Men, molecules and machines

TIPS -

inaugural

issue, I979

1

Men, molecule machines A. W. Cuthbert Lkpwtmiwro/PhormacoloR.vpv, Unrversrr.v oJ Camhrrdw.

Camhrrdge CSZ 2QD. L’ h

Viewpoint is an occaGonal series in which authors muse about the pharmacological world at large and assess major trends, past, presenf and furure. In the first article of this kind A. W. Cuthbert begins wrth an assessment of three pharmacological specialities, bioassay, pharmacokinetics and structure-activity relationships and concludes with his thoughts on receptors. Recently I had the pleasure of listening to Harold Burn when he came to give a seminar in the department at Cambridge. BUI n, em&u; professor of pharmacology at the University of Oxford, has maintained a vital interest in pharmacology in the two decades since his official retirement. His subject was acetylcholine as a driver of the heart, and he described work he and his colleagues had started in the late ‘fifties. We were told, or reminded, how under appropriate conditions acetylcholine could start a stopped heart. Twenty years ago this was a burning issue, and Rurn came close to pinpointing the effect of acetylcholine on potassium permeability. When talking afterwards to graduate students, I was struck by how different their approach would have been, and how unfamiliar they were with the techniques Burn had used. Such was the stimulus for these thoughts about the changing face of pharmacology during the period in which I have been able to observe it at first hand. I started pharmacology at the time Burn was experimenting with acetylcholine and automaticity in cardiac tissue; indeed, some of my first experiments were concerned with the automaticity of small pieces of embryo chick heart in culture. -_

* . . bought in Woolworths . . . At a superficial level manipulative skills have probably declined in the last 20 years. How many graduate students can now make a heart-lung preparation, and if they could do so would it be of much use to them? On the other hand, our machinery has now become very sophisticated, compared with the gadgets which dominated experiments at that time. Gaddum is reported as saying all that is required for experimental

pharmacology

can be made

from things found in the home or bought in Woolworths. Gadgetry is not special to pharmacology, and a glance at the exhibits in the Cavendish Laboratory will show that the same is true of early experimental physics. What is important is to look behind the machines, at the men ard women and their ideas, and try to see what has changed. I do not find pharmacology an easy subject to teach; it is not possible to present it as a sustained and coherent set of arguments derived from a set of basic principles. Contrast this with crystallography, perhaps the most exact of the experimental sciences, where if the basic principles are mastered and the appropriate technology and analysis carried through the answers are absolute. In genleral our conclusions are tentative ones, our literature peppered with words like ‘indicate’, often prefixed by may, might, could or possibly. A recent commentator” searchrng for a collective noun for pharmacologists came up with ‘bodi’. a word listed in “The boke of St Albans (1486)” and defined as “a number of persons taken collectively, usually as united and organtsed in a common cause”. a fine sentiment indeed, but at times one feels imbroglio might be more appropriate. The same writer points out that over the years our e:uamination questions have remained the sa.me while the answers have changed -perhaps this is progress. I wonder how man). who like me hate been in pharmacolo,gy for some time. get the impression that everything used IO be a great deal easier, What discoveries Le should and could have made, but didn’t. 1 suspectthis is an illusion. as it is impossible to look back and discard at the same time a]] the ideas accumulated over the period. Perhaps the illusion is an indication of progress.

Bioassay - old rnd new i cdn identify only three type\ of actrvitg which were Feculiarly and distincti! pharmacological in the late ‘firtie<. The+e are bioassay, pharmacokinetics and a study of strucrure-activity relationships. Bioassay provided an exqutsitely sensitive way IO measure activity when adequate chemical o: physical techniques were unavailable. II was partly because of critrcisms of the ogtdatedners of the Britirt Pharmacopoeia by H. H. Dale and A. J. Clarke that the Pharmaceutical Society set up its Pharmacologtcal Laboratories in 1926 with Burn as it< director. Thi\ Ha\ 5 years before the Bnti\h Pharmacological Society was founded, jnd the prime aim of the laboratories was to devise tunable ways for the bioassay of galenical drugs then in use. Suggest to a graduate studer.r today that digoxin might be assayed b,, pigeon emesis or that the vitamin 1) content of cod liver oil be measured on rachitic chick femurs and see the incredulous look of disbelief. W’hat about N!vlR. IR, mass spectrometry and gas shromatography, they czclaim. 411 very well if the sample is in pure solution, but nor if it i\ in blood, a piece of hypothalamus or cod liver oil.

. . prostacyclins from rings of artery It is easy to show that bioassay 13 not death indeed it contmues to make an important contribution to our science. with a good deal of spill-over into other >~asctences. Consider the ‘cascade’ of >uperfused preparations, each one sensitive to a different autocoid as developed bg \.ane. This method provided an e\tremrly economic and instantaneous u,lg of examining the liberation and elimmation in the body of a number of cndagenouc materials of pharmacological and pathological importance. V’ane. too. was responsible for showing the liberation of pro3tacyclins from rings of arter) b) miubatmg them together wrth plateler~. using aggregation as a measure of effect. Both the methods have led IO important fmdmgs, but in prmaple neither mstholi 1sdtlferent from that useri by 1 orwi vvhen he superfused two hearts in +zrtrs. II IS the \ensitivity and \pesil’iar) vvhr;h rnake~ bioassay a powerful and important tool. In his Nobel Lecture in IY’ Cutllemin’, descrtbmg the isolatron of attributed success to the c‘)bernins. development of novel bioassa)b - .md remember he obtained only I mg of TRF from 3OO.OOOsheep hypothalami. E~acrl) the same sort of situation applied IO the isolation oi vitamm B,: from liver 3)

TlIcFS - inuugurul

2 yea?< before,

when it was discovered it

co~,;$ be conveniently and rapidly assayed by its effect on the growth of Luc~oh~i/l~~.~ cosei. Bioassay is a set of methods, an approach to quantitative IT xasurement. Whether a science can be constituted by Inethods alone is a philosophical point ithich I shall not pursue here, but bioassay remains a peculiarly pharmacological approach. The first clue for the exist,ence of enkephalins came when Hughes applied a brain extract to an isolated mouse vas dejerens, caused to contract by indirect electrical stimulation. What a bizarre experiment this must seem to a nonpharmacologist. I heard an eminent physiologist exclaim that he could not understand how what was going on in the brain could be examined on the vus deferens, but to those who measure substance P on goldfish intestine, angiotensin on the rat colon strip or adrenaline on the chick rectum, it is the stuff of our trade.

where correlations between structure and activity were reported. Looking back at SAR I can feel only a disappointment; twisting the cheinical tail for therapeutic advantage has been a poorly rewarded enterprise. We do not yet know the rules, if indeed there are rules to be learned. In his Harter Memorial Lecture in 1972, Alfred Spinks divided the total cost of research and development in the drug industry by the number of new drugs appearing on the market. The result was a staggering ti0 million, but as he remarked, “This amount is not, of course, the likely cost of discovering a new drug: some unskilful manufacturers spend much and discover little and others spend little and discover much”. Nevertheless the cost of drug development is very high, and only one in every several thousand new compounds reaches the market-place. Some individuals, however, have been unusually successful, for example G. H. Hitchings and J. W. Black. Perhaps not only chemical

issue, i’979

design of the antngonists. However,

cvcn

as I write this art ~clea paper has appeared on impromidine’ which intuitively one might expect to be a Hz-antagonist. yet it is an agonist more potent than histamine itself-the rules are still baffling. Thus, in spite of Hansch analysis and Hammett functions we remain rather like chemists who study complex bimolecular reactions, but knowing only about one of the reactants. III total terms Nature herself has been as successful with new drugs as we have. Consider what five compounds you would choose for survival, a sort of kit of desert islard drugs. My choice would include three of natural origin, Even for many of our successful drugs the honours are not all ours. The introduction of tubocurarine or quinine into medicine owes as much to the South American Indian and 1.0 the Corragidor of Loxa as to pharmacology. Smce the discovery of enkephalins !there has been vigorous activity to produce synthetic materials with similar effects. The cynic might argue that when a suitable compound is found, its structure made into a rigid analogue, groups added IO protect agamst peptidases, and physical properties conferred to allow passag: into the brain, morphine will have been rediscovered. However, a negative, gloomy approach such as this is not helpful: what we need is to know more about the other half of the interaction, which brings me to the receptor.

Locks for keys

In the last LO )ears pharmacokinetics has become an important part of our discipline, particularly in relation to drug action in man. Four *‘actors have perhaps contributed to this, nnmel!; the appearance of increasing nurr.be:rs of potent, &erapeutically important drugs, the availability of radiolabelled drugs. the emergence of clinical pharmacology as 3 discipline, and production of simple analogue and digital devices for modelling experiments. Twisting the chemiwl tait My first love was chemistry, and my introduction to pharmaco’iogy was through classic papers of thje Barper and Dale type

and biological skills aye required, but more importantly a shrewd sense of how to attack the problem in a unique way. For a long time a variety of reasons why antihistamines did not biock gastric secretion were given until Schild pointed out that the recepto- type was different. Black attacked the problem of Hz-antagonists by considering the structure-activity relationships in the histamine analogues. The recognition that HI-activity of histamine analogues resided in compounds with a high mole fraction of the NT-H tautomer was a piece of pharmacological detective work in the highest tradition. but more importantly it provided a rule for the

If pharmacology has eras then the last two decades ?aave been the age of the receptor, or at least its coming of age. What a transformation there has been. As late as 1943 Dale was still doubting the usefulness of ;.he receptor concept, others denied their existence, while the majority considered they woul’d only be demonstrable indirectly, by the biological tffects of activation. The smooth muscle preparation which has been central to bioassay methods and for receptor classif,cation may have actually deterred us from getting to grips with the receptor itself and the long period of ‘drugs moving levers’ was broken only when Stephenson introduced the ideas of spare receptors. At this juncture 1 must comment on the work of A. J. Clark, a man of vision whose remarkable volume on “Mode of Drug Act.ion of Cells” in 1926 is a classic. He was a good deal more cautious about the meaning of the quantitative relationship between drug concentration and response than was apparent in the fifties, and who Ican tell

TIPS - inaugpral what

issw, 1979

contributions

Clarke

1

may

have

made but for his untimely death. The demonstration by Waser of curare binding at the neuromuscular junction by autoradiography in the early ‘fifties came close to satisfying the skeptics, but the real honours must go to Paton and Rang who, in 1965, first demonstrated the presence of a saturable binding component in intestinal muscle with “C-atropine. The properties of this component were such as to leave little doubt that they had detected the muscarinic receptor by complex formation with a drug. It may be disputed that this was the first time that receptors had been measured by binding, as a few years earlier Jansen and Jacob had detected cytosolic binding proteins for oestrogens now often called the oestrogen receptor. Steroid binding proteins are not receptors in the pharmacological sense, and we all GROWwhat a pharmacological receptor is even though discussion of definitions often occupies inordinate amounts of time at symposia. If the honours go to the muscarinic receptor, then the prizes so far have gone to the nicotinic. A recent review’ will show just how much information is now available about the nicotinic receptor, and rather dcoressingly just how much there is still to be learned. A great number of receptors have now been detected by ligand binding techniques!, and I do not propose to review them here. Binding of radioligands has become an established pharmacological technique, but reports are now appearing at such a rate that one might expect receptor binding to be an ephemeral activity. However, already a variety of second- and third-order types of binding study are appearing which are not simply concerned with the number and location of receptor types. It is clear that an alteration in receptor numbers and sensitivities provides a further dimension of regulatory control of the biological response, for example the low density of insulin receptors on fat cells in obese mice compared with

normal mice, the diurnal rariation in a-receptor density in the pineal, and our o~ln findings on the effect\ of hormone\ and ions on the propertie\ aodIum channels.

of epithehal

It might have been expected that binding studies would remain a rather esoteric activity, but the merhodologq has been applied already to problem\ of drug development. Binding met hods can be uxd to predict whether antiprychottct are liable to produce a Parkinsonian syndrome, to differentiate morphine agor%ts from antagonists and to gauge the potency of new benzodiazepines. Such binding assays are carried out with brain homogenates. even human brain homogenates remoled at post mortem. Screening tects of this type are very cheap and clearly more acceptable from a variety of standpomtc. . , . learn how h~.v rrck

. .

On other fronts the chemical attack of the receptor is unabated. Modification by enzymes, development of covalent. fluorescent and photo-affinity labels and a concerted effort to isolate receptors which retain the binding characteristics are all being investigated. What is to be done with these isolated receptors? One thing at least is to put them back, either where they came from or into other cells. vesicles and possibly lipid bilayers. We can then begin to learn how they tick, but this IS for the future. The other thing ae can do with receptors is to study them bg some of the techniques which the physical scientists have drteloped. The foundarrons for this have already been laid, and in England none more than Arnold Burgen has prepared the ground-so that iin, widths, field shifts. and doublets are no longer totally foreign Holds to the pharmacologist. I think the report uhich moit es&d my interest in recent years H’ab that on electrical noise at the neuromuscular junction by Katz and Miledi”. The idea was brilliant. but simple -thar ar stead>

state m the presence of acetklsholine the electrical noise \*a\ generated h: the probabilistic nature of coll~.l~nr heinzen acetylcholine molecules and rhelr recepror\. I rrmember excitedly exalamtng this to an electrlcat engineer, and rra5 comev.hat creslfallen to be told that noise anall\i\ had been widely ured to rtud) elcirrochemical processes in engineering for some time. Is there nothing neu under rhr sun?! New or not, noise anal3515 has had a tremendous impact on the study of drug action at the molecular level. Even come %ery fixed ideas are being mndifled; for example it is noa known that rubocurarme not only competes Hlth acetklch4ne for the binding rite. but can aloc’ler> has groan from a feu hundred in the late ‘fifties to around 1400 m 1979. E\rn earlier there *as onl) a handftil *ho had vision to see that a proper ytudy ot drug\ and their actions uai a uorth\\hile adlenrure. Reading list