Inflammatory process and screening methods for anti-inflammatory agents- a reviewSR Naik, UK Sheth
C.S.I.R. Pharmacology Research Unit, Department of Pharmacology, Seth G. S. Medical College, Bombay 400 012, India
In the last few years a number of anti-inflammatory compounds have been introduced with a view to find out a potent and safe anti-inflammatory anti-arthritic drugs which would come near enough to steroids without any deleterious effects.
Compounds of synthetic and indigenous origin have to be screened in laboratory animals before they can be tried in man. A number of laboratory models are available for screening anti-inflammatory agents. These models try to stimulate biological and biochemical characteristics of inflammation and particularly arthritis. However, none of these could be considered as hundred per cent correct so that the results of which can always predict the human usefulness.
In order to screen new potential anti-inflammatory-anti-arthritic compounds, one must have clear understanding about the prime cause of inflammation, the nature of inflammation, target organ involved, various stages of inflammation, biochemical and other systemic changes due to inflammation. Are any endogenous triggering and controlling factors involved? and if involved what is the end result of inflammation?
Considering the above mentioned questions, we have made an attempt to review the inflammation, various factors involved in the inflammatory process, available methods for screening potential anti-inflammatory agents and finally future trend of research in the field of inflammation or connective tissue disorders.
Inflammation has been variously defined. Houck (1963) has called it a vital response of tissue injury. Considering Houck's definition one might be tempted to describe it as a protective and normal response to any kind of noxious stimulus. This stimulus may alter the normal physiological process of the host, varying from the acute transient and highly localized response to simple-mechanical injury or to the complex persistent response involving the whole organism. This initial response may initiate further a series of biochemical, immunological and cellular events, which may range in time from recognition of the noxious stimulus through mobilization of natural defence mechanisms ending with physical repair and restoration of function of the injured tissue. Thus inflammation can be defined simply by summing up all these processes, as a complex, vascular lymphatic and local tissue reaction elicited in animals by the presence of viable and non-viable irritants.
Classification o f Inflammation
Inflammation may broadly classify into three categories:
(1) Acute inflammation.
(2) Chronic inflammation.
(3) Miscellaneous kinds of inflammation.
This third category may include allergic and dermatological disorders.
1 Acute Inflammation
When a tissue injury is caused by a single event such as mechanical trauma, a thermal or chemical burn or a single exposure to non-replicating antigen the protective phenomena results in inflammation and repairative process proceeds smoothly from injury to recovery.
Thus whole inflammatory process at least in acute inflammation examplifies a beneficial homeostatic mechanism trying to restore the affected tissue to its normal healthy state.
2 Chronic Inflammations
There are many diseases which are distinguished by signs and symptoms characteristic of response to chronic inflammatory process of unknown etiology. Some of the rheumatic disorders are characterized by a lack of detectable anti-globulin (rheumatoid factor) and antinuclear antibodies in the serum. These are often loosely called as collagen disease which is suggestive of involvement of structure and/or metabolism of collagen in the diseased process. Several classifications have been suggested (Kulonen, 1971). The main members include rheumatic fever, rheumatoid arthritis, ankylosing spondylitis and osteoarthritis, but many other disorders exhibiting chronic inflammatory changes such as periarteritis nodosa, scleroderma and systemic lupus erythematosus are frequently included in general classification (Goodman and Gillman 1970). However we have included these disorders in miscellaneous group.
There are some other types of chronic inflammations caused by self replicating parasite like bacterium, virus or neoplasm. Such inflammation may become much more complex because of persisting injurious agents or their degraded products. When noxious agents cannot be destroyed or early eliminated, the inflammatory responses try to isolate them from the rest of the organism by forming granuloma (e.g. in pulmonary tuberculosis and silicosis or gumma as in syphilis). The ultimate response of this type is classified as Gohn complexes which contain viable but imprisoned tubercule bacilli functionally isolated from the host.
Gout is characterized by acute and chronic inflammatory response to the deposition of microcrystals of sodium urate in the joints and tissues. Now the etiology of gouty arthritis are totally well understood.
3. Miscellaneous kinds o f inflammation
This group of disorders is not essentially inflammatory but its components are of inflammatory origin. Many of the dermatological conditions consist of acute, subacute and chronic inflammatory reactions to various known and unknown prime causes. All these disorders mainly involve skin and can readily be assessed. Some examples of these skin diseases are pemphigus, pemphigoid and discoid lupus. These, however, apparently seem to be immunologic but they hardly respond to immunosuppressive therapy (Ebringer and Mackay, 1969). Contact dermatitis on the other hand is a manifestation of delayed hyper-sensitivity, Encapheli mylites involving spinal cord and motor incoordination. Rejection of the transplanted organ is clearly initiated by normally protective immunological reactions but it is mediated by the familiar sequelae of inflammatory stimulus leading to cardinal signs of inflammation and, above all the loss of function.
Probable inflammenogenic factors
Lysosomal Enzymes-It is postulated that lysosomal components such as hydrolytic enzymes or cationic proteins play important roles in the initiation of inflammation, tissue injury and connective tissue breakdown (Weissman and his co-workers, 1964, 1969; Shen, 1967, Janoff and Zweifach, 1964). Anderson (1970) found higher levels of catalytic enzymes in inflammed tissue or serum of arthritic rats as comperd to normal animals. In rat adjuvant arthritis, it has been stressed that the potential destructive capacity of connective tissue is acid hydrolases and is liberated within the endogenous cellular elements of connective tissue or derived from migrating leukocytes (Anderson, 1970). However Paulous and Whitehouse (1973) have stressed that the presence of potentially destructive enzymes in serum is not the sole factor in injury, because Collin and Lewis (1971) have found no correlation between maximum enzyme activity and presence of tissue damage. This is further supported by the fact that, in rheumatoid arthritis the catabolic activity is not the sole problem, but there is also the articular damage which may be due to adopted leukocytes in synovial tissue.
Recent studies revealed the presence, within human polymorphonuclear leukocyte lysosomes, of enzymatic activity capable of degrading the non-collagenous proteoglycon matrix of hyaline cartilage at neutral pH (Ignarro et al., 1973). Rapid breakdown of the sulfated mucopolysaccharide constituents of cartilage was enhanced in a neutral pH or balanced salt solution by lysosome granule lysates derived from human, but not from rabbit or guinea pig, polymorphonuclear leukocytes. Thus human leukocytes are remarkably different from other species with regard to the capacity of their lysosomal enzymes to degrade intact cartilage under conditions of neutral pH and balanced ionic movements. The neutral protease activity of human leukocyte lysosomes to degrade hemoglobin was demonstrated (Ignarro, 1973). The presence of elevated lysosomal contents namely neutral proteases, acid hydrolases, chemotactic factors, kinin generating factors, vascular permeability factors, pyrogens in the synovial fluid of arthritic patients has been clearly documented (Ignarro, 1974a).
Lysosomal enzymes secretion occurs as a result of interaction between the leukocyte or macrophage plasma membrane f and immunologic reactant or stimulus. Endocytosis of particulate reactants is not a prerequisite for enzyme secretion however, secretion occurs in the absence of phagocytosable particles, I such as non-phagocytosable immune a complex surfaces (Henson, 1971; Hawkins, d 1972; Oronsky et al., 1973; and Ignarro 1974). This immunologieally-provoked d selective secretion of lysosome granule contents from neutrophils appears to result from leakage into the extracellular, environment of primary lysosomes contents at precisely the time when newly-formed heterophagic vacuoles are still open to the extracellular compartment while merging at their surfaces with the lysosomes. All this important data indicate that there may be some kind of regulation by autonomic neurohormones, glucocorticoids, prostaglandins and cyclic nucleotides in immunologically-provoked secretion of lysosomal mediators of inflammation by human neutrophils. We found in our laboratory that Aspirin (high doses), phenylhutazone and indomethacin inhibit the increase of acid phosphatase in liver and inflammed tissue during various types of experimental inflammation in rats (Naik, 1973). There are also conflicting reports regarding action of anti-inflammatory agents on these lysosomal enzymes (Weissman, 1968). However, we feel that these catabolic enzymes, which are degraded from connective tissue or cellular elements make a common pathway for inflammatory process. Hence one should consider these enzymes for the evaluation of anti-inflammatory agents.
There are some reports that a-globulin like macromolecules are present in pregnancy serum or adjuvant arthritic rats and are capable of stabilizing isolated liver lysosomes (Hempel et al., 1970). Thus one may presume that these substances may be endogenous natural antiinflammatory substances.
The future research on the effect of lysosomes stabilization, enzyme deficiency or inhibition may probably lead to find out factors involved in chronic inflammatory process.
B. Prostaglandins (PGS)
Recontly Ramwell and Pharris (1972) have claimed that prostaglandins of E series are involved in cellular injury and inflammation. Most of the non-steroidal anti-inflammatory agents are active inhibitors of its production from its precursor, arachidonic acid. These prostablandins E 1 ,E 2 often induce the increase of vascular permeability in animals and flare response in human beings (Horton, 1963; Crunkhorn and Wills, 1971; Kaley and Weiner, 1971, 1971a). PGE type has been identified in the inflammatory exudate of carrageenin induced inflammation in the rat (Wills, 1969). Prostaglandins occur relatively late in inflammatory process and are often associated with migration of leukocytes into inflammed site (Willoughby, 1971). In rabbits it has been shown that during phagocytosis, prostaglandins are released from leukocyte lysomome (Higgs and Youlten, 1972) and also during endocytosis (Anderson; 1971). Aspinall and Cammarata (1969) and Zurier and Quagliata (1971) have reported that PGE 2 , elicits potent anti-arthritis effects in a model of adjuvant polyarthritis in the rat, but prostaglandin was not anti-inflammatory in acute inflammatory model. PGE 2 , PGA 1 , and PGA 2 , inhibited whereas PGF 2 -α increased the immunologic release of betaglucuronidase from human leukocytes (Zurirer et al., 1973).
Further, PGE 1 , PGE 2 , PGA 1 , PGA 2 , and PGF2-α were reported to depress phagolytosis by polymorphonuclear leukocytes (Cox and Karnovsky, 1973).
Aspirin, indomethacin and salicylates lave been shown by a number of workers to inhibit synthesis or release of PGE 2 and PGF 2-α from arachidonic acid from variety of tissue (Vane, 1971; Smith and Wills, 1971; Ferreira et al. 1971; Smith and Lands, 1971). The inhibition of prostaglandin synthetase by antinflammatory agents depends on its order of potency in carrageenin induced inflammation (Tomlinson et al., 1972). Though non-steroidal anti-inflammatory drugs suppress prostaglandin synthesis, recently it has been shown that PGE 1 and PGE 2 at high doses suppress local inflammation of arthritis and carrageenin induced inflammation (Aspinall et al., 1969; Glenn et al., 1972) with some side and toxic effects like hyperplasia, prostavation and diarrhaea which may contribute to its anti-inflammatory effect.
Denko (1974) has shown by his beautiful experiments the involvement of prostaglandins in urate crystal inflammation. He explained that the prolonged inflammation of urate crystals is due to the constant formation or release of prostaglandins from antecedent phospholipids in the membrane. These released prostaglandins may occur with membranolysis. He concludes that urate crystal inflammation may be a membrane disease.
In many cases the mechanisms by which prostaglandins elicit 'some of their actions are thought to involve cyclic AMP (Kahn and Lands, 1973). Similarly with regard to their potential anti-inflammatory effects certain prostaglandins have been reported to stimulate the synthesis and elevate the levels of cyclic AMP in human leukocyte (Scott, 1970; Bourne and Melmon, 1971. Bourne et al., 1971).
In addition, PGE 1 , PGE 2 and PGF 2 -α were reported to stimulate adenylate cyclase activity in human mixed leukocytes (Poigar et al., 1973). Endogenous cyclic AMP and cyclic GMP might mediate the opposing effects of prostaglandins on lysosomal enzyme secretion from neutrophils. The prostaglandins, especially the biphasic actions of PGF 2 on neutrophil function argue in favour of a modulatory function for these tissue hormones in the inflammatory process. Glucocorticoids reduce the rapid accumulation of cyclic GMP' provoked by immune reactants but have no effect on levels of cyclic AMP.
C. Complement System.
Paulus and Whitehouse (1972) have mentioned some of the complements which together form approximately 10% (w/v) of human serum globulins. These, some people often call reactive proteins, are continually available to all body tissues and play a vital role in the protective mechanism of the organism against exogenous or endogenous injury agents. These complement constituents when set into action against injurious agents often cause damage to the host.
The complement activation further stimulates many other reactions by which many more pathogenic and inflammatory factors are formed for example anaphylatoxin which causes smooth muscle contractions, increases capillary permeability, accumulation of migrated leukotcytes, releasing histamine from tissues, formation of high molecular kinins, lysis of platelets and thereby releasing vasoactive amines and other catalytic enzymes (Muller-Eberhard, 1969). These eleven complements are formed separately by different organs. (Paulus and Whitehouse 1972). Hence one must eventually find out some chemicals or agents which can selectively control the biosynthesis of these different types of complements in various organs.
Trypsin inhibitors are irreversible complement inhibitors in vitro and some of them are also active in vivo but only at subtoxic dose level. Baker and Hurlbut, 1969; Cory et al., 1972). Cobra venom has long been used as complement inhibitor and is now successfully used as suppressor of immune response in organ transplatation.
D. Protein Breakdown Process
Local protein breakdown process may bring about perpetuation or the reduction of inflammation. Hence it will be advisable to think of the use of some anti-proteolytic agents in inflammatory conditions. If anti-proteolytic drugs can prevent the process like histolysis, kinin, kallikrein formation, fibrin deposition, then it will be a real therapeutic use.
However, it has also been shown that exogenous proteases may act as anti-inflammatory agents and increased synthesis of glycoproteins in the liver during inflammatory process. Hence, it is very difficult to say whether the proteolysis may aggravate or inhibit the existing inflammatory process.
Extracellular proteases from plasma transudate infiltrating leukocytes, chondrocytes and synovial cells may degrade albumin (Barnnart et al., 1968) which perhaps enhance the protein synthesis and wound repair by delivering aminoacids and peptides from mobile aminoacid pool.
Our preliminary experiments with treatment of aminoacids namely glycine, glutamic acid, phenylalanine and aspartic acid, in carragenin edema and cotton pellet-granuloma showed good anti-inflammatory effect (Naik et al., 1974a).
The question, however, remains unsolved since the local regulation of proteolytic activity has some advantages and also disadvantages.
Calcium is required for numerous secretory processes (Rubin, 1970). Woodwin et al., 1973) have reported that calcium is required for rabbit granulocytes to discharge f3-glucuronidase in the presence of leucocidin. Smith and Ignarro (1974) have indicated that calcium influx from the extracellular medium into human neutrophils occurs during cell contact with immune reactants at 37°C. Calcium influx was associated with, but preceeded, by lysosomal enzyme secretion from human neutrophils (Ignarro, 1974). Immune reactants such as zymosan treated serum or certain activated complement components operated as calcium ionophores in triggering lysosomal enzyme secretion from human neutrophils (Ignarro 1974). The knowledge that extracellular calcium is required for many reactants to provoke enzyme release suggests that calcium entry into the cells is important. Organic substances which provoke lysosomal enzyme secretion from neutrophils do so by first promoting calcium entry into the cells. Increased intracellular calcium could bring about the accumulation of intracellular cyclic GMP, which in turn could signal the secretion of lysosome granule constituents into the extracellular environment.
F. Cyclic Nucleotides
Advanced research in the bio-regulation of cell function has revealed that cyclic GMP and cyclic AMP serve as a second messenger role in the cellular actions of numerous primary hormones or effective substances. Accumulated evidence suggest that these two naturally occurring cyclic nucleotides play a vital role in expressing the actions of autonomic neurohumors, prostaglandins, glucocorticosteroids and calcium translation on lysosomal enzyme secretion from human neutrophils.
Epinephrine, prostaglandins (PG) E l , norepinephrine, isoproterenol, glucagon or adrenocorticotrophin were reported to stimulate cyclic AMP synthesis in isolated and mixed human leukocytes (Scott, 1970; Bourne and Melmon, 1971). This catecholamine effect was inhibited by beta adrenergic blockers but not by a adrenergic blockers. Ignarro (1974) reported that effect of epinephrine is mediated by intracellular cyclic AMP by virtue of the capacity of this neurohormone to stimulate leukocyte adenylcyclase activity and thereby elevate the levels of cyclic AMP.
Acetylcholine, other cholinergic agents and cyclic GMP analogs markedly accelerate lysosomal enzyme secretion (Ignarro, 1973). Atropine, a muscarin receptor blocker blocks the action of cholinergic agents and neither choline nor guanosine 5' - monophosphate affects enzyme secretion. Cholinergic agents and cyclic GMP enhance the secretion of lysosomal neutral protease activity from purified human neutrophils during phagocytosis of complex or altered IgG and rheumatoid factors (Ignarro 1974) Weissmann and his colleagues (Weissmann et al., 1971, Zurier et al., 1973, Zurier et al., 1973a and Stossel et al., 1972, Cox and Karnovsky, 1973) have shown that cyclic AMP and/or analogs of cyclic AMP have inhibitory effect on phagocytosis induced by polymorphonuclear cells. Epinephrine and isoproterenol were found to reduce phagocytosis by human neutrophils (Ignarro 1974b). Hence, it appears that at least two independent functions of neutrophils can be inhibited by cyclic AMP and catecholamines.
Certain vasoactive hormone-mediators of inflammation and cAMP help in protecting host from the dangerous chain of an unregulated immune respose. cAMP is in control of leukocyte functions, mainly histamine release due to antigen-antibody reactions, hypersensitivity etc. (Bourne et al., 1971, 1972). Beta-adrenergic catecholamines, E series of prostaglandins and histamine itself stimulate accumulation of cAMP in leukocytes by activating adenyl cyclase (Lichtenstein and Gillespie, 1973). Relation of cAMP to regulation of immune and inflammatory response in vivo through the receptor in leukocytes namely neutrophils, thymus derived T cells and lympocytes derived from bone marrow (Bourne et at., 1974).
Hence, nucleotides, both exogenous or endogenous consistently inhibit the secretory events which are responsible for immune response.
Ignarro (1974d) reported that calcium mobilization into cells would stimulate guanyl cyclase and thereby elevate the levels of cyclic GMP. This high level of cyclic GMP would then signal the secretion of lysosomal contents.
Bourne et al., (1974) stressed that the future line of research should be directed towards the implications of receptors of vasoactive amines in leukocytes and effects of cAMP on the early phase of immune response.
Methods for evaluating anti-inflammatory Agents.
Acute inflammatory conditions can be produced in laboratory animals by using various phlogistic agents; however, duration of these inflammatory conditions is quite transitory and these inflammatory conditions can easily be controlled by using currently available antiphlogistic agents. Our main difficulty is dealing with chronic inflammatory conditions which include a large number of diseases and syndromes, caused by one or more unknown factors. Most of the available drugs (steroidal and non-steroidal) to treat these various chronic inflammatory conditions have their limitations due to toxic effects. Hence one should seek a truely non-toxic, yet potent, broad spectrum anti-inflammatory-antiarthritic drug.
Ideally speaking anti-inflammatory drugs should have: (1) effect on prime causative factors, (2) inhibitory effect or blocking effect on initial reaction set in a biological model by the prime cause and thereby inhibit the established inflammation, (3) effect on end results of established inflammation which probably modifies non-specifically the underlying symptoms of inflammation or enhances the repairing process.
Now the next important question is what models might be appropriate and which way they can be developed? These two questions will continually face the workers in this field of research until more satisfactory answers are available.
In vivo Methods.
In a laboratory animal, we often artificially induce a diseased state with foreign substances which can very easily be attacked pharmacologically. We are mentioning some models of inflammatory conditions involving some known and unkown etiological factors with some agents in large and small animals.
Edema Assay -Each one of the cardinal signs of inflammation has been used at one time or other in the search for new anti-inflammatory drugs. Various edemagenic agents have been used viz dilute formalin (Selye, 1949), eggwhite (Winder et al., 1957), Kaolin (WagnerJauregg et al., 1964), Carrageenin (Winter et al., 1962). Besides these, many other irritants like brewer yeast, dextran, serotonin, creatine complex, compound 48,/80, hitamine and mustard are used for producing edema in rats (Winter et al., 1964). Proteolytic enzyme like trypsin (Vogel and Marek, 1963) and glasspowder (Riesterer et al., 1971) are also used for producing local swelling.
Carrageenin edema is quite simple, rapid and gives a constant result with most of the clinically active rheumatic drugs namely aspirin, phenylbutazone, indomethacin, hydrocortisone, etc. Carrageenin edema is mostly involved with prostaglandin liberation and leukocyte migration' and these anti-inflammatory or anti-rheumatic drugs mostly inhibit the prostaglandin liberation and cellular migration of leukocyte and thereby inhibit the inflammatory process (Wills, 1969). Some of the antihistaminics and anti-serotonin also show a slight inhibition which probably indicates involvement of histamine and serotonin in the early phase of inflammation (DiRosa and Willoughby, 1971).
Bradykinin, SH dependent protease and histamine have been shown to be involved in inflammatory process and pain. However, such a picture can be obtained in thermal edema and pleurisy and most of the antibradykinin and antihistaminics effectively inhibit the above mentioned inflammatory process.
Erythema Assay -Erythema has been widely used to screen anti-inflammatory drugs in laboratory animals. Erythema can be induced by ultraviolet light. However, this gives a picture of acute response of injury which is assessed by erythema and local increase of temperature. It is difficult to ascribe this type of response to inflammatory process. Most of the non-steroidal drugs and inhibitors of glycolysis are effective against ultraviolet induced erythema. Most of the steroidal drugs are ineffective.
Considering that the analgesic and antiinflammatory action might be representing two different aspects of a single phenomenon, Randall and Selitto (1957) have reported a technique which demonstrates the effect of anti-inflammatory drugs on pain induced by the injection of silver nitrate into the ankle joints of rats. This method gives some idea regarding pain, swelling of the joints and its improvements with the anti-inflammatory therapy. Aspirin shows a better effect than phenylbutazone and indomethacin. This may be attributed to the better analgesic propery of aspirin, which is also true for rheumatic disorders where aspirin gives more relief than other nonsteroidal anti-inflammatory drugs.
D'Arey and Howard (1967) have described a new method of placing filter paper disc on the chorio-allantoic membrane of the eight day old chick embryo incubating at 37°C for four days and measuring the inflammatory reaction on the adjacent membrane.
There are two animal models of granuloma which have been widely employed.
(a) Implantation of cotton wool pellets (Winter and Portar, 1957)
(b) Granuloma Pouch Seiye, 1953)
In cotton pellet method most of the anti-inflammatory drugs are active at very high doses (e.g. cytotoxic drugs and steroidal drugs). Granuloma pouch is mainly used for assessing the drugs for their topical use rather than parenteral use. Granulation formation is one of the key features in chronic inflammation (Wilhelm, 1966; Shen, 1967) and hence has been employed for screening antiarthritic drugs. By employing granuloma as an experimental model it is possible to study the process of inflammation in detail both from the point of view of pathophysiology and therapeutic approach.
Kulonen (1970) has suggested the following parameters:
(I) Fibroblast population.
(II) Connective tissue which, metabolizes collagen and carbohydrate.
(III) Differentiation of protein synthesis.
(IV) Development and Aging.
(V) Wound healing and repair.
(VI) Pathology of granulation tissue and fibroblast.
(VII) The effect of chemical and physical factors on connective tissue.
Our laboratory experience indicates that granuloma pouch model is often suitable for agents/drugs which are topically active. Recently, we found that in granuloma pouch, there were hardly any systemic changes (biochemical changes in other tissues barring local tissue) except in liver glycogen and blood sugar in initial stages (Rupawalla, 1976). Most of the steroidal drugs are effective at low doses but non-steroidal drugs are active only at doses whihc are ten times the steroidal doses (Naik and Sheth, 1974).
Experimental gouty arthritis model in animals: Urate iinduced inflammationTravsky and Kopecky (1966) employed urate crystals to study the acute phase of inflammatory process. In this procedure, inflammation was induced in the right foot pad of rats by injecting 0.1 ml of 2% suspension of sodium urate crystals in saline, the rear foot pad of the animal serving as control. This method would be of help in the evaluation of anti-gout agents. Van Arman et al., (1970) injected sodium urate or ellagic acid into the synovial space of the stifle joint of the dog and measured synovial fluid and pressure exerted by the appropriate foot. They have compared this with various other methods and stressed that this method offers a better means of comparing the relative potencies of anti-inflammatory anti-gout drugs.
The measurement of delay in the onset of standing on one leg in pigeon after intra-tarsal injection of the talc suspension has been suggested as a method to study anti-inflammatory and antigout drug activity (Benzi et al., 1965).
Experimental arthritis in laboratory animals: Gardner 1960 has reviewed, in detail methods which have been described in the literature to produce arthritis in small animals by injecting various infective, chemical, hormonal, immunological or physical agents into the joints.
Formaldehyde arthritis can be produced by the injection of formalin (Brownlee, 1956).
In formaldehyde arthritis, we found most of the non-steroidal drugs ineffective; however, steroidal anti-inflammatory drugs show good anti-arthritic activity (Naik, 1973). Our laboratory experience indicates, that one should not use this type of model for screening anti-inflammatory drugs of non-steroidal origin unless they have a steroidal type of activity for e.g. methyl glycyrrhetic acid and glycyrrhetic diacetate (Tangri et al, 1965). Poly arthritis has been induced in rats by injecting various biological preparations of diverse nature. The arthritic syndrome is induced by injection of Freunds adjuvant consisting of dead and fast mycobacteria in liquid paraffin without any additional antigen (Wakesman et al., 1960; Ward and Jones, 1962; Newbould, 1963 and Pearson, 1964). This syndrome was characterised by the appearance of inflammed lesion remote from the injection site approximately after 10 days. However, we found that all the rats do not show the secondary and tertiary lesions with this adjuvant. This may also be equally true in human-disease like Rheumatoid varients or Rheumatoid arthritis which occur sporadically in certain more susceptible members of an out bred population and does not occur in some others.
Paulus and Whitehouse (1972) have mentioned two types of experimental arthritis (a) Catheptic arthritis, induced by lysosomal enzymes mainly from leukocytes and liver into the joints of the rabbits, (b) Immune arthritis, induced by the second injection of antigen into the joints of the rabbits which are preimmunized.
Mycoplasma arthritis L 4 was originally isolated from the Murphy-Sturn lymphosarcoma of the rats. This substance can now be taken from the arthritic joints of infected animals (Klieneberger, 1962).
While evaluating the effect of antiphlogistic or anti-rheumatic agents on the above mentioned arthritis one can divide the effect into two parts.
(1) Effect during onset of arthritic syndrome.
(2) Effect on established arthritis.
Although this method is preferred by everybody, most of non-steroidal antiinflammatory agents do not suppress the secondary and tertiary lesions. However, they delay the onset of the lesions and inhibit the local inflammations (Naik, 1973). In Freunds adjuvant induced arthritis we have also considered some other tests like motility of peripheral joints (determined by grip function test), pain threshold of the joints and general health condition of the animal (Naik, 1973).
Many immunosuppressants and antimetabolites suppress the secondary and tertiary lesions in adjuvant arthritis in animals but they have very high toxic effects (Paulous and Whitehouse, 1973).
These experimental models have primarily dealt with inflammatory conditions; as we have already pointed out earlier in the introduction, one should consider the inflammatory condition in other types of diseases like allergy hypersensitivity reactions and immunological disorders. We mention briefly some of the experimental models which perhaps may be beneficial for screening potential drugs for chronic inflammatory processes e.g. systematic lupus erythematosus (SLE) (Hollander 1966). This is an immunologically mediated disease accompanied by inflammation and attacks mainly kidneys, skin, joints, heart and blood vessels. Despite the extensive immunological research the prime cause of S.L.E. remains unsolved. Recent experiments with Aleutian minks and NZB mice showed the possibility that S.L.E. is of viral origin (Williams, 1968). Many cytostatic immunosuppressants, salicylates, antimalarial drugs are also used. Canine systemic lupus erythematosus has been described by Lewis (1968) as a spontaneously developing disease in animals which often respond to high doses of corticosteroids.
Mycoplasmal infections of animals (rats, swine, turkeys) may be associated with chronic inflammation of joints, pericardium, respiratory and genital organs (Walton, 1968).
Rejection of transplanted organ is often mediated by protective immunological reaction which is manifested by sequelae of inflammatory reactions and loss of function. Hence it is very useful to use such model to screen the anti-inflammatory anti-arthritic activity of new compounds. A pharmacologist should always seek to extinguish the inflammatory state altogether by deleting either this histocompatibility of antigen of the donor (the cause of inflammation in homograft) or recognition by the recipient.
Most of the dermatological conditions comprise of acute, subacute or chronic inflammatory reactions to many unknown prime causes.
Contact dermatitis is an example of delayed hypersensitivity. This can be induced in the guinea-pig foot pads by painting with picrylchloride. The symptoms can be seen after 2-5 days. One should employ this as a screening method for anti-inflammatory agents because this often is accompanied by inflammation. Our initial experiments failed to give positive effect with non steroidal antiinflammatory drugs on contact dermatitis. However, steroids are very effective in contact dermatitis. The dermatological manifestations are due to bacteria, or virus and are often a part of some generalized chronic inflammatory diseases (rheumatic fever, ulcerative colitis, etc). Inflammation of the small blood vessels or capillaries and inflammatory cells infiltrates are prominent aspects of many skin diseases.
All these above mentioned animal models of chronic inflammatory diseases could be employed for screening compounds for efficacy in attaining any goals of therapy which have been mentioned in the earlier part of the review.
Screening methods in "in vitro" Systems
In vitro studies may throw some light on the mode of action of anti-phlogistic agents at molecular level. These studies are often carried out in isolated organ system, or cellular or subcellular preparations with an idea that both of these preparations are drug sensitive and sufficiently retain the required characteristics (Pacemaker events) which sustain the on-going disease processes.
These in vitro procedures are quit simple, rapid and affords an empirica. screening procedure for many compounds
Now let us consider some of the available in vitro procedure for screening anti phlogistic agents.
Protein denaturation has been employee as an in vitro screening method for anti-phlogistic agents by Mizushima and his co-workers (Mizushima, 1964; Mizushima and Kobayashi, 1968). Grant et al. (1970) also confirmed their work and reported that anti-inflammatory drug., inhibit protein denaturation. Drug binding to plasma albumin may inhibit thermal denaturation of albumin which perhaps block Ǿ -NH 2 groups in case of histidine decarboxylase or may displace urate from albumin (Skidmore and Whitehouse, 1965).
Red cell aggregation induced by using various agents like gelatin, carrageenin, nucleotides etc, have proved to be effective screening non-steroidal anti-inflammatory drugs, (Gorog et al., (1970). Actomycin like contractile protein having ATPase activity is situated on the outer surface of erythrocytes. It probably plays an important role in maintaining the form of erythrocyte and distribution of the surface charge on the outer membrane. The non-steroidal anti-infiammatory drugs bind to this contractile protein and inhibit its ATPase activity and its contractile property (Gorog et al., 1970). They have further stated the existence of a relation between the effect in the connective tissues and that exerted on the erythrocyte membrane (Gorog et al., 1970). Famaey and Whitehouse (1975) have suggested that non-steroidal anti-inflammatory drugs may affect various membranes differently, depending upon the concentrations of drugs available and on the natural composition and function of the membrane.
Lysosome membrane stabilization property of drugs can also be determined by using erythrocyte lysis by heat, hypotonic solution or by some other means (Grant et al., 1970).
Effect of anti-inflammatory drugs on synthesis biopolymers in intact cells like fibroblasts, lymphocytes or tissue slices like cartilage etc., may probably be located in the site and mechanism of action.
Van Arman et al., (1974) have reported the leukocyte activation and its involvements in initiation of inflammatory process. Such types of in vitro reaction can be carried out using lymphocytes and its response to antigen or mitogen or polymorph response to urate crystals, latex particles and chemotactic agents.
Aggregation of red cells and erythrocyte sedimentation rate.
The erythrocyte sedimentation rate (ESR) is a reliable index of red cells adhesion, rouleaux formation, inflammatory process, changes in electrostatic charge on the membrane and a variety of other experimental and clinical situations (Ziff and Baum, 1966). Non-steroidal antiinflammatory agents inhibit red cell hemolysis caused by heat, hypotonicity and heterologous anti-serum to red cell membranes. (Brown et al., 1967, 1971; Glenn et al., 1969). Phenyl butazone and other anti-inflammatory drugs inhibit red cell aggregation induced by high molecular dextran, fibrinogen and gelatin. Most of the non-steroidal antiinflammatory drugs are acidic in nature and hence, positive charges are available on damaged or altered cell membranes and thereby prevent their tendency to adhere, aggregate, clump or pull together.
Though red cell aggregation is a convenient method for the assessment of nonsteroidal anti-inflammatory agents it fails to give consistent results like many other isolated methods. Many diverse chemical agents give false positive results with this method (Glenn et al., 1971). However, recently Famaey and Whitehouse (1974) have stressed that an acidic function is not required for membrane activities of non-steroidal anti-inflammatory drugs.
These in vitro methods give many false positive results which may lead to wrong conclusions. Hence one must select the in vitro method more carefully and interpret the results accordingly. However, in our mind these in vitro tests do give certain clues regarding the possible site and mechanism of action and can readily be employed for screening a large number of compounds as a preliminary test.