New analgesic assay utilizing trypsin-induced hyperalgesia in the hind limb of the rat.

Subplantar injection of 250 micrograms of trypsin in the rat resulted in a biphasic increase in pain sensitivity (hyperalgesia) with peaks at 10 and 150 min separated by a period of decreased sensitivity to pain (hypoalgesia). Hyperalgesia was assessed by a decrease in response latency to a 3.0-kg force applied to the injected hind limb. Response latencies at 150 min were increased in a dose-dependent manner by pretreatment at 90 min with acetaminophen; phenacetin; the arachidonate cyclooxygenase inhibitors aspirin, indomethacin, and ibuprofen; and the opiate analgesics codeine and morphine. ED50s of 17, 13, 10, 0.48, 1.6, 3.9 and 1.2 mg/kg p.o. were obtained for these drugs, respectively. The hyperalgesia present at 150 min was not affected by pretreatment with antiinflammatory steroids, an antihistaminic, an antiserotonin agent, and an anticholinergic. We recommend measurement of drug-induced increase in response latencies produced 150 min after injection of 250 micrograms of trypsin as the basis for a new sensitive and selective analgesic assay. ED50s obtained in this assay correlate well with doses that are used clinically to produce analgesia. Development of the hypoalgesic component was selectively inhibited by pretreatment with an antiserotonin agent. Additional drug studies indicated that the algesic response to the subplantar injection of trypsin is the resultant of independent, temporally overlapping hyperalgesic and hypoalgesic components.

Nucleosides of azathioprine and thiamiprine as antiarthritics.

Azathioprine [Imuran; 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)thio]-1H-purine] is a widely used immunosuppressive and antiarthritic drug. For the sake of comparison, the riboside, the 2'-deoxyriboside, and the arabinoside of azathioprine and its 2-amino congener, thiamiprine, were prepared by an enzymatic method. In vitro, the cytotoxicities of these aglycons and their nucleosides were similar (ED50 = 0.8-2 microM), except for the arabinosides, which were nontoxic (ED50 greater than 100 microM). In vivo, their activities were compared in the rat adjuvant arthritis model. The ribosides and 2'-deoxyribosides were less potent than their corresponding aglycons. The safety indexes of these nucleosides were comparable to those of the corresponding aglycons except for the 2'-deoxyriboside of azathioprine, which had an appreciably lower safety index than did azathioprine. Both arabinosides were inactive and nontoxic. All of the aglycons tested (6-mercaptopurine, azathioprine, 6-thioguanine, and thiamiprine) were of similar potency. However, azathioprine had a more favorable therapeutic index than did 6-mercaptopurine. Similarly, thiamiprine was safer than was 6-thioguanine. In this model, the S-(1-methyl-4-nitro-1H-imidazol-5-yl) moiety imparted greater safety to these thiopurines by decreasing toxicity but not affecting potency.

Pharmacological characterization of the algesic response to the subplantar injection of serotonin in the rat.

Subplantar injection of 0.10 micrograms of serotonin in the rat resulted in a brief period (0-20 min) of increased pain sensitivity to an applied force (hyperalgesia) which preceded a longer period (40-120 min) of decreased pain sensitivity (hypoalgesia). The magnitude of each of these changes and the duration of the hypoalgesia were dose-dependent. The development of hyperalgesia was selectively and dose dependently reduced by inhibitors of arachidonate cyclooxygenase. The hypoalgesia was selectively and dose dependently reduced by the serotonin antagonist methysergide. Selective inhibition of the hyperalgesia by aspirin and of the hypoalgesia by methysergide revealed that components of both hyperalgesia and hypoalgesia were present in the 10-120 min interval. These findings, the level of serotonin reported to be released in rat dermal tissue, and selective drug inhibition studies suggest that some irritant-induced changes in algesia measured in the rat hindlimb result from release of dermal stores of serotonin. Selective inhibition of the hypoalgesic component of the hindlimb irritant trypsin by the antiserotonin agent methysergide supports this hypothesis. The principal conclusion derived from these studies is that the algesic response to the subplantar injection of a single agent can be the resultant of independent, temporally overlapping hyperalgesic and hypoalgesic components each of different intensity and pharmacological sensitivity.

Pathway to carrageenan-induced inflammation in the hind limb of the rat.

A sequential 43-step pathway scheme for the inflammatory response of the rat to interdermal injection of carrageenan (C) was devised. It consisted of a nonphagocytic inflammatory response (NPIR) followed by a phagocytic inflammatory response (PIR) in the dermis and an epidermal NPIR. The dermal NPIR comprised edema, hyperemia, and hyperalgesia followed by hypoalgesia. Antiserotonin agents inhibited the hypoalgesia and part of the edema. These findings and histological observations suggested that dermal mast cells were injured by C. The hyperalgesia and part of the edema were sensitive to arachidonate cyclooxygenase inhibitors (AACOIs). It is speculated that injured mast cells metabolize arachidonic acid and reactive intermediates, not prostaglandins, mediate the NPIR hyperalgesia and part of the edema. The dermal PIR consisted of mobilization of neutrophils, edema, hyperalgesia, mobilization of monocytes, and proliferation of fibroblasts and vascular tissue. Selective drug actions revealed that the edema, hyperalgesia, and monocyte mobilization of the PIR depended on the mobilization of neutrophils. After the mobilization of neutrophils, AACOIs reduced edema formation and hyperalgesia. Arachidonic acid metabolism by neutrophils is speculated to produce the mediators of phagocytic inflammatory (PI) edema and hyperalgesia. Monocyte function was associated with cessation of PI edema formation and phagocytosis of neutrophils and cellular debris. Interleukin 1 is speculated to mediate the adherence of neutrophils to injured dermal endothelium. The epidermal NPIR consisted of edema, hyperplasia, and hyperkeratosis. These parameters were not studied mechanistically. There was no evidence for histamine, bradykinin, platelets, clotting factors, or complement mediating any events in the pathway.

Imidazo[4,5-c]pyridines (3-deazapurines) and their nucleosides as immunosuppressive and antiinflammatory agents.

A variety of imidazo[4,5-c]pyridines (3-deazapurines) were synthesized. With use of these aglycons as pentosyl acceptors, the corresponding ribonucleosides and 2'-deoxyribonucleosides were prepared by an enzymatic method involving transfer of the pentosyl moiety from appropriate pyrimidine nucleosides. With most of the imidazo[4,5-c]pyridines, the products obtained from the enzyme-catalyzed reactions were pentosylated exclusively in the 1-position. However, some 3-pentosylation occurred with aglycons that had H or N3 in the 4-position. In addition to the 2'-deoxy congener of the ribonucleoside of 4-amino-1H-imidazo[4,5-c]pyridine, the 5'-deoxy and 2',5'-dideoxy congeners were synthesized. All of the aglycons and their nucleosides were tested for toxicity to mammalian cells in culture. None were markedly cytotoxic. These compounds were also evaluated for their ability to inhibit lymphocyte-mediated cytolysis in vitro. 3-Deazaadenosine (23) and its 2'-deoxy congener (38) were the most potent inhibitors (ED50 = 20 microM). In addition to these two in vitro tests, in vivo inhibition of the inflammatory response in the rat carregeenan pleurisy model was determined. 3-Deazaadenosine (23) was the most potent compound (ED50 = 3 mg/kg) in this in vivo test.

Arachidonate metabolic pathways in cells harvested from rat pleural cavity at various times after carrageenan administration.

Cells were harvested from rat pleural cavity before and during the inflammatory response stimulated by carrageenan injection. The conversion of [14C]arachidonate by intact cells into products of the cyclooxygenase and 5-lipoxygenase pathways was studied in the absence and presence of ionophore. Incorporation of arachidonate into phosphatidic acid was also followed. In the absence of ionophore, the principal arachidonate metabolites of resident macrophages were the cyclooxygenase products, prostacyclin and thromboxane, while mobilized monocytes produced thromboxane as a major product, but very little prostacyclin. Arachidonate-metabolizing enzymes in mobilized monocytes were, in general, less active than those of resident macophages. Cells harvested at times when mobilized neutrophils were the predominant cell type were capable of converting arachidonate into thromboxane and prostaglandins, but not prostacyclin. These cells exhibited the most active turnover of arachidonate into phosphatidic acid, and the extent of this turnover appeared to be temporally related to the presence of edema in the pleural cavity at the time of cell harvest. Enzymatic formation of 5-lipoxygenase products was dependent on calcium and was markedly stimulated by ionophore A23187 in both resident and mobilized pleural cells. Among several non-steroidal drugs tested, cyclooxygenase inhibitors were the most effective in preventing the inflammatory response in the carrageenan model of inflammation.

Pathway of onset, development, and decay of carrageenan pleurisy in the rat.

A sequential 37-step pathway scheme has been devised that describes the actions and events responsible for the onset, development, and decay of carrageenan pleurisy. It is postulated that the subpleural cytotoxicity of absorbed carrageenan initiates the response by producing a biphasic subpleural inflammation, the first phase of which precedes any sign of pleural exudation. Pleural exudation began 1 h after the injection of carrageenan and consisted of mobilized neutrophils and a barely detectable exudate volume. The time course of intrapleural neutrophil mobilization was monophasic (S shaped). Monocyte mobilization began after neutrophil mobilization and was also monophasic. Pleural exudate formation was biphasic. The first exudative phase was sensitive to inhibitors of neutrophil mobilization and arachidonate acid cyclooxygenase (AACO). Drug studies revealed that although neutrophils were required to initiate the first exudative process, the cells of the pleura produced a postulated reactive prostaglandin intermediate that increased vascular permeability and resulted in exudate formation. The etiology of the second exudative phase is unknown. This phase is insensitive to AACO inhibitors but is highly sensitive to steroids. Inhibition of monocyte mobilization by colchicine revealed that these were not associated with any exudate formation. Monocytes are postulated to stop the exudative process. These cells phagocytose the mobilized neutrophils and return the pleural cavity to normal. Thus, in this model of acute inflammation, monocytic function is related solely to anti-inflammatory activities.

Development of carrageenan pleurisy in the rat: effects of colchicine on inhibition of cell mobilization.

Colchicine produced three effects which modified the acute inflammatory response to carrageenan in the rat pleural cavity: (i) inhibition of neutrophil mobilization and concomitant exudate formation (3 hr); (ii) inhibition of monocyte mobilization (21 hr); and (iii) augmented exudate formation (3 and 21 hr). The 1st effect was related to the intraperitoneal dose of colchicine and occurred only at leukopenic dose levels This effect could not be produced by intrapleural injection of nonleukopenic doses of colchicine. The second effect, on the other hand, was produced by intraperitoneal leukopenic doses and to a lesser extent by intrapleural administration of nonleukopenic doses of colchicine. Importantly, the normal biphasic exudative response to carrageenan developed fully in the absence of monocytes. The third effect, a dose-dependent augmentation of both exudative phases of carrageenan pleurisy, was produced by low, nonleukopenic, intrapleural doses of colchicine. The augmented exudate was sensitive to prostaglandin synthetase inhibitors but not to anti-inflammatory steroids. Neither neutrophils nor monocytes were responsible for the augmented exudate. Colchicine, injected into the rat hindlimb or pleural and peritoneal cavities did not elicit the mobilization of neutrophils or a pleural effusion. In addition, colchicine did not affect the magnitude, temporal development, or decay of the potent edematogenic action of serotonin in the rat hindlimb. Thus irritancy was not responsible for any of the effects of colchicine.

Azathioprine treatment of adjuvant arthritis.

Administration of azathioprine in the diet to Lewis rats reduced the lethality of this drug relative to various regimens by gavage. This mode of administration made it possible to administer effective doses with reduced toxicity. Azathioprine blocked the early development of adjuvant arthritis and decreased the joint scores of animals with established adjuvant disease. A combination of azathioprine and prednisolone produced an additive reduction of both developing and established joint scores. A 10 day pulse regimen of prednisolone resulted in a strong, rapid decrease in established joint scores. This decrease was sustained by continuous administration of azathioprine. In all cases removing azathioprine and/or prednisolone from the diet of animals with established adjuvant arthritis resulted in recurrence of disease. The results of these studies support the validity of the adjuvant model for prediction of anti-arthritic activity.

Quantitative studies of the pathway to acute carrageenan inflammation.

A 12-step scheme representing initiation and development of acute carrageenan inflammation in the rat has been devised. Simultaneous quantitative temporal measurement of the number of inflammatory cells mobilized and edema volume produced as carrageenan pleurisy developed helped elucidate several of the early steps, In the pleurisy a 20-min lag phase preceded both the mobilization of neutrophils and edema formation. Temporal histological studies suggested that the lag phase represents the time needed for the neutrophil chemotactic factor to be generated and/or released (steps 2 and 3) in addition to the time required for these cells to move through the walls of the capillaries into the pleural space (steps 4 and 5). The neutrophil chemotactic factor is unknown. The complement system is not involved since cobra venom factor-treated animals produced a normal edematous and cellular response to carrageenan in spite of severely depressed complement levels. The mobilized neutrophils are believed to phagocytize the carrageenan(step6). In this process lysosomal enzymes are released (step 7). Drug inhibition studies suggest that lysosomal enzymes are not the edemagenic agents in carrageenan inflammation. In the scheme these enzymes are responsible for activation of the prostaglandin biosynthetic chain (step 8). Prostaglandin biosynthesis (step 9) leads to the release of an intermediate (step 10), as yet unidentified, which is responsible for increased tissue permeability (step 11) and edema formation (step 12). Histamine, serotonin, bradykinin, arachidonic acid, and prostaglandin's E1 and E2 are not deemagenic in the rat pleural cavity and therefore cannot be responsible for edema formation. Aspirin and indomethacin reduce the edema produced by carrageenan in a dose-related manner without affecting the magnitude or the time course of neutrophil mobilization. These findings led to the concept that edema formation is not the result of inflammatory cell mobilization but is rather a consequence of cellular activity, presumably phagocytosis, after mobilization.

Quantitative comparison of the analgesic and anti-inflammatory activities of aspirin, phenacetin and acetaminophen in rodents.

The mild analgesic activities of aspirin, phenacetin and acetaminophen have been compared in the trypsin, kaolin and carrageenan hyperalgesic assays as well as in the acetic acid writhing test. The trypsin and kaolin hyperalgesic assays were designed to be unaffected by drugs with anti-inflammatory activity. Aspirin and acetaminophen were inactive in these two tests at dose levels devoid of side effects. Phenacetin was active in the trypsin and kaolin assays with oral ED50's of 114 +/- 36.2 and 107 +/- 11.5 mg/kg, respectively. Non-steroidal anti-inflammatory drugs as well as phenacetin and acetaminophen were active in the acetic acid writhing and carrageenan hyperalgesic assays. This led to evaluation of phenacetin and acetaminophen as anti-inflammatory agents. Both of these latter drugs were active in the carrageenan pleurisy and adjuvant arthritis models of inflammation. In all studies phenacetin was equipotent to or more potent than acetaminophen. The data suggest that the analgesia produced by aspirin and acetaminophen results from their anti-inflammatory activity whereas the analgesia produced by phenacetin has two components, one dependent on and one independent of anti-inflammatory activity.

Potentiation of the anti-inflammatory and analgesic activity of aspirin by caffeine in the rat.

Caffeine has been found to potentiate the acute anti-inflammatory activity of aspirin, indomethacin, and phenylbutazone, but not the activity of sodium salicylate or hydrocortisone, in the carrageenan pleurisy or hindlimb models of inflammation in the rat. The mobilization of inflammatory cells was not affected by aspirin in the presence or absence of caffeine. The mild analgesia produced by aspirin was confined to a hyperalgesic test in which this drug was able to reduce inflammation and concomitant hyperalgesia and thereby produce an "apparent" analgesic effect. This "apparent" analgesia produced by aspirin was potentiated by caffeine. The mechanism responsible for the potentiated anti-inflammatory and mild analgesic activity of aspirin remains unknown since caffeine did not alter the plasma salicylate levels or prostaglandin synthetase inhibition produced by aspirin.