The molecular pharmacology of anti-inflammatory drugs: Some possible mechanisms of action at the biochemical level

Biochemical Pharmacology, Supplement, pp. 293-307. Pergamon Press. 1968. Printed in Great Britain

THE

MOLECULAR

ANTI-INFLAMMATORY MECHANISMS

OF ACTION

PHARMACOLOGY DRUGS: AT THE

SOME

OF POSSIBLE

BIOCHEMICAL

LEVEL

MICHAEL W. WHITEHOUSE* Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T., Australia Abstract--The biochemical properties of drugs currently used in the clinical treatment of rheumatism and chronic inflammatory disease are examined and it is concluded that: (1) They are polyvalent (able to inhibit several diverse enzyme reactions). (2) At the molecular level, the steroid drugs may act in a rather different manner from the acidic drugs (phenylbutazone, indomethacin, fenamic acids) although the net result of steroid interaction with key events in inflammation may resemble the effects of the acidic drugs in many respects. (3) They can suppress the formation of inflammatory mediators, notably histamine, 5-hydroxytryptamine and perhaps the kinins, and moderate the action of inflammatory proteases. (4) They can deprive inflamed tissue of essential metabolic energy, in the form of adenosine triphosphate (ATP), needed to promote the inflammatory response. (5) The acidic drugs, steroids, and indoxole may affect protein and R N A synthesis in circulating lymphocytes---cells which mediate the immune response and play a role in the pathogenesis of certain chronic inflammatory (and autoimmune?) states. Some molecular determinants of drug activity are discussed briefly.

"Science tends to generalize, and generalization means simplification . . . . And generalizations are also more satisfying to the mind than details . . . . Of course, details and generalizations must be in proper balance: generalization can be reached only from details, while it is the generalization which gives value and interest to the detail." A. Szent Gyorgyi (1964) 1 THIS contribution will be a rather speculative survey of the way in which some of these drugs appear to act at the molecular level, as we (mis)understand it today, in altering the characteristics of the inflammatory response howsoever caused. In the light of this knowledge we might then inquire what are the principal physical and chemical factors which would appear to determine the biological activity of these particular drug molecules. To-day we are fortunate in having available a number of recent reviews which discuss various aspects of the pharmacology of inflammation and some of the properties of the more notable anti-inflammatory drugs currently used for treating rheumatic disease and arthritis. 2-10 I shall therefore only refer in detail to original work not cited in these particular reviews, being either of more recent origin or generally overlooked. * Present address: College of Pharmacy, The Ohio State University, 500 W.12th Ave., Columbus, Ohio, 43210, U.S.A. 293

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It is clear that what we normally designate as inflammation is the sum of many molecular events and physical changes which we might attempt to list as: (1)The formation or release of pro-inflammatory agents such as histamine, 5hydroxytryptamine (5-HT, serotonin), kinins, and other widely recognized but as yet poorly characterized factors. (2) The response of the tissue adjacent to the site of injury to these local " h o r m o n e like" agents (e.g. increased vascular permeability, granuloma formation, and, ultimately, w o u n d repair). (3) The response of other, distant tissues, such as leukocyte chemotaxis, increased biosynthesis o f glycoproteins by the liver, and, if an immune response is involved, immunoglobulin synthesis by lymphoid tissues. A drug designated as "anti-inflammatory" might influence any one, if not all, of these events. It is rather remarkable that m a n y o f the currently available anti-rheumatic drugs are able in fact to regulate more than one of these individual molecular events. This brings us to the first generalization. G E N E R A L I Z A T I O N 1. T H E S E D R U G S A R E P O L Y V A L E N T Table 1 shows that these drugs can profoundly influence the in vitro activity o f several different, seemingly disparate, enzyme systems and, by extrapolation, more TABLE 1. POLYVALENT PROPERTIES OF SOME ANTI-INFLAMMATORY(AI) DRUGS AT MOLECULAR LEVEL

Drug-sensitive enzyme systems Pro-inflammatory enzymes Hydrolases: thiol esterases (cathepsins ?) trypsin-like (kinin-forming ?) chymotryptic other proteases (cartilage, skin) L-Amino acid decarboxylases: acting on 5-HTP~. specific for histidine Key enzymes for synthesis of Amino acids (transaminases) Mucopolysaccharides (amino-sugar formation, sulfation) Nucleic acids (in lymphocytes) (in other tissues) ATP (mitochondrial energy conservation)

AI Acids?

P only ? !

E

Drugs* Hydroortisone

Ref.

:

11,12 13,14,42 15,16 12,17

--t- ~ ~+ :

16 4,18,29

@

3,8,19

~-

-]

Chloroquine

, -!@ ~

2,4,20 21-25 26-28 4

* -i- -- inhibits; - = no effect; :~ = may actually increase enzyme level ("negative inhibition"). 7"AI acids = (aromatic) anti-inflammatory acidic drugs exemplified by salicylates, 4-isobutylphenylacetic acid, phenylbutazone (P), indomethacin, and the fenamic acids. ++5-HTP = 5-hydroxytryptophan. than one physiological event. This generalization would appear to be true for each o f three different classes of anti-inflammatory/anti-rheumatic d r u g s - - n a m e l y , the aromatic acids exemplified by salicylic acid, the (gluco) corticosteroids exemplified by hydrocortisone (cortisol), and the heterocyclic bases (antimalarials) exemplified by chloroquine (Resochin).

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295

We might therefore surmise that it would be highly improbable that these drugs owe their clinical activity to any unique mode of action at the molecular level. Rather, it would seem that their beneficial action in supressing the symptoms, if not the cause, of disease is due to a rather fortunate combination of individual effects at more than just one stage (molecular event) in the development and maintenance of the inflammatory/rheumatoid state. Put metaphorically, these drugs probably mute several instruments of the pre-inflammatory or pro-inflammatory orchestra to achieve the consequent diminuendo; they do not assassinate the conductor or destroy all the parts of the musical score. This somewhat clumsy analogy may be useful at least in cautioning us from expecting that a new drug, patterned after those currently available, would be able to switch off (as it were) all the numerous and complex molecular events which, happening in concert, underlie the inflammatory state. Extending this idea, we could only hope to attain a sustained pianissimo but not silence. In fact, I will venture to predict that the elusive ideal drug, completely effective at a ridiculously small dose and selectively controlling the pacemaker event(s) in inflammation, will not be a supersalicylate and probably not even a super-steroid. Perhaps it would be more profitable, literally, to search for combinations of new (or even old) drugs to improve upon those presently available, which are apparently polyvalent in their action. GENERALIZATION 2. THE STEROIDS ARE A SPECIES APART This is perfectly evident to any chemist or endocrinologist; it may not be so obvious to the patient or physician. Table 1 again shows that the biochemical properties of hydrocortisone (and other neutral steroids) in many ways contradict those of the other two types of anti-inflammatory drugs considered here (the aromatic acids and heterocyclic bases)--the steroids often stimulating those enzyme systems which are inhibited by the other (ionized) drugs. So we might expect that the effective steroids do not primarily interact with charged active centers of key macromolecules, whether these are nucleic acids, enzyme proteins, or other biological receptors still to be specified, in the manner that the other (charged) drugs may. The molecular pharmacology of the corticosteroids will therefore be distinct from that of other anti-inflammatory drugs. Another distinctive and contrary feature of steroid action is that their effects on many enzyme systems, such as those listed in Table 1 (and which are steroid-stimulated), are not manifested by addition of steroid drugs in vitro; they must be preadministered in vivo. In these biochemical studies, we only see the end effects of a chain of events initiated by steroid drugs in the whole animal. This contrasts with the direct action of the aromatic acids and the antimalarials, which rapidly inhibit many enzymes in vitro. Quantitatively, the steroids are rather more effective drugs in vivo than are the currently available charged drugs. The lipophilic character and low water solubility of the more potent steroids contrasts with the rather hydrophilic character of the charged drugs. The steroids can readily penetrate through lipid-rich cell membranes, even into cell nuclei; for comparable penetration of charged drugs, very much higher extracellular drug concentrations must be attained. These physical considerations do point toward the possibility that anti-inflammatory steroids may act as nuclear regulators of cell metabolism. Recent studies have indicated that the action of other hormonal steroids may be mediated through steroid-activated synthesis of a particular

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MICHAEL"W. WHITEHOUSE

species of nucleic acid in the cell nucleus: for example, the template activity of the nuclear RNA in rat liver is increased by injection of hydrocortisone into the intact animal. 25 A baffling feature of the steroids is the very variable response they elicit in different organs. Thus, in lymphoid tissues hydrocortisone inhibits nuclear RNA synthesis, which contrasts with the effect of this same molecular species in promoting nuclear RNA synthesis in the liver. The confusing and contradictory reports that hydrocortisone does or does not inhibit glucose uptake and glycolysis in different cell lines only emphasize the fact that anti-inflammatory steroids have no single "blanket action" on all cell types. We must first establish what are their true target tissues h7 vivo (as for the classical humoral agents) before we can adequately discuss the molecular pharmacology of these powerful and fascinating steroid drugs. This situation contrasts with that of the anti-inflammatory acidic drugs; for example, salicylates have been found to uncouple oxidative phosphorylation in a great number of cell types (brain, muscle, liver, kidney, cartilage, HeLa cells, yeast, etc.). I therefore feel on much safer ground in mainly confining my further generalizations to the action of nonsteroid drugs. GENERALIZATION 3. INHIBITION OF PRO-INFLAMMATORY REACTIONS To an outsider, this must seem to be an obvious property of an anti-inflammatory drug, almost by definition. It does not appear to be so obvious to certain workers within the field. Repeatedly it is stated, both in the literature and at conferences, that the mechanisms of action of these drugs is unknown. I would just ask if that viewpoint is defensible in the light of some recent and some not-so-recent studies which indicate that the formation of at least two inflammatory mediators, namely histamine and 5-HT, is profoundly influenced by many anti-inflammatory drugs. Amine formation

Histamine is formed continuously in the body by decarboxylation of L-histidine, not only at those sites where it is stored (mast cells) but also in many other tissues which fail to store it in any significant quantity. The so-called antihistaminics inhibit only the early stage of the response to an experimental injury. This finding does not preclude the possibility that histamine is being continuously formed during the perpetuation of the inflammatory state, especially if the activity of the antihistaminics is directed almost exclusively against the release of stored histamine and the binding of exogenous administered histamine to specific receptors (e.g. the guinea pig ileum) not involved in maintaining the inflammatory state. Schayer29 has argued that locally produced (and presumably short-lived) histamine regulates the microcirculation, acting as a dilator. He has adduced considerable evidence to indicate that histamine formation is in turn regulated by anti-inflammatory corticosteroids which reduce the activity of the (inducible) histidine decarboxylase present in the vessel wall. Skidmore and 1is have shown that all the known acidic anti-inflammatory drugs and two commercial preparations of g31d salts (used therapeutically to treat rheumatic disease) are effective but reversible inhibitors in vitro of histamine formation by histidine decarboxylase(s) present in the fetal rat liver and mature rat stomach (pyloric). We elucidated the mechanism of drug action to be competition between the drug anion

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and the loosely bound co-enzyme, pyridoxal-5-phosphate, for the co-enzyme-binding site on the enzyme apoprotein, which we believe to be a lysine e-amino group (Fig. I). There is an excellent correlation among drug potency in inhibiting histamine formation in vitro, potency in inhibiting the binding of certain aromatic aldehydes (pyridoxal phosphate, 2,4,6-trinitrobenzaldehyde) to reactive protein (lysyl) amino groups, as and tl

~ ~J,_'H,

('! ]

-

0 P O,H, " -

--~O OH

Apocnzymc

Drug~mion

]ysincrcsiduc

NH ~ [CI'H2] ~

-OR

/

Aclive enzyme

Drug-apoenzymc

FIG. 1. Proposed mechanism for inhibition of histidine decarboxylase by acidic drugs. (Reprinted from Skidmore and Whitehouse.TM) relative anti-inflammatory 3 and anti-rheumatic 8° activity. Corroborative evidence that anti-inflammatory acidic drugs and hydrocortisone may inhibit histamine formation in vivo is available. 4, 81, 82 We should therefore consider these acidic drugs and gold preparations as another class of anti-histamine drugs, in this case, inhibiting histamine biogenesis. Tannic acid is also a good inhibitor of histamine formation in vitro and of the reaction of trinitrobenzaldehyde with reactive protein amino groups. This may explain the effectiveness of this time-honoured medicament in the topical treatment of burns, because it is in fact acting as a local anti-inflammatory agent to suppress histamine formation. We also found that the formation of 5-HT from 5-hydroxytryptophan (5-HTP), which is catalysed by dopa decarboxylase, is also susceptible to inhibition by acid and aromatic anti-inflammatory drugs (Fig. 2) but is not affected by gold salts or nonaromatic anti-inflammatory acids such as glycyrrhetic acid. Drug activity in inhibiting 5-HT formation in vitro paralleled the anti-inflammatory activity of these drugs in conventional pharmacological assays. Kinetic studies showed in this case that the aromatic drugs competitively antagonized the binding of substrate to the enzyme. 16 This enzyme has a fairly broad substrate-specificity and, for this reason, is sometimes described as the L-aromatic amino acid decarboxylase. It will even form histamine from histidine, although it has a very low affinity (more precisely, a rather high Kin) for histidine. 8a It is therefore not surprising to find that this enzyme can bind other aromatic acids which, not being metabolized, will partially inhibit the binding of natural amino acid substrates and, consequently, reduce the amount of amine formed from these substrates. The actual efficacy of a drug of this type depends on both the relative binding of substrate and inhibitor and the actual rate of reaction of bound substrate. The formation of dopa-amine by this enzyme may be less susceptible to drug inhibition than is the formation of 5-HT (or histamine) because the enzyme reacts much more rapidly

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MICHAELW. WHITEHOUSE]

with dopa than with other naturally occurring aromatic amino acids, aa This is of importance because dopa-amine is an obligatory precursor of the catecholamine hormones which have been postulated to be natural anti-inflammatory agents,34 inhibiting the initial increase in capillary permeability normally accompanying inflammation. While catecholamines will inhibit the early vascular response to heat injury or applied histamine,a4, a5 nevertheless one of these amines, noradrenaline, I Ill)

I11t:

I)OPAdccarhoxylasc

CIDmotryp+m 4-

t 1 ( ) ~

/NH '112-('ItICOO-

Substralc

Ac)I)N tt ~,B,/('I t :--CH--cotx Ho x'~..7

5-Hydroxytryptophan

N-Acyltyrosineesters (~

/n-Butyl C - -

{.tt~~

"

NHOC{)()-

Mcl'cnamic acid

Pilonvll~u!8.zono

FIG.2. Metabolismof aromaticaminoacidderivatives. is reported to potentiate the delayed phase of an acute inflammatory reaction,as If this is true, then any drug action on dopa decarboxylase would effectively diminish the supply of not one but two pro-inflammatory amines, namely, 5-HT and noradrenaline, which may promote the early and the late phases, respectively, of an inflammatory response. These aromatic acidic drugs do not inhibit rat liver homogentisate oxidase, indicating that they do not inhibit the metabolism of all phenolic derivatives of aromatic amino acids. Unlike dopa decarboxylase (and chymotrypsin, vide infra), this oxidase will only act on one substrate, 2,5-dihydroxyphenylaceticacid. Thus it would seem that only enzymes with a fairly broad (binding) specificity for aromatic substrates may suffer competitive inhibition by aromatic anti-inflammatory acids.

Proteases The association of proteolytic enzymes with inflammation has been discussed by other contributors to this symposium. I just wish to add a few comments concerning possible drug action on these enzymes, particularly on (1) those that release histamine from tissue stores, (2) those implicated in kinin formation, and (3) those implicated in tissue destruction. There is evidence that trypsin, thrombin,86 and chymotrypsinz7 will stimulate the contraction of smooth muscle by releasing histamine. The enzyme implicated in the

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degranulation of mast ceils and concomitant release of histamine, "chymase," closely resembles chymotrypsin in its substrate specificityz8 but it is reported to be less susceptible than chymotrypsin to irreversible inhibition by a substrate-specific analogue (a-N-tosylphenylalanine chloroketone),z9 This observation indicates that, while pancreatic chymotrypsin may provide a useful model for studying possible drug action on tissue histamine-releasing enzymes, its molecular properties probably do not correspond in all respects with those of nonenteric enzymes of the same substratespecificity presumed to participate in the inflammatory response. However, we have found that crystalline bovine a-chymotrypsin is inhibited by the anti-inflammatory acids. Kinetic studies indicated that there is competition between the substrate, N-acetyltyrosine ethyl ester (ATEE), and the individual drugs (other than indomethacin) for the substrate-binding site on the enzyme.16 Indomethacin does not compete with the substrate but apparently reacts itself with the enzyme, to inactivate it. The drug levels needed to inhibit chymotrypsin in vitro seem to be somewhat higher than those reported by MSrsdorf and Cornelissen15 for effective inhibition of ATEE hydrolysis by an enzyme present in rat's paw and activated by local inflammation. Nevertheless, the same order of drug potency was observed for both these enzyme systems, with mefenamic acid being the most potent and salicylic acid the least potent of the anti-inflammatory drugs investigated. On theoretical grounds we might expect the hydrolysis of peptide linkages adjacent to a lysine residue (contributing the carboxyl group to the peptide bond), by enzymes resembling trypsin in their substrate specificity, to be inhibited by anti-inflammatory acidic drugs if these drugs are able to bind to these lysyl (amino) residues of protein substrates in the same fashion as they bind to certain lysyl residues on plasma albumin and histidine decarboxylase (apoprotein). The affinity of a tryptic enzyme for its protein substrate is normally rather high, with many secondary forces such as hydrogen bonding and hydrophobic associations being brought into play to reinforce the charge-directed union of the active center of the enzyme with a charged center on the substrate (lysyl-ammonium ion or arginine-guanidinium ion) (Fig. 3) and it does not seem very likely that simple drug anions, with an affinity for the lysyl e-amino groups, would be very effective inhibitors of tryptic enzymes involved in establishing the inflammatory state. This is borne out by experimental studies with kallikrein which, despite previous reports to the contrary, is apparently not inhibited by these acidic drugs. 40 Kallikrein probably hydrolyzes an arginyl linkage in kininogen to form kinins; if this is so, arginine analogues such as benzamidine might be expected to selectively inhibit kallikrein action. However, events in kinin formation earlier than action of kallikrein also involve tryptic enzymes, such as the Hageman factor and activation of prekallikrein. Should any of these biochemical antecedents of kallikrein involve the scission of a single lysyl linkage in a protein substrate, for which the tryptic enzyme concerned has only a low affinity, then it might be predicted that anti-inflammatory acidic drugs would have some ultimate effect on kinin formation. Our own studies have shown that the hydrolysis of simple substrates, such as esters of lysine and aminohexoic acid, by trypsin is insensitive to these particular drugs. Evidently, in the competition between drug and enzyme for association with the cationic group of these small substrate molecules, the association of the substrate with the enzyme is favoured. This need not be the case when the substrate is a protein with a large molecular weight favouring strong drug binding by hydrophophic bonding BIO. SUPP.oU

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MICHAEL W. WHITEHOUSE

as well as by coulombic interaction; the protein-drug combination might then be a rather poor substrate for the (tryptic) enzyme if the adsorbed drug were to act as a physical impediment to the union of (protein) substrate and enzyme protein. This might also apply to the union of antigen with antibody, especially where a lysyl amino group primarily determines the association as in the union of myoglobin antibody with myoglobin. 41 Lysyl-ammonium ion

Arginyl-guanidium ion

NII~

Nil,

I

i

('tl,

I

I

(('| 12)3

A :,l

+

('--NII:

Nil

I

t

Acyl NH--CH--COt X X = N H (amino acid, amtde) or O-alkyl(ester)

Inhibited by 6-Ammohexoic acidl ECA) N t4~

Benzamidine NH,

L

C|I~

I

CII~---Nit

(('tl ,)~ ( ' t | ,-- C'OO? A-1 anions ? FIG. 3. Substrates for tryptic enzymes.

So far, I have argued the hypothetical case that anti-inflammatory acids might be expected to inhibit some stages in the complex chain of molecular events leading to kallikrein activation following injury. I am not aware of any definitive experimental evidence to suggest or refute this hypothesis though there is some circumstantial evidence available: salicylates retard blood clotting, a process involving certain molecular transformations which also precede kallikrein activation. Cline and Melmon 42 recently reported that glucocorticoids inhibit kinin formation in vitro. This exciting and provocative finding certainly suggests that this type of drug action would be associated with anti-inflammatory activity. Furthermore, this might explain the association between non-narcotic analgesic activity and conventional anti-inflammatory activity which is frequently observed---consider for example, the salicylates, mefenamic acid, indomethacin, and phenylbutazone which are all useful (peripheral) analgesics, and even the steroids manifest this property, z, a The sensation of pain is in part mediated by local kinin production and also by the release of acetylcholine, serotonin, and histamine. The formation of these amines is susceptible to the antiinflammatory acids, but is this sufficient to account for their analgesic activity or must we look for the explanation in drug action on kinin production ? Another important class of proteolytic enzymes associated with the overall course of inflammation are those concerned with tissue destruction, in the course of clearing

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up tissue debris, rejecting foreign cells, or even rejecting self as occurs in an autoimmune (or, to be more exact, autointolerant) state. The enzymes primarily involved are cathepsins, normally contained in the lysosomes. One obvious site of drug action in suppressing gross tissue destruction is stabilization of the lysosomal membrane to prevent release of the catheptic enzymes in injury. This is discussed in detail by Dr. Weissmann. However, there may be other autolytic enzymes in tissues, not necessarily within lysosomes, which become activated in inflammation and one such enzyme resembling chymotrypsin, found in the inflamed rat's paw, has already been discussed (vide supra). Another example is provided by the family of chondrolytic enzymes present in various types of cartilage which are normally latent but become activated when the ambient pH decreases (as in anoxia or by excessive lactate production) and release mucopolysaccharide-peptides from the cartilage matrix. Some of these chondrolytic enzymes are inhibited by sodium salicylate 43 and chloroquine.12These findings suggest that another beneficial property of current anti-rheumatic drugs is to retard connective tissue destruction brought about by endogenous catabolic enzymes, although it must be added that none of these drugs is particularly potent in this respect. Nevertheless, it may be hoped that when these chondrolytic enzymes have been further characterized, drugs will be found which may be more effective "anti-erosion/ anti-autolytic" agents and serve as models for the future development of new therapeutic agents for use in degenerative arthritis and other states involving gross tissue destruction. Finally, before leaving the subject of proteolytic enzymes, I should like to raise the question of using such enzymes as therapeutic agents to suppress inflammation. We have already considered how enzymes closely related to trypsin and chymotrypsin may release tissue-bound histamine and form kinins, thereby increasing the levels of pro-inflammatory agents. How do we reconcile this with the use of similar enzymes as anti-inflammatory agents? One resolution of this dichotomy is to suppose that exogenous proteases suppress inflammation by rapidly destroying the kinins. This principle "that if you cannot successfully prevent the formation of an undesirable humoral agent, then try and hasten its destruction" might be usefully extended to other areas of pharmacology including, perhaps, the suppression of inflammatory mediators other than the kinins. GENERALIZATION 4. REDUCTION OF CELLULAR ENERGY-YIELDING REACTIONS Anti-inflammatory steroids and acidic drugs can affect the energy-yielding potential of animal cells either by inhibiting certain oxidative enzyme reactions or by selectively inhibiting (uncoupling) the phosphorylation of adenosine diphosphate (ADP) in respiring mitochondria.4 This last process, commonly termed "oxidative phosphorylation," is the principal biochemical mechanism for trapping the energy liberated by cellular oxidation. Mervyn Smith9, 44 and his colleagues have found that many NAD-linked oxidative reactions are inhibited by relatively high concentrations of salicylates (> 1 raM) and have shown, in some instances at least, that the salicylate anion competes with the coenzyme for the NAD-binding site on the appropriate dehydrogenase apoenzyme. The corticosteroids are moderately powerful inhibitors of cellular respiration but there is a paradox here. Of the simple C21 compounds (pregn-4-en-3-ones), those which are

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the most highly oxygenated and potently anti-inflammatory (e.g. cortisol) are lnuch weaker respiratory inhibitors than those which have little systemic anti-inflammatory activity (such as desoxycorticosterone and progesterone). This paradox has yet to be resolved satisfactorily, although one probable explanation is that the latter steroids do not gain access to those peripheral tissues which are the seat of inflammation. When applied topically these desoxysteroids and other inhibitors of cellular oxidation or glycolysis have proved to be moderately potent inhibitors of inflammation.4, 45 The relative potency of anti-inflammatory acids and gold salts as uncouplers of oxidative phosphorylation in vitro closely parallels their potency as anti-inflammatory and anti-rheumatic drugs in vivo. Some evidence has been obtained that these drugs and other so-called uncoupling agents, such as the dinitrophenols and pentacholorphenol, may bind to an essential amino group which is intimately associated with the energy conservation process in mitochondria.4a-4s At the molecular level then, drug action on ATP biosynthesis, like that on histamine formation, is probably another consequence of drug binding to essential lysyl amino groups on key enzymes involved in inflammation. The question may be asked, "Why should drug action on oxidative phosphorylation determine anti-inflammatory activity in vivo when this energy-trapping process is not confined to those tissues suffering the inflammatory insult but is common to nearly all the cells of the body?" There is no simple answer to this question, but the following factors must certainly be considered. (1) Inflamed areas of tissue may contain more ATP and are often synthesizing ATP more rapidly than the adjacent "normal" tissue. 49, ~0 (2) Inflamed tissues may have a lower pH than the surrounding tissue, which facilitates concentration of circulating acidic drugs in the inflamed area because drug anions usually partition much less readily into cells and subcellular organelles than do the unionized drug species (the latter form proportionately increases on reducing the ambient pH). The local vasodilation and increase in capillary permeability will also favour drug accumulation at the inflammatory focus. (3) An inflammatory stimulus initiates many energy-dependent reactions which in turn sustain the inflammatory response. These include vasodilation, histamine release, movement of leukocytes into the inflamed area, and increased biosynthesis of kininogens and glycoproteins in the liver and of immunoglobulins in lymphoid tissues, which all require a continuous supply of ATP. (4) Belleau and others 51 have presented an attractive theory that the tissue response to catecholamines at an adrenergic receptor is triggered by the production of either ADP or cyclic AMP from ATP prebound to the receptor. The receptor is considered to be a latent phosphorylytic enzyme (ATP-ase or adenyl cyclase) with its substrate already attached, which is only activated by combination with these amines; the product of the enzyme reaction (a phosphoprotein or cyclic AMP) then propagates the catecholamine-mediated response. In this way an amplification would be achieved with many molecules of the enzyme product being formed per molecule of activator amine (as in the cascade process in blood clotting). If this concept can be extended to other biological amines with the ethylamine sidechain (histamine, 5-HT) then we can visualize how a biologically amplified response to very small quantities of the pro-inflammatory amines might be highly dependent upon the lacM availability of ATP for combination with the histamine/5-HT receptors.

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Depriving both the local and the distant tissues (liver, lymph nodes) of their full energy-yielding potential by drug action on ATP synthesis would significantly affect the rapidity with which the whole inflammatory response, including the ultimate reparative processes, will proceed. We might even coin the dictum "No ATP, no inflammation." This is perhaps self-evident from the failure to elicit inflammation in dead animals and also from the effects of local cooling, which may considerably reduce the rate of energy production in inflamed tissue, the cellular infiltration, and the severity of inflammation. In chronic inflammatory disease, proliferation of connective tissue frequently accompanies the degenerative erosive phase. Two examples are the well-known thickening of the vascular intimal tissue in response to the lipid deposition in atheromatous lesions and the new collagen synsthesis which is a primary process in the pathogenesis of joint stiffness.~2 Anti-inflammatory drugs inhibit some phases of connective tissue proliferation and polymer biosynthesis,2, 4 partly because they diminish the metabolic energy supply supporting these anabolic and endergonic reactions in these tissues with rather limited rates of respiration. GENERALIZATION 5. DRUG ACTION ON LYMPHOID CELLS The striking effect of anti-inflammatory steroids in causing involution of the rat thymus has provided a very useful method for assaying the potential anti-inflammatory/ anti-rheumatic activity of these steroids. It perhaps is not so well known that both the steroids and the nonsteroid anti-inflammatory drugs may have a profound effect on other lymphoid tissues including the circulating lymphocytes. For example, we have observed that the cell count in sheep popliteal lymph is approximately halved within 2 hr after injection of ACTH (20 I U porcine origin) into the jugular vein of adult ewes, which represents a very rapid response to endogenous steroids released from the adrenal cortex of these animals. It has been established only quite recently that the small lymphocytes participate in mounting an immune response to natural (particulate) antigens. After antigenic stimulation, a local lymph node elaborates a population of ceils which disseminate throughout the body and function (1) as messengers in conveying the immune response to other nodes and (2) as memory cells which are capable of responding to a secondary dose of antigen. Drs. J. B. Smith and B. Morris (of this Department) have recently shown that a local injection of particulate antigen gives a much more vigorous immune response in sheep than when the same antigen is given intravenously. This is due to the fact that the lymphatic system propagates and amplifies the immune response through production and dissemination of "activated" lymphocytes. This does not occur to the same extent when only the spleen and bone marrow are involved after i.v. administration of antigen. The importance of the circulating lymphocyte in the genesis of an autoimmune state is clearly shown by studies of the model disease, adjuvant arthritis in rats. Extirpation of the lymph nodes53 or treatment with an anti-rat-lymphocyte serum54 effectively prevents the secondary (arthritic) response to complete Freund's adjuvant. In this connection we consider it rather significant that all the anti-inflammatory drugs, steroids and nonsteroids alike, rapidly "switch off" nucleic acid synthesis in circulating lymphocytes obtained from sheep, rat, and rabbit lymph (Whitehouse,

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unpublished data). The drug action was measured by the inhibition of uridine-5-H 3 and thymidine-6-H ~ incorporation, which is manifested within 10 min or less of addition of the drugs to either normal or activated lymphocytes (the latter cells were collected after stimulating the popliteal node in sheep with locally administered antigen). The acidic anti-inflammatory drugs probably act on these cells primarily by uncoupling (nuclear) oxidative phosphorylation. Nucleic acid synthesis in these cells was certainly more susceptible to a given concentration of uncoupling drug than was nucleic acid synthesis in other unicellular organisms examined (yeast, Tetrahymena pyriformis, Aerobacter aerogenes, guinea pig polymorphonuclear leukocytes). The mechanism of action of the steroids remains to be established although it was notable that, in this in vitro system, desoxycorticosterone again proved to be a more potent inhibitor of metabolism than cortisol. Indoxole a new anti-arthritic drug, '55 was particularly potent in suppressing nucleic acid synthesis by lymphocytes isolated from lymph, being effective at only 2-10 tzM in a protein-free medium. However, fixed lymphocytes taken from thymus tissue (of rats and rabbits) or from the bursa of Fabricius (of chickens) were remarkably less sensitive to this drug (which is reported to be not thymolytic in rats:'5). The importance of drug action on RNA and DNA synthesis in lymphoid cells is indicated by repeated observations that antibody production is preceded by intense mitotic activity in lymphoid tissues. Furthermore, antibody levels are maintained in the serum after antigenic stimulation only when there is continued cellular proliferation within the lymphoid tissues. Certain anti-turnout drugs such as 6-mercaptopurine will inhibit adjuvant arthritis in rats ~6 and have beneficial anti-rheumatic activity in patients (K. Trnavsky, personal communication) even though these drugs do not manifest much (if any) anti-inflammatory activity in acute biological assays (involving edema formation or response to ultraviolet irradiation). The therapeutic value of these drugs in these inflammatory states is probably attributable to their action in suppressing the proliferation of lymphoid cells. Perhaps the well-known but puzzling effect of colchicine on gout can be attributed to the same cause. DETERMINANTS OF DRUG ACTIVITY Our preliminary studies with indoxole certainly show that not all lymphoid cells are similar in their drug sensitivity. These, with other observations, point toward the possible development of a new class of anti-lymphoid drugs with a narrower spectrum of activity than is manifested by the steroids. What determines the relatively selective drug action of indoxole on one type of lymphocyte and not on another (e.g. thymocyte) remains to be elucidated. We might profitably enquire what determines the potency of the other anti-inflammatory drugs which are less selective in their action. Our own and other studies have shown that lipid solubility, pKa, and affinity for protein amino groups are three important determinants of the uncoupling activity4, 7, 16, 4v of acidic drugs and presumably determine their anti-inflammatory activity as well. To these three factors must be added certain subtle stereochemical considerations, illustrated by the remarkable anti-inflammatory potency of one geometrical isomer of an indomethacin analogue 57 and one optical isomer of 3-chloro-4cyclohexylphenyl-a-propionic acid 58 or the powerful uncoupling activity of the least

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acidic (i.e. the 3,5 isomer) dinitrophenol. 48 Drug action on the amino acid decarboxylases is largely determined by these same characteristics, notably hydrophobic character and acidity. Molecular features determining the anti-inflammatory activity of the steroids are most competently reviewed by the Merck scientists, s There is, however, a disconcerting lack of agreement between the relative inhibitory potency of various steroid drugs in vitro and their anti-inflammatory activity. I have already referred to the fact that desoxycorticosterone may be several times more potent than cortisol in vitro; and very high levels of cortisol (100-250 tzM) may be required to elicit an inhibitory effect on normal cell metabolism in short-term experiments. Even in those few instances reported to date in which very low levels of cortisol (0.1-10 tzM) have consistently inhibited metabolism in vitro, the significance of the results may be questioned because mixed or aged cell populations from lymphoid tissues were used as hormone substrates.~2, 28 The apparent inhibitory effects of these steroids could in fact be just "nonsense results", of little physiological or pharmacological significance, if the prime action of cortisol and its analogues is to "switch on", rather than "switch off" or inhibit, certain metabolic events; for example, induction (or release) of autolytic enzymes in the thymus or induction of a repressor of the inducible histidine decarboxylase (the enzyme postulated to regulate the microcirculation29). This recalls Ingle's permissive theory of endocrine action and it is now possible to conceive a permissive action of cortisol in molecular terms. May I just remind you of Belleau's hypothesis that the adrenergic receptors are latent enzymes awaiting stimulation by the catecholamines. 51 I f this is really the case, why should there not be extrahepatic enzymes or nucleic acids (patterned after, or perhaps different from, the cortisol receptor(s) in liver nuclei) which are activated by association with the steroid and initiate the "true" molecular (and suitably amplified) response to these drugs ? REFERENCES 1. A. SZENT-GY6RGYI,Science 146, 1278 (1964). 2. R. DOMENJOZ,Adv. Pharmac. 4, 143 (1966). 3. C. A. WINTER,,4nn. Rev. Pharmac. 6, 151 (1966); Progr. Drug. Res. 10, 139 (1966). 4. M. W. WHITEHOUSE,Progr. Drug. Res. 8, 321 (1965). 5. Y. MIZUSHIMAand T. NAKAGAWA,Rheumatism 22, 1 (1966). 6. W. G. SPECTORand D. A. WILLOUGHBY,Pharmacology of Inflammation. English Universities Press (1967). 7. I. F. SKIDMOREand K. TRNAVSKY,Acta Rheumat. Balneol. Pistiniana, 3 (1967). 8. L. H. SARETT,A. A. PATCHETTand S. STEELMAN,Progr. Drug Res. 5, 11 (1963). 9. M. J. H. SMITHand P. K. SMITH,The Salicylates: .4 Critical Bibliographic Review. Wiley, New York (1966). 10. H. K. VON RECHENBURO(Ed.) Phenylbutazone (Butazolidine) 2nd edn. Edward Arnold, London (1962). 11. M. W. WHITEHOOSEand J. E. LEADER,Biochem. Pharmac. 16, 537 (1967). 12. F. K. COWEYand M. W. WmTEHOUSE,Biochem. Pharmae. 15, 1071 (1966). 13. M. W. WHITEHOUSEand I. F. SKIDMORE,J. Pharm. Pharmae. 17, 668 (1965). 14. D. BELLAMYand R. A. LEONARD,Biochem. J. 98, 581 (1966). ! 5. K. MORSDORFand G. CORNELISSEN,Med. Pharm. exp. 15, 399 (1966). 16. I. F. SKIDMOREand M. W. WHITEHOUSE,Biochem. Pharmac. 16, 737 (1967). 17. J. C. HoucK and Y. M. PATEL,Nature, Lond. 206, 168 (1965). 18. I. F. SKIDMOREand M. W. WmTEHOUSE,Biochem. Pharmac. 15, 1965 (1966). 19. B. J. GOULD,P. D. DAWKINS,M. J. n. SMITHand A. J. LAWRENCE,eolec. Pharmac. 2, 526 (1966). 20. P. SCHONHOFER,Med. Pharmae. exp. 15, 491 (1966).

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W. STEVENS,G. COLESSIDESand T. F. DOUGHERTY,Endocrinology 76, 110 (1965). C. K1DSON, Nature, Lond. 213, 779 (1967). M. H. MAKMAN, B. DVORKIN and A. WHITE, J. biol. Chem. 241, 1646 (1966). V. G. ALLEREY, B. G. T. POGO, A. O. POGO, L. J. KLEINSMITH and A. E. MIRSKY, in Histones. Their Role in the Transfer o f Genetic Information. CIBA Foundation Study Group No. 24, p. 50. J. & A. Churchill, London (1966). 25. D. C. TORMEY, H. H. FUDENBERG and R. M. KAMIN, Nature, Lond. 213, 281 (1967). 26. P. P. DUKES, C. E. SEKERIS and W. ScnMro, Biochem. biophys. Acta 123, 126 (1966). 27. S. N. COHEN and K. L. YIELDING, Proe. HatH. Acad. Sci. U.S.A. 54, 521 (1965). 28. R. L. O'BRIEN, J. G. OLENICK and F. E. HAHN, Proc. HatH. Acad. Sci. U.S.A. 55, 1511 (1966~. 29. R. W. SCHAYER,Fedn. Proc. 24, 1295 0965). 30. D. E. BARNARDO, H. L. F. CURREY, R. M. MASON, W. R. FOX and M. WEATHERALL, Br. Med. J. 2, 342 (1966). 31. S. NORN, Aeta pharmac, tox. 22, 369 (1965). 32. O. V. SJAASTADand O. SJAASTAD, Actapharmac. tox. 23, 303 (1965). 33. W. LOVENBERG,H. WEISSBACHand S. UDENFRIEND, J. biol. Chem. 237, 89 0962). 34. D. A. WILLOUGHBYand W. G. SPECTOR, J. Path. Bact. 88, 159 (1964). 35. B. GOzsY and L. KATO, Nature, Lond. 212, 1049 (1966). 36. K. O. HAUSTEIN and F. MARKWARDT, Archs int. Pharmacodyn. Thdr. 163, 393 (1966). 37. T. M. GILFOIL and C. A. KELLY, Br. J. Pharmac. 27, 120 (1966). 38. I. PASTAN and S. ALMQVIST, .]'. biol. Chem. 241, 5090 (1966). 39. Z. DARZVNKIWIECKZ and E. A. BARNARD, Nature, Lond. 213, 1198 (1967). 40. G. E. DAVIES, G. HOLMAN, T. P. JOHNSTON and J. S. LOWE, Br. J. Pharmac. 28, 212 (1966). 41. M. Z. ATASSt, Nature, Lond. 209, 1209 (1966). 42. M. J. CLINE and K. L. MELMON, Science 153, 1135 (1966). 43. D. P. SIMMONSand O. D. CHRISMAN, Arthritis Rheum. 8, 960 (1965). 44. J. A. STURMANand M. J. H. SMITH, Bioehem. Pharmac. 15, 1857 (1966). 45. P. GOROG and L. SZPORNV, J. Pharm. Pharmac. 16, 635 (1964). 46. M. W. WHITEHOUSE and I. F. SKIDMORE, Biochem. J. 100, 52P (1966). 47. E. C. WEINBACH and J. GARBUS, J. biol. Chem. 240, 1811 (1965). 48. J. F. BURKE and M. W. WHITEHOUSE, Biochem. Pharmac. 16, 209 (1967). 49. D. A. KALBHEN, Archs int. Pharmacodyn. Thdr. 143, 337 (1963). 50. H. K o c a and D. A. KALnHEN, in Die Entzundung (Eds. R. HHSTER and H. F. HOFFMAN), p. 148. U r b a n and Schwarzenberg, Munich (1966). 51. B. M. BLOOMand I. M. GOLDMAN, Adv. Drug. Res. (Eds. N. J. HARPER and A. B. STMMONDS)3, 89 (1965). 52. E. E. PEACOCK, JR., Ann. Surg. 164, 1 (1966). 53. B. B. NEWBOULD, Ann. Rheum. Dis. 23, 392 (1964). 54. H. E. F. CURREY and M. ZIEF, Lancet ii, 889 (1966). 55. E. M. GLENN, B. H. BOWMAN, W. KOOYERS, T. KOSLOWSKE and M. L. MYERS, J. Pharmac. 115, 157 (1967). 56. S. J. PILtERO, M. L. GRAEME, E. B. SIGG, G. CmNEA and C. COLOMBO, Life Sci. 5, 1057 (1966). 57. L. H. SAREa~rand T. Y. SHEN, in Die Entziindung (Eds. R. HEISTER and H. F. HOFEMAN), p. 291, Urban and Schwarzenberg, Munich (1966). 58. T. Y. SHEN, Chem. Engng News 10, February 13 (1967).

21. 22. 23. 24.

COMMENTS DR. ERD6S: I assume that you have proof that it is the e-amino group that is reacting on the protein and not some other positively charged group. May I ask also about the rather high concentrations of the various drugs which were needed to cause inhibition of the decarboxylase ? DR. WHI~HOUSE: In the case of salicylate, we did not need a concentration higher than 1 m M for it to be effective, and such a concentration is of course required clinically. In the case of flufenamic acid, much lower concentrations would suffice. DR. GLENN: Since you alluded to indoxole, it should be mentioned that this drug can produce severe phototoxicity. So, in this case, a drug which inhibits inflammation can also produce it under the proper circumstances.

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DR. WEISSMANN: One of the things that is interesting to me about the uncoupling effect of the nonsteroidal anti-inflammatory agents is the fact that, in thyrotoxicosis associated with thyroiditis, one is not impressed with the anti-inflammatory action of thyroxine. Since thyroxine is an uncoupling agent, should it not be a superb anti-inflammatory drug as well ? DR. WHITEHOUSE" I know of no experiments which would show that one could get high enough concentrations of thyroxine across the various membranes into the coupling site within the mitochondria. Besides, much of the work on the uncoupling effect of thyroxine was done on the liver, which is a very bad model to choose. DR. DIENER" Could you tell us more about the differential effect of indoxole on lymphocytes ? DR. WHITEHOUSE: If one takes lymphocytes out of tissues such as the thymus and compares them with circulating lymphocytes, one finds that the latter are much more drug-sensitive. DR. MELMON: Could you elaborate on a few points in relation to the effect of anti-inflammatory drugs on decarboxylases? First of all, the investigators who studied /-amino acid decarboxylases, which include tryptophan decarboxylase, were unable to show that salicylate or phenylbutazone actually were inhibitors. Likewise, if your theory is correct, why are not some of the well-known decarboxylase inhibitors, such as ct-methyl-DOPA, good anti-inflammatory agents ? DR, WHITEHOUSE" I think you missed the point of my very first remark, which was that these drugs probably did not act by any exclusively unique mechanism. They probably act by coincidentally affecting many enzyme systems. DR. EVERETT: Relative to the differential effect of drugs on lymphocytes, I might add that in our experience the corticosteroids have a much more selective effect on the short-lived lymphocytes than they do on the long-lived cells.