messages than that one. However, I would like to preface the main part of my address with some thoughts on the question of whether reasoned debate- and I stress the word debate- is possible in this area. Certainly, the people who have attacked my home and those of a growing number of my colleagues have no interest in debate or they would not remain clandestine. Was the person who sent a letter bomb to Professor Roy Calne interested in debate? Or the ones who recently broke into the home of the Managing Director of The Wickham Toxicology Laboratory and floored him with an iron bar? I do not think so.

 

The criminal behaviour of militant activists is escalating around the country, perhaps encouraged over the past few months by the numbing brutalisation of society seen night after night on television screens as confrontation between pickets and police. The burning of buses, the stoning of police, the increase in damage to people and to machines seems to be becoming acceptable through incessant exposure. No wonder, in a climate of habituation to brutality the animal liberation front is able to violate and damage the laboratories and the homes of scientists with apparent impunity. Clearly values are changing and it is against a background of transition that those of us who believe in the necessity and rightness of animal experimentation need to keep arguing our case. In response to a recent letter of mine in The Times, the distinguished parliamentarian lord Houghton suggested that the current intensity of feeling in some quarters about the rights of animals was an extension of the general philosophical movement towards defining and extending ‘rights’.

 

‘Sir John hopes to counter the growing demands for stricter controls’ Lord Houghton wrote, ‘by presenting the impressive catalogue of benefits to man and animals from the use of living things in laboratories. He is appealing to reason, though more and more this is becoming a moral issue which is as little open to argument as the Pope’s stand on abortions.

 

‘What is happening is that human rights are becoming so fashionable and acceptable that they are spilling over to cover species other than ours’.[1]

Lord Houghton was not putting forward a very new idea. One has only to turn back the pages of Conquest, the Society’s journal, to find a decade ago Dr Harold Hillman - experimentalist and vegetarian — arguing that 'the advance of civilisations involves human beings becoming more sensitive and attempting to prevent all suf­fering . . . Nevertheless, as a research worker, I suspect that in a century's time we will still have many difficult questions, which can only be answered by sacrificing animals[2].

My main purpose tonight is to look at why Dr Hillman's suspicion that we will still need animal experiments in 2074 is likely to prove true. But I do want to spend a minute or two first on the philosophical problem. To return to Lord Houghton, he said 'Unless Sir John and his fellow researchers will meet the weight of responsible moderate opinion and accept controls and restrictions which will spare animals the worst excesses of pain and suffering, no matter for what purpose, they will encourage the accelera­tion of extremism'. Lord Houghton, animal experimentalists are humane and they always have accepted those controls and restrictions. We are not brutes. We shall also continue to look for ways of reducing the use of animals and increasing the use of alternatives. But unless the weight of responsible moderate opinion takes centre stage and distances itself from the extre­mists it will, by default, no longer be seen to want to enter into responsible and informed debate, through its implicit association with violence.

 

Over the ages, western man has relied on animals as workhorses, for transport, for clothing and for food. Ironically, it is only the scientific and technical progress of the last century, that has allowed a climate to develop in which the question of animal rights can be voiced.

 

And this includes our progress in medicine. Medicines available today have helped to ensure that life here in Britain is no longer in a state which Thomas Hobbes described as 'nasty, brutish and short' and has given people the life­span and leisure to concern themselves with problems which would have been seen by our forefathers as having the same relevance to life as that famous medieval debate about the num­ber of angels which can be accommodated on a pinhead.

 

Today's medicines are based upon the accumu­lation of knowledge of thousands of years from folklore, serendipity and science - including this century's explosion in knowledge of the bio­sciences. The medicines of tomorrow will depend upon experiments being done today. End animals experiments now and we shall lose the cures that we are entitled to expect in the next 50 years for illnesses that afflict hundreds of mil­lions of people such as cancer, heart disease, viral diseases, malaria, schistosomiasis and sickle cell anaemia.

 

It has become fashionable to belittle the achievements of the pharmaceutical industry. Animal 'liberators' often suggest that changes in lifestyle would be more effective in promoting health than our chemotherapeutic armament­arium. Often, they go further and suggest that chemotherapy actually promotes ill-health through adverse drug reactions etc. This, of course, is arrant nonsense. The Drug Surveillance Research Unit at SouthamptonUniversityhas recently calculated that the removal of all drugs from use would increase the average Briton's life expectancy by 37 minutes, due to abolition of side effects or adverse reactions. On the other hand, the resultant lack of therapy would decrease life expectancy by 20 years.[3]

 

I do not accept, and society cannot afford to accept, that we already have enough drugs, medi­cines and vaccines. Such an argument belies the continued advancement of scientific knowledge and the exciting discoveries yet to be made. It is like saying that the development of cars could have stopped with the Model T Ford because that provided adequate transport.

 

To continue with this analogy, there have been substantial improvements in design, efficiency and acceptability since the Model T Ford, but not without some hazards. Cars kill and maim many people (and animals) but we go on using them. Despite stringent quality control, some new models have been recalled because of unsuspec­ted faults. But we do not use this as an argument to stop the development of new cars. The same applies in the development of drugs. I hope, by example, to illustrate to you that the drugs of today are superior to the models that existed years ago. As with cars, there are unforeseen hazards that need to be addressed. However, just as invention, design, development, improvement through new scientific knowledge are all essen­tial ingredients for a modern car, so they are for a modern drug. And just as cars need the build­ing blocks of steel, plastics and glass so do drugs need building bricks, including the results of animal experimentation, in order to prove their safety and efficacy. I am going to take examples from three thera­peutic areas in order to try to convince you of this statement. The first is hypertension, or high blood pressure, the second is antiviral therapy and the third malaria.

 

Hypertension

High blood pressure is known as the silent killer for there are few observable symptoms before disaster strikes in the form of a heart attack or a stroke. It is only recognised when the doctor takes your blood pressure. Many factors contri­bute to hypertension, including stress, over­weight and diet but even when these are adequately controlled, a condition known as essential hypertension is still widely prevalent. This can be controlled by drugs and various studies have indicated that such treatment does prolong life. Before the development of drug treatment, surgical intervention was the only way to control blood pressure. Treatment, however, was available to few and did not have a high suc­cess rate.

In the late 1940s, WDM Paton and Eleanor J Zaimis at the National Institute for Medical Research in Londonwere studying the mode of action of curare, as were R B Barlow and H M Ing in Oxford[4]. Curare is widely known as the substance used by some South American tribes as a poison for their arrow heads. Its active com­ponent, d-tubocurarine, blocks the nerve impulse to skeletal muscles, thus inducing paralysis. TheOxford andLondon groups both studied simple analogues of tubocurarine, notably a series of substances containing two quaternary nitrogen compounds (Figure 2).

Figure 2 Formulae of methonium compounds

Barlow and Ing were concerned with the rela­tionship between curare-like activity and inter-quaternary distance - that is the distance between the two nitrogen atoms, which depends on the length of the carbon backbone separating them. They cut the diaphragm and its nerve from rats and measured contractions of the isolated tissue induced by nerve stimulation in a bath of salt solution, an in vitro system. They found that the nerve-muscle blocking activity was highest with decamethonium, the compound with ten carbon atoms separating the nitrogens.

 

As the carbon chain shortened, this blocking activity decreased sharply between nine and eight carbon atoms and was almost negligible for the six-carbon hexamethonium.

Paton and Zaimis were also interested in the general pharmacology of the methonium com­pounds. They did their tests in whole animals and found the same results. However, they noted that the smaller molecule hexamethonium caused a fall in blood pressure. This unexpected observation, which could come only from whole animal experiments, was shown to be the result of the compound blocking transmission of impulses across the ganglionic synapses - the peripheral relay stations through which all the activity of the sympathetic and parasympathetic nervous system passes. At the time, one known ganglion-blocking agent, tetraethylammonium, had been recently introduced into clinical prac­tice but was bedevilled by side effects - paresis, fibrillation and muscular paralysis.

 

Hexamethonium avoided most of these draw­backs and became the first successful drug treat­ment for hypertension. It has of course been superseded by better drugs, but I think its origins are worth recounting here because it opened up what A E Doyle called the beginning of the modern era of therapeutic enthusiasm in the management of this illness'[5]. It also shows how much important information may be missed by using in vitro techniques rather than whole animals.

 

Basically, our treatment of hypertension is symptomatic, because we do not know the under­lying cause. The immediate cause is increased peripheral vascular resistance and the effects of this can be countered in several ways. In addition to using substances which block the sympathetic nervous system, we can also relieve hypertension by acting on vascular function through the use of vasodilators, by acting on renal function through the use of diuretics, or by acting on the reninangiotension enzyme system.

 

It would take a lecture in itself to describe the development of all these over the past 30 years. So let me pass briefly over the introduction of hydralazine and reserpine in the early 1950s and the very important thiazide diuretics in the late 1950s, to pick up my theme again with the introduction of adrenergic blocking agents in1961.

 

The difficulty with ganglion blocking agents was their lack of selectivity. They blocked ganglia in both the sympathetic and parasympathetic nervous system, leading to unwanted side effects such as dry mouth. The next advance came from studying ways of improving selectivity towards the sympathetic nervous system which, when stimulated, generally increases vessel resistance. As a result, drugs such as bretylium, bethanidine and guanethidine were developed (Figure 3). These prevent the release of noradrenaline, the transmitter substance which is responsible for bringing about the vessel-shrinking effects of sympathetic stimulation. Literally hundreds of compounds were tested in animals. Bethanidine, for example, was the 85th out of nearly 300 guanidine derivatives examined at the Wellcome Research Laboratories.[6]

 

Activity in other parts of the pharmaceutical industry was no doubt as intense. James Black, then with IC1, tells of testing a simple analogue of guanethidine which appeared to have some advantages. ‘Mice, rats and cats tolerated larger doses of this analogue than of guanethidine,’ Black says.

 

Figure 3 Formulae of bretylium, guanethidine and bethanidine.

 

‘As a pilot experiment, two beagles were given 200mg/kg of the drug by mouth…Nothing untoward was seen for nearly 90 minutes. At this time, the animal attendant inspected the animals and was greeted, as usual, by the dogs jumping up in their pens, their tails wagging; but this time one of them sudden, moaned and, literally, dropped dead.[7]

 

The second dog dropped dead 20 minutes later. It turned out that ventricular fibrillation had been the cause, resulting in part from sensiti­zation of the heart muscle to circulating cate­cholamines.

 

The substance was abandoned, one of the many potential drugs whose safety - or lack of it - needed a whole animal to demonstrate.

 

James Black is, of course, associated with one of the most successful developments in hyper­tensive therapy based on modification of the nervous system, the beta-blockers. At the time, he was working on anaesthetised dogs to try to develop a drug which would reduce the heart's demand for oxygen and thereby reduce attacks of angina in people with coronary heart disease. Basing his work on R P Ahlquist's proposal that there are two distinct types of catecholamine receptor (called alpha and beta), Black investiga­ted a number of compounds, of which the best known is propranolol.

In this context, it is interesting that such drugs do not lower a normal blood pressure and that the antihypertensive effect in many is demon­strable only after several days of treatment. Thus, in the initial experiments on anaesthetised animals, the effect was not observed. It was first picked up by Brian Prichard at UCH when he was using pronethalol - a precursor of propranolol -clinically.[8]

Why then, you may ask, bother with animal experiments at all when an important effect can first be seen in man? The safety aspect demon­strated by Black's cautionary tale of the guanethi­dine analogue is one good reason. But there is also the wider principle that experiments on whole living organisms (whether animals or man) will always yield more important informa­tion than expected, especially when the right model of a disease entity is available. At this stage, it is worthwhile to digress briefly to catalogue the biological experiments which are required before a drug company (or a government) is satisfied with the safety and efficacy of a new drug. There are five stages which I will deal with in turn.

  1. Discovery

Discovery of a new activity depends upon testing the hypothesis. For some hypotheses it »S simple to design a test, for mammals and man have very similar autonomic nervous systems, relying on the same chemical transmitters. Thus, measuring in some way the results of stimulating sympathetic or parasympathetic or motor nerves will be a test of blocking agents. In the case of propranolol the hypothesis was that decreasing the workload of the heart would reduce anginal pain. The only relevant test was to measure the workload of the heart in a whole animal to find out whether it was reduced by propranolol, for anginal pain is not measurable in animals. An enzyme screen or an in vitro test on live cells to yield adequate information was not possible in 1960 and is still not possible today for this type of work. There are other areas, which I shall men­tion later, where such screens are being deve­loped and used.

Once a test has been devised, validated as well as possible and the desired activity has been detected in a test substance, the synthetic chemist prepares a series of analogues to maximize the activity. This may increase the potency of the potential drug hundreds if not thousands of times, thereby selecting for the activity required and hopefully reducing other pharmacological effects. Nowadays, the very powerful tool of computer graphics is increasingly used to help the chemist design new molecules to fit to the drug 'receptor' (Figure 4). As this technique develops, it will surely reduce the number of chemicals that need to be made and tested to achieve the opti­mum effect, thereby also reducing the number of animal experiments needed.

2. Secondary Evaluation

In the search for new drugs, we seek many dif­ferent interventions - vasodilators for vascular diseases, bronchodilators for asthma, anti­secretory drugs for duodenal ulcers, uterotropic substances and so on but a compound which has all these activities (like some of the prosta­glandins) does not make a useful drug. We have to have selectivity. For confirmation of selectivity many tests are required to show that the drug does not have an undesirable effect on other organs. Detection of central nervous activity is particularly important and the complexity of the whole organism is essential for this sorting out process.

3. Drug Metabolism

It is also necessary to know whether the drug is absorbed orally and how long it lasts in the body. At the same time, we need to know the route of metabolism to make sure [hat toxic or active metabolites are not formed. Some ancillary knowledge can be obtained by isolating enzymes from dead animals or dead people to find out if they produce modifications to the compound under study. Nevertheless, full animal experi­ments are necessary in order to see what hap­pens to systems which may be quite distinct from those under study. Let me give you an example from recent work in our laboratories.

For several years, we have been working on a novel anti-epileptic compound, which is now-ready for multicentre clinical trials. The com­pound is a complex organic molecule, with a triazine ring structure. Three years ago, we were carrying out animal tests to check, before going into clinical trials, that the compound had no adverse effects on the cardiovascular system. These tests were done in several species. In the

dog, there were changes in the ECG after a few-hours which indicated a delay in the spread of the contraction of the heart muscle with each beat.

 

The time delay between administration of the test substance and the ECG effect indicated the formation of an active metabolite which we had not found in the other test species. We identified and synthesized the metabolite - it was the original compound with just one of its triazine nitrogen atoms alkylated. When that was admini­stered to our other test species, it produced the same ECG effect. Further study showed that the other species do in fact produce some of this compound, but only a- very small amount. The balance of liver enzymes which can react with this compound in the dog favours this particular metabolite in a way not found in other species, including some human volunteers.

 

I have related this incident not just to provide a further example of my principle of always learning more than we expected. This example goes beyond that, for one simple reason. A very close analogue of the metabolite we isolated from the dog is about to go into human volun­teer studies as a potential anti-arrhythmic agent. Heart arrhythmias are a common problem and can, as in the case of ventricular tachycardia, endanger life. Had we been looking for an anti­arrhythmic, we might have suspected that anti-epileptic activity would be a pointer. In both cases, therapy depends on altering electrical activity in part of the organism. However, our anti-epileptic, as is implicit in what I have said already, does not have anti-arrhythmic activity and nor is the metabolite important in man. Had we decided, on empirical grounds, to try to induce such activity, we would have had a large number of sites on the molecule which could be chemically modified in order to change the bio­logical activity. Without a hypothesis in favour of one site, the whole exercise would have been too expensive and time-consuming to bother with.

 

On the other hand, presented by our experi­mental animals with an active metabolite, we were able to target a little chemistry around that and come up with a potential new drug. There is another twist to this story, which may already have occurred to you. Alkylation of a triazine nitrogen changes the basicity of the molecule significantly; as a result, it will no longer cross the blood-brain barrier.

Had it still crossed this barrier - which, of course, its precursor must do to have an anti­epileptic effect — then it might have shown the disadvantages of existing anti-arrhythmics, such as lignocaine, which enter the brain and produce undesirable side effects. As it is, we may - and I stress that there are still hurdles at which this compound could fall - have discovered in an extraordinarily short time a new drug. More than any other example in my experience, I think this shows that animals can be said to discover drugs. Without the use of a whole animal in test­ing the related anti-epileptic, there is no reason to believe that men would have stumbled on this compound in the foreseeable future.

 

4. Safety

The safety of drugs is a prime concern and remains under public-scrutiny after unexpected tragedies such as thalidomide and more recently benoxaprofen. When unexpected toxicity occurs in man, there is a strong and vigorous response from government and from industry to prevent it happening again. New standards are quite rightly devised, always depending on animal tests. It is ironic that some of the opponents of animal experimentation cite thalidomide as an argu­ment against the use of animals. Interestingly, the Sunday Times Insight team concluded that a major cause of that tragedy was insufficient animal experimentation.[9]

In case you believe that our safety testing is lax, let me enumerate those tests which are at present required by sophisticated regulatory authorities in countries such as theUKand theUSA(Table 1). These range from the in vitro Ames test on bacteria through to life-time studies requiring daily dosage of the new substance to rodents over two or three years. The safety tests required depend upon the type of drug. A sub­stance which you are likely to receive just once, say as an adjunct to anaesthesia at an operation, clearly requires less safety testing than an anti­hypertensive pill which you may take three times a day for the rest of your life.

 

Of course there is a debate between govern­ments and industry with respect to the magni­tude of these safety tests. Some, including myself, would say that groups of animals dosed for six months on a daily basis will give you as much practical information as dosing the same groups for two or three years. However, there is no debate as to the need for this type of safety test. Thalidomide showed that safety tests then in general use did not adequately address the effects of drugs on the unborn. Since then, addi­tional methods have been devised to cover this deficiency. It is, however, of no value to decry all safety testing in animals because of one unexpec­ted tragedy. Safety tests, as part of the process of drug discovery, have filtered out many hundreds of toxic compounds before they have ever reached man. Recently, an MP suggested to me (perhaps with his tongue in his cheek) chat we should call for volunteers from amongst the animal rightists to replace the animals used in these tests, but this would be irresponsible and unethical, for I believe that the life of man is more important than that of animals.

 

5. Experiments in Man

All of the animals tests so far described are used to predict the safety and efficacy of the substance in man. When the clinicians concerned are satis­fied with these predictions, the new compound is given carefully and under very strictly controlled conditions to volunteers starting with an almost homoeopathic amount and working upwards (if still safe) to a dose which gives the expected therapeutic concentration. It is in these experi­ments that the predictability of the animal experimentation begins to be rigorously tested.

As with all experiments, those in man some­times give unexpected results. The life of the drug in the body of man may be different from that in the body of animals. The absorption characteristics may also be different. Some further potential drugs are eliminated because of problems in this phase but others come through and then enter rigorous double blind clinical trials in order to test the efficacy of the sub­stance. This clinical testing can take up to five years but eventually, if the researchers, deve­lopers and clinicians are all satisfied with the results, a bulky dossier is composed and submit­ted to the regulatory authorities. Scrutiny of this dossier may take a further three years before the government concerned is satisfied with the results and there is often an ongoing dialogue between government and company, sometimes leading to further animal experiments.

 

I would now like to return to the anti­hypertensive story briefly, primarily to illustrate that progress did not stop with the introduction of the beta-blockers in the late 1960s.

One of the claims made against the pharmaceuti­cal industry is that we waste countless animals by developing 'me too' drugs. Such claims are frequently made by committed individuals who do not like to be confused with facts. If there is a drug which can treat hypertension, they say, why waste a single animal life trying to find another? Well, my next example will show that drug discovery can sometimes arise from animal experiments conducted in academia for the advancement of science. In 1965, when I was at the Royal College of Surgeons inLondon, a Brazilian - Sergio Ferreira — came to my labora­tory on a post-doctoral fellowship. He brought with him a venom preparation from a Brazilian snake called Bothrops jararaca, which had been shown by Professor Rocha e Silva to be capable of generating the potent vasodilator peptide bradykinin. At the time, I was studying the intri­cacies of the renin-angiotensin system and suggested to Ferreira that he should look at the effects of his snake venom on the different enzymes involved. In fact, he turned the tables on me and persuaded me to join him in his work on bradykinin and we made some interesting dis­coveries there. It was only later that another of my colleagues looked at the effects of the snake venom on angiotensin-converting enzyme.

 

As you probably know, angiotensin-converting enzyme changes the relatively inactive deca-peptide angiotensin I into the much more potent vasoconstricting substance angiotensin II. My colleague Dr Mick Bahkle found that the snake venom was a potent inhibitor of the enzyme and could thus be a useful tool to find out whether the renin-angiotensin system was important in high blood pressure.

 

At the time, I was a consultant to the drug firm Squibb and, after much persuasion, managed to get them to work in this area. They isolated and purified a nonapeptide (Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro) and developed it to a stage where the hypothesis could be tested in man.[10] As a result of this programme, they went on to develop the orally acting drug Captopril. Subsequently a number of pharmaceutical companies have shown keen interest in developing similar sub­stances for anti-hypertensive use. The search is by no means over and this is possibly one of the most competitive areas in drug design at present.

 

I do not think any reasonable person could call anti-hypertensives based on the renin-angiotensin system 'me too' drugs by comparison with the beta-blockers. To return again to my analogy of cars, it would be the same as calling the car a 'me too' railway train.

 

The focus of research interest in anti­hypertensives is now turning to renin inhibitors and to the calcium channel blockers, yet another method of tackling this widespread condition. So, for over 30 years, we have had a succession of therapeutic developments all springing, in a sense, from Bill Paton's choice of the whole animal rather than an in vitro system for his-work on the methonium compounds.

In elaborating in some detail on the develop­ment of anti-hypertensive drugs, I believe I have illustrated several points.

1. Although in vitro systems are useful and have been used both as crude and refined instruments, they cannot be relied upon now to predict anti-hypertensive activity. Even organs isolated from whole animals are insufficient. The only way now and in the foreseeable future to test for hypertensive activity is in an animal model.

2. Each of the classes of drug I have illustrated is built upon the knowledge obtained from the previous one but each pharmacological intervention has been singular in its method of attack. Many different systems can contribute to normality of blood pressure, or homoeostasis. The body never relies simply on one control mechanism so when things go wrong, there are many avenues to explore. It is also interesting that poisons from South America, one from a plant and one from a snake, have twice given leads to important new antihypertensive drugs in a totally unexpected way. As Comroe and Dripps have pointed from their painstaking and erudite study on the scientific endeavour that leads to a major advance in medicine,[11] there is a whole pyramid of enabling work essential to provide the overall knowledge for such a major advance, which is often attributed to one man. Of the key work in this pyramid more than half is 'basic' physiology, pharmacology, etc and more than two thirds takes place in Universi­ties. Our knowledge of the underlying mechanisms of hypertension and of many other diseases is increasing daily through basic studies in academia and industry using animal experimentation. New pharmaco­logical interventions are at present being evaluated which, if successful, will lead to important new anti-hypertensive drugs with different types of action in the next ten years.

3. In the examples used so far, the use of animal models relies upon similarities in the physio­logical organisation of animals and man. Common laboratory animals and man all have kidneys, hearts, lungs, brains, muscles, intestines and so on. This motley array of organs is also orchestrated in the same way by similar hormonal and nervous systems and it is the pharmacological intervention with these hormonal and nervous systems which allows us to try to correct with drugs elements of the system which go wrong.

 

Viruses and vaccines

Hypertension is a disease which has attracted the derogation of some sections of the community as a 'lifestyle' disease, a disease which could be eliminated if we all changed our habits and would thus not require drugs. The arguments for this viewpoint lack rigour, but that of course does not make them of less value in the eyes of the animal liberators. Any half-truth or over­simplification seems to be grist to their mill -provided it supports their case. Nevertheless, if one studies the anti-medical literature of today -and there is more than enough of it about - it usually concedes that the pharmaceutical indus­try has made a contribution in one area, the control of bacterial infections.

Within a few years, we could have added to that the control of viral infections. As you may know, my company, Wellcome, was responsible for introducing acyclovir, a molecule which acts in a very specific way against the herpes viruses.

In the past, the problem with tackling viral diseases has been the way in which a virus mobilises the biochemical machinery of its host for its own ends. To attack the virus without attacking the host has been an intractable prob­lem until recently - except by the use of vaccines, to which I shall return shortly.

A brief explanation of how acyclovir (Figure 5) works may indicate the level of sophistication we need to achieve in this field. The drug probably needs a diffusion gradient to cross cell mem­branes. As it is unchanged by uninfected cells, the gradient rapidly disappears. In a herpes infec­ted cell, however, acyclovir is phosphorylated by an enzyme specific to the virus. This prevents the new substance from escaping, but also maintains the gradient, so that more acyclovir enters the infected cell. Uptake is about 3,000 times higher in infected than uninfected cells. This combina­tion of phosphorylation and accumulation in the virally invaded cells gives acyclovir a unique and highly selective toxicity to the herpes virus. .

When viral DNA replicates, the phosphoryla­ted acyclovir substitutes for deoxyguanosine. Because of its acyclic structure, this brings the growing DNA chain to a halt and viral replication is inhibited.

The initial indication that acyclovir might be a useful antiviral agent came from tests in cell cul­ture. Our ability to grow isolated cells in culture has improved enormously in recent years. In some respects, they make good test systems. They are a lot less expensive to keep than whole animals, yet they are still capable of significant metabolic activity.

The problem is that they are not so complex as whole animals, and this can have two dis­advantages. The first is that a compound which works in an isolated cell may not work in the body. It may be converted to an inactive metabolite before it reaches the infected cells. Alterna­tively, it may never reach them. Given the huge number of enzymes throughout the body, we were fortunate that the initial phosphorylation of acyclovir could not be carried out by any of them. Getting a drug to its target is one of the major hurdles that any pharmaceutical company has to face. Secondly, compounds which do not work in cell culture may work in whole animals through being metabolised to active compounds. I am sure that I do not need to remind you that a sub­stantial part of the revolution in antibacterial therapy can be traced back to Prontosil, a sub­stance inactive by itself, but converted to an active antibacterial by mammalian metabolism.

The other way in which we have tackled the problem of viral disease is through vaccination -a term which, of itself, indicates our dependence on animals. The word is derived from the Latin for 'cow', for reasons with which I am sure you are all familiar. When vaccination was first introduced, it had its opponents, but at least James Gillray used humour as his weapon, not the iron bars and bombs preferred by some of today's animal activists (Figure 6). The value of viral vaccines is evident from their success. One hundred years ago in 1885, Pasteur used the first rabies vaccine derived from virus grown in the spinal cord of the rabbit. Now the disease can be prevented in man and animals using virus grown in cell cultures as the vaccine.

Travel to countries in Africa andSouth Americawhere yellow fever is still endemic is safe because of yellow fever vaccine. Most impressive of all, smallpox has been eradicated from the globe using vaccinia virus introduced by Jenner nearly 200 years ago.

Poliomyelitis is now controlled in all countries where vaccination campaigns using either living or killed vaccines have been widely implemen­ted. Measles and rubella can also be controlled by vaccines. Similarly, animal diseases due to viruses are controlled by vaccines, both diseases in food animals like foot and mouth disease, and diseases of cats and dogs.

Animals have been essential for the develop­ment of these vaccines. First, they were required to elucidate the cause of the disease. Second, to grow the virus to make the vaccine. Vaccinia used for the smallpox eradication campaign was grown in the skin of animals, mainly calves or sheep. Later, chick embryos were introduced for growing viruses, for example, yellow fever vac­cine is made in eggs, although it was derived by serial propagation in mice to attenuate it. Similarly, influenza viruses are grown in eggs.

More recently, there has been an increasing use of cell cultures for vaccine production. This is a great advance because high titres of virus can be obtained of a higher degree of purity. Also many more viruses can be grown in the labora­tory. At first, cell cultures were derived directly from animals and this, of course, increased the use of animals to provide tissues. Later, cells that could be grown continuously in the laboratory were substituted wherever possible. For example, human diploid cells have replaced monkey kidney cells for poliovaccine production and rabbit or chick embryo cells for rubella vaccine production.

Even so, animals are essential for monitoring vaccine quality. The most conspicuous use is for controlling the level of attenuation of oral polio-vaccines. They are also required to assess the safety and potency of killed vaccines. One of the required tests for demonstrating the safety of an attenuated live virus polio vaccine is the 'neuro-virulence' test in primates. InBritain, we now use cynomolgus monkeys for this, which seem more sensitive than the rhesus, monkey, used formerly in theUKand still used in theUSA.

Batches which fail the test do so because the virus is too strong and causes polio. But which is better - to test the safety of the vaccine in monkeys, or to allow it to cause polio in children, or to put whole populations at risk by abandon­ing vaccination altogether? Make no mistake, polio vaccines have changed the quality of life of millions of people in the western world who would otherwise have been at risk

 

Figure 6 Gillray’s cartoon opposing vaccination in 1802 (Courtesy Wellcome Institute Library, London)

 

Malaria

I would like to turn finally to an area which the pharmaceutical industry is frequently accused of neglecting, that of tropical medicine. To take malaria alone, there are two hundred million cases of malaria in the world and resistance to currently available antimalarial drugs is increas­ing.

 

At least a million children die from the disease each year. One third of the world's population -more than a billion people - live in malaria-risk areas. Clearly, a new, effective cure or a new, effective prophylactic for malaria is of major importance. Attempts to root out the problem by using insecticides led to the development of resistant mosquitos, as a result of which WHO had to abandon its vector eradication programme.

 

With the discovery some ten years ago that one form of malarial parasite could be incubated and kepi alive in human red blood cells in vitro, an alternative method of testing became pos­sible. This has been developed so that new chemicals can be tested against a human cell system carrying the very parasite against which they are expected to be active. This is a welcome alternative method but it is limited in its predic­tive ability. Any new drug coming out of this type of screening test still has to be tested extensively for secondary pharmacology, for absorption, dis­tribution and metabolism and for safety in a whole battery of animal tests. Despite the inventiveness of the modern chemist, there are precious few new antimalarials around. Over a period of years, the Walter Reed Army Research Institute in theUSAscreened more than a quarter of a million compounds and found only seven possible antimalarial drugs.

 

The other approach to the problem is through development of an antimalarial vaccine. This is now possibility of anti­genic change to overcome a new vaccine. Life of any sort is a terrible struggle for survival. Bacteria, viruses and parasites modify their makeup to survive, through evolving strong resis­tance to man's killer-drugs. Mosquitos and other vectors of disease do the same. Wild animals do not have the benefit of modern drug or vaccine treatment and continue to be scourged by disease, as they eat each other to survive.

 

Animals domesticated by man, such as the horse, pig and cow for food and the dog and cat for company have a healthier life, sharing with man the medical benefits brought about by animal experimentation.

 

The relationship between man and his domes­tic (and experimental) animals is a symbiotic one, but there should never be a question of which comes first. This is highlighted with crystal clarity by the recent transplant of a baboon heart into a dying baby. Put out of your minds the side issues and distractions and make the simple but realistic assumption that this could herald an era in which animal tissues can be implanted as a means of saving infants' lives. If it does, then I believe that most people would not side with the baboon (or pig or whatever), if that meant deny­ing their own baby the chance to grow into a thinking, learning and reasoning adult able to accumulate experience by spoken, written or printed word, able to ask moral questions and feel moral obligations and, as a consequence, able to contribute to the further development of human society.

 

Conclusion

In this lecture, I have described some therapeutic advances and have tried to show how they have been connected intimately with the use and the help of whole animals. I hope you do not feel that I have laboured this point unduly. If you do, I would suggest that you have fallen into a trap that confronts many of us. We who use animals and appreciate their role in the discovery of drugs are tempted to feel that the connection is sufficiently obvious that chapter and verse are not required. That is not so. Our opponents speak always in generalities, which have little substance but frequent emotional appeal.

For example, there are alternatives - why aren't they being used? The answer of course is that they should be and are being used where they provide a real alternative. But often they do not. What we need to provide are examples of how an alternative in one area provides no alter­native in another. This can only be done by being thorough and clear in our explanations.

The second point about alternatives is that they can only be used if regulatory authorities will permit their use. For some years now, scien­tists in several countries have been pressing for the adoption of pyrogen tests based on horse­shoe crab blood. The blood, by the way, is taken from crabs in the same manner as blood donations from humans, and does them no more harm. This test could replace the one in which suspected pyrogens are injected into rabbits and their temperature measured. Although Wellcome's rabbits have a longer lifespan than the average pet rabbit, no-one can doubt that photo­graphs of rows of rabbits under restraint do not endear us to most people. Again, we must explain that we are willing to change and have spent research money on facilitating a change. Sometimes it is the regulators who hold us back.

 

I urge experimentalists to devote as much care to the task of clear explanation as they would to devising an animal experiment. I do not expect to convince the Pope to change his views on abortion and I do not expect to convince the 'animal rightists' to change their beliefs either. But let them proselytise with words, not violence! There is a moderate majority which understands the needs for animal experiments. We must not, by default, allow the public to be swayed away from their common sense by the transient news-worthiness of violent extremists.

In last year's Paget lecture Roy Calne exhorted the liberationists to be consistent and eschew such commodities as meat and leather and to adopt a Job-like stance when plagued with boils. I would prefer to exhort the nine out of every ten people inBritain, who, according to a recent ABPI survey, accept the necessity of animal experimentation. To them, I say: You are right, but be vigilant. Continue to abhor violence and support progress. Believe that there will be tremendous advances in the future which can only come from scientific endeavour. Condemn the extreme minority and show them that reason and reasonableness will prevail. Help us to go forward towards the conquest of disease, in the expectation that animal experiments will be limited to those which are necessary, but with the realization that many are going to remain essential and that future progress in the bio­logical sciences and in the process of drug dis­covery would be tragically crippled without them.



[1] Lord Houghton, The Times, 24 September 1984

[2] Hillman, H, Conquest, 1974, 165, 21-22

[3] Scrip, 1984, 939, 3

[4] Paton, M W D, Brit J Clin Pharmacol, 1982, 13, 7-14.

[5] Doyle, A E, idem, 1982, 13, 63-65

[6] Green, A F, idem, 1982, 13, 25-34.

[7] Black, J W, in Drug responses in man, ed G Wolstenholme and R Porter,London: Churchill, 1967, 111-118.

[8] Prichard, B N C, Brit J Clin Pharmacol, 1982, 13, 51-60.

[9] Sunday Times Insight Team, ‘Suffer the children’,London: Deutsch, 1979.

[10] Collier, J G, Robinson, B F, and Vane, J R, The Lancet, 1973, i, 72-74; Ondetti, M A, Rubin, B and Cushman, D W, Science,1977, 196, 441-444

[11] Comroe, J H, and Dripps, R D, The top ten clinical advances in cardiovascular-pulmonary medicines and surgery, 1945-1975, Washington: US Government Printing Office, 1977.


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