Pharmacological Research, Vol . 24, No . 4 . 1991
377
CARBONIC ANHYDRASE INHIBITORS ARE ANTIARTHRITIC IN THE RAT JOSEPH C . NOLAN*, CAROL E . GATHRIGHT, CYNTHIA H . RADVANY, RICHARD J . BARRETT* and LAWRENCE F . SANCILIO Department of Pharmacology, A . H . Robins Co ., 1211 Sherwood Avenue, Richmond, VA 23220, USA Received in final form 2 May 1991
SUMMARY Adjuvant-induced arthritis in rats was attenuated by the therapeutic administration of carbonic anhydrase inhibitors . Female Lewis rats with established disease were treated daily (day 18 through day 50) with various carbonic anhydrase inhibitors ; oedema and joint integrity (X-ray) were determined post-treatment . Acetazolamide, ethoxzolamide, methazolamide, and dichlorphenamide reduced paw oedema and attenuated the deterioration of the joints of rats with adjuvant arthritis . However, no carbonic anhydrase inhibitor tested possessed significant, acute, anti-inflammatory activity in the carrageenan-paw oedema test . The activity of carbonic anhydrase inhibitors in the chronic model of inflammation may be due to their reported inhibition of bone resorption . KEY WORDS :
arthritis, carbonic anhydrase inhibitors .
INTRODUCTION Inflammatory arthritis is characterized by the loss of the integrity of connective tissue structures of the joint . This degeneration is mediated by a variety of inflammatory cells and soluble factors released from those cells [1] . Interleukin-1 and other cytokines have been shown to stimulate neutral protease and collagenase production in cultures of chondrocytes and human synovium [1], thus suggesting a rational hypothesis for the destruction of collagenous structures of the joint . Bone resorption is also an important clinical feature of the arthritic diseases [2, 3] . Osteoclasts in the vicinity of mononuclear cells are activated [4] . Potent bone resorbing agents, such as PGE 2 , are available to induce resorption [5] . Together, these results suggest that the cells and mediators potentially responsible for cartilage and bone destruction are present in the inflamed joint . Acetazolamide, a potent carbonic anhydrase inhibitor, inhibits bone resorption induced by disuse in the rat [6] and, in vitro, inhibits bone resorption induced by *Present address : Whitby Research Incorporated, 2801 Reserve Street, Richmond, VA 23220, USA . 1043-6618/91/080377-07/$03 .00/0
© 1991 The Italian Pharmacological Society
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Pharmacological Research, Vol. 24, No . 4, 1991
parathyroid hormone, PGE 2 , or by 1,25-dihydroxyvitamin D 1 [7, 8] . Recently, these effects on bone resorption have been extended to other carbonic anhydrase inhibitors, such as ethoxzolamide and HTS (5-3-(hydroxybenzoyl)-2thiophenesulphonamide [9] . The availability of various carbonic anhydrase inhibitors and their inhibition of bone resorption in vivo and in vitro permitted us to determine the effects of these drugs in a chronic model of arthritis, the adjuvant arthritic rat . In the studies presented here, we report that carbonic anhydrase inhibitors show antiarthritic activity in rats .
MATERIALS AND METHODS Adjuvant arthritis
Female Lewis rats that weighed approximately 180 g (Charles River Laboratories, Wilmington, MA) were used in these studies . The animals were kept on a 12-h light/dark cycle with food and water available ad libitum . On day 0, 0 .05 ml of a suspension of 1 .5% Mycobacterium butyricum in mineral oil was injected into the subplantar surface of the right hind paw . On day 18, the hind paw volume of the uninjected hind limb was determined by mercury displacement ; rats with significant paw swelling (>2 .4 ml) of the uninjected hind limb were randomized by block design into groups of 7 . All drugs were dissolved or suspended in the vehicle (0 .5% Tween 80) and were given daily by gavage (10 ml/kg) to the rats from day 18 through day 50 after adjuvant injection (excluding weekends) . Oedema was determined by the difference (from day 18 paw volume) on days 29 and 50 . At the termination of the experiment, the rats were sacrificed by CO2 inhalation and the uninjected hind limb was removed, Xrayed, and scored on an arbitrary scale (1, no damage ; 10, maximum damage) in a blinded manner .
Carrageenan oedema
Carrageenan-induced oedema was determined in female Lewis rats (150-175 g) . Groups of six fed rats were dosed orally with test compounds on four consecutive days prior to injection of 0 .1 ml of a 1% carrageenan solution (in saline) into the subplantar surface of the right hind paw . The last dose of drug was given 1 h prior to the injection of the carrageenan solution . Oedema was determined by the difference in paw volumes measured (by mercury displacement) prior to and 3 h post-carrageenan challenge .
Carbonic anhydrase assay
The inhibitory activity of the compounds against carbonic anhydrase (from bovine erythrocytes) was determined in the following manner . Approximately 10 units of bovine carbonic anhydrase in 6 ml of barbitone (barbital) buffer were incubated for 10-15 s with various amounts of the test drugs . The reaction was initiated by the addition of substrate (4 ml C0 2 -saturated H 2 O) . The rate of
Pharmacological Research, Vol. 24, No . 4, 1991
379
enzyme-catalysed change in pH was followed, and the IC 50 was calculated (graphically) from the concentration-% inhibition curves .
Materials and statistical analyses
Acetazolamide, dichlorphenamide, indomethacin, and carbonic anhydrase were purchased from the Sigma Chemical Company, St Louis, Missouri . Ethoxzolamide and methazolamide were synthesized in the Chemical Research Department of the A . H . Robins Company, Richmond, Virginia . Data were analysed by using the Dunnett's t-test to compare the effects of drug treatments to a control .
RESULTS Various carbonic anhydrase inhibitors (acetazolamide, ethoxzolamide, methazolamide, and dichlorphenamide), as well as the reference non-steroidal anti-inflammatory drug (NSAID), indomethacin, were given to groups of seven rats with established adjuvant arthritis . The results in Table I represent the pooling of several experiments . All of the carbonic anhydrase inhibitors tested produced anti-inflammatory activity as determined on day 29 or on day 50 (Table I) . They also attenuated the changes seen in the X-ray score of the arthritic joint on day 50 (Table I) . Significant activity was observed with the lowest dose of each drug tested . From the data presented, it is apparent that the potent carbonic anhydrase
Table I Antiarthritic activity of carbonic anhydrase inhibitors and indomethacin in adjuvant-arthritic rats Treatment
Vehicle Indomethacin Acetazolamide
n
55 55 28 35 7 7 7 7 14 7
Dose (mglkg) p .o .
3 .16 3 .16 10 .0 31 .6 1 .0 3 .16 3 .16 31 .6 100
Oedema' day 29 (ml±so)
Oedema" day 50 (ml±so)
X-Ra v score (mean±so)
C .A ." iC :,, (pM)
0 .19±0 .28 -0 .05±0 .43 8 .57±1.03 3 .80±0 .72` -0 .73±0 .22` -0 .63±0.30` >34 -0 .21±0 .23` -0 .42±0.20` 6 .30±1 .49` 0 .072 -0 .32±0 .21` -0 .53±0 .28` 6 .37±0 .79` -0 .43±0 .20` -0 .69±0 .18` 6 .79±0 .74` Ethoxzolamide -0 .03±0 .20` -0 .36±0 .17` 6 .89±1 .07` 0 .016 -0 .29±0 .20` -0 .43±0 .35` 6 .11±1 .31` Methazolamide -0 .24±0 .35` -0 .43±0 .24` 6 .71±I .01` 0 .040 Dichlorphenamide -0 .10±0 .20` -0 .34±0 .27` 7 .93±1 .30` 0 .233 -0 .29±0 .25` -0 .54±0 .25` 5 .89±0 .45` Compounds were given to the rats daily (excluding weekends), by gavage, beginning on day 18 after adjuvant injection and continuing through day 50 after adjuvant injection . Vehicle-treated animals received 0 .5% Tween in H 2 O (10 ml/kg) . X-rays of the uninjected hind limb were made on day 50 . Oedema=difference between paw volume on day 29 or day 50 and paw volume on day 18 (first day of drug treatment) . hC .A .=carbonic anhydrase in vitro . `P
377
CARBONIC ANHYDRASE INHIBITORS ARE ANTIARTHRITIC IN THE RAT JOSEPH C . NOLAN*, CAROL E . GATHRIGHT, CYNTHIA H . RADVANY, RICHARD J . BARRETT* and LAWRENCE F . SANCILIO Department of Pharmacology, A . H . Robins Co ., 1211 Sherwood Avenue, Richmond, VA 23220, USA Received in final form 2 May 1991
SUMMARY Adjuvant-induced arthritis in rats was attenuated by the therapeutic administration of carbonic anhydrase inhibitors . Female Lewis rats with established disease were treated daily (day 18 through day 50) with various carbonic anhydrase inhibitors ; oedema and joint integrity (X-ray) were determined post-treatment . Acetazolamide, ethoxzolamide, methazolamide, and dichlorphenamide reduced paw oedema and attenuated the deterioration of the joints of rats with adjuvant arthritis . However, no carbonic anhydrase inhibitor tested possessed significant, acute, anti-inflammatory activity in the carrageenan-paw oedema test . The activity of carbonic anhydrase inhibitors in the chronic model of inflammation may be due to their reported inhibition of bone resorption . KEY WORDS :
arthritis, carbonic anhydrase inhibitors .
INTRODUCTION Inflammatory arthritis is characterized by the loss of the integrity of connective tissue structures of the joint . This degeneration is mediated by a variety of inflammatory cells and soluble factors released from those cells [1] . Interleukin-1 and other cytokines have been shown to stimulate neutral protease and collagenase production in cultures of chondrocytes and human synovium [1], thus suggesting a rational hypothesis for the destruction of collagenous structures of the joint . Bone resorption is also an important clinical feature of the arthritic diseases [2, 3] . Osteoclasts in the vicinity of mononuclear cells are activated [4] . Potent bone resorbing agents, such as PGE 2 , are available to induce resorption [5] . Together, these results suggest that the cells and mediators potentially responsible for cartilage and bone destruction are present in the inflamed joint . Acetazolamide, a potent carbonic anhydrase inhibitor, inhibits bone resorption induced by disuse in the rat [6] and, in vitro, inhibits bone resorption induced by *Present address : Whitby Research Incorporated, 2801 Reserve Street, Richmond, VA 23220, USA . 1043-6618/91/080377-07/$03 .00/0
© 1991 The Italian Pharmacological Society
37 8
Pharmacological Research, Vol. 24, No . 4, 1991
parathyroid hormone, PGE 2 , or by 1,25-dihydroxyvitamin D 1 [7, 8] . Recently, these effects on bone resorption have been extended to other carbonic anhydrase inhibitors, such as ethoxzolamide and HTS (5-3-(hydroxybenzoyl)-2thiophenesulphonamide [9] . The availability of various carbonic anhydrase inhibitors and their inhibition of bone resorption in vivo and in vitro permitted us to determine the effects of these drugs in a chronic model of arthritis, the adjuvant arthritic rat . In the studies presented here, we report that carbonic anhydrase inhibitors show antiarthritic activity in rats .
MATERIALS AND METHODS Adjuvant arthritis
Female Lewis rats that weighed approximately 180 g (Charles River Laboratories, Wilmington, MA) were used in these studies . The animals were kept on a 12-h light/dark cycle with food and water available ad libitum . On day 0, 0 .05 ml of a suspension of 1 .5% Mycobacterium butyricum in mineral oil was injected into the subplantar surface of the right hind paw . On day 18, the hind paw volume of the uninjected hind limb was determined by mercury displacement ; rats with significant paw swelling (>2 .4 ml) of the uninjected hind limb were randomized by block design into groups of 7 . All drugs were dissolved or suspended in the vehicle (0 .5% Tween 80) and were given daily by gavage (10 ml/kg) to the rats from day 18 through day 50 after adjuvant injection (excluding weekends) . Oedema was determined by the difference (from day 18 paw volume) on days 29 and 50 . At the termination of the experiment, the rats were sacrificed by CO2 inhalation and the uninjected hind limb was removed, Xrayed, and scored on an arbitrary scale (1, no damage ; 10, maximum damage) in a blinded manner .
Carrageenan oedema
Carrageenan-induced oedema was determined in female Lewis rats (150-175 g) . Groups of six fed rats were dosed orally with test compounds on four consecutive days prior to injection of 0 .1 ml of a 1% carrageenan solution (in saline) into the subplantar surface of the right hind paw . The last dose of drug was given 1 h prior to the injection of the carrageenan solution . Oedema was determined by the difference in paw volumes measured (by mercury displacement) prior to and 3 h post-carrageenan challenge .
Carbonic anhydrase assay
The inhibitory activity of the compounds against carbonic anhydrase (from bovine erythrocytes) was determined in the following manner . Approximately 10 units of bovine carbonic anhydrase in 6 ml of barbitone (barbital) buffer were incubated for 10-15 s with various amounts of the test drugs . The reaction was initiated by the addition of substrate (4 ml C0 2 -saturated H 2 O) . The rate of
Pharmacological Research, Vol. 24, No . 4, 1991
379
enzyme-catalysed change in pH was followed, and the IC 50 was calculated (graphically) from the concentration-% inhibition curves .
Materials and statistical analyses
Acetazolamide, dichlorphenamide, indomethacin, and carbonic anhydrase were purchased from the Sigma Chemical Company, St Louis, Missouri . Ethoxzolamide and methazolamide were synthesized in the Chemical Research Department of the A . H . Robins Company, Richmond, Virginia . Data were analysed by using the Dunnett's t-test to compare the effects of drug treatments to a control .
RESULTS Various carbonic anhydrase inhibitors (acetazolamide, ethoxzolamide, methazolamide, and dichlorphenamide), as well as the reference non-steroidal anti-inflammatory drug (NSAID), indomethacin, were given to groups of seven rats with established adjuvant arthritis . The results in Table I represent the pooling of several experiments . All of the carbonic anhydrase inhibitors tested produced anti-inflammatory activity as determined on day 29 or on day 50 (Table I) . They also attenuated the changes seen in the X-ray score of the arthritic joint on day 50 (Table I) . Significant activity was observed with the lowest dose of each drug tested . From the data presented, it is apparent that the potent carbonic anhydrase
Table I Antiarthritic activity of carbonic anhydrase inhibitors and indomethacin in adjuvant-arthritic rats Treatment
Vehicle Indomethacin Acetazolamide
n
55 55 28 35 7 7 7 7 14 7
Dose (mglkg) p .o .
3 .16 3 .16 10 .0 31 .6 1 .0 3 .16 3 .16 31 .6 100
Oedema' day 29 (ml±so)
Oedema" day 50 (ml±so)
X-Ra v score (mean±so)
C .A ." iC :,, (pM)
0 .19±0 .28 -0 .05±0 .43 8 .57±1.03 3 .80±0 .72` -0 .73±0 .22` -0 .63±0.30` >34 -0 .21±0 .23` -0 .42±0.20` 6 .30±1 .49` 0 .072 -0 .32±0 .21` -0 .53±0 .28` 6 .37±0 .79` -0 .43±0 .20` -0 .69±0 .18` 6 .79±0 .74` Ethoxzolamide -0 .03±0 .20` -0 .36±0 .17` 6 .89±1 .07` 0 .016 -0 .29±0 .20` -0 .43±0 .35` 6 .11±1 .31` Methazolamide -0 .24±0 .35` -0 .43±0 .24` 6 .71±I .01` 0 .040 Dichlorphenamide -0 .10±0 .20` -0 .34±0 .27` 7 .93±1 .30` 0 .233 -0 .29±0 .25` -0 .54±0 .25` 5 .89±0 .45` Compounds were given to the rats daily (excluding weekends), by gavage, beginning on day 18 after adjuvant injection and continuing through day 50 after adjuvant injection . Vehicle-treated animals received 0 .5% Tween in H 2 O (10 ml/kg) . X-rays of the uninjected hind limb were made on day 50 . Oedema=difference between paw volume on day 29 or day 50 and paw volume on day 18 (first day of drug treatment) . hC .A .=carbonic anhydrase in vitro . `P
