SHORT COMMUNICATION
Clin. Drug Inve st . 10 (3): 179-1 82, 1995
1 173-2563/95/0009-{) 179/ S02.rfJ/O
© Adls International Limited. All rights reserved.
Dapsone as an Immunomodulator In Vivo Using the Tuberculin Skin Test as a Parameter 1.R. Snyman,1 WK. lacyk,2 D.J. Lizamore 3 and De K. Sommers1 1 Department of Pharmacology, University of Pretoria, Pretoria, South Africa 2 Department of Dermatology, University of Pretoria, Pretoria, South Africa 3 Department of Anatomy, University of Pretoria, Pretoria, South Africa
Dapsone has been in extensive clinical use for infectious diseases such as leprosy and the prophylaxis of malaria for many years.[l] This agent also possesses anti-inflammatory properties that have been employed successfully in the treatment ofbullous pemphigoid and dermatitis herpetiformis.[l] Dapsone a~ts as an anti-infective agent by inhibiting the synthesis of dihydrofolic acid through competition with para-arninobenzoate for the active site on the enzyme dihydropteroate synthetase pi Its anti-inflammatory action is, however, still not clear despite numerous animal and in vitro studiesp-7] Reports on its seemingly dose-related[4,8] immunomodulatory effects are conflicting. In one study, dapsone in doses of 100 to 200 mg/kg suppressed experimental arthritis in laboratory animals to the same extent as prednisolone;[6] however, in another study 5 mg/kg but not 2 mg/kg suppressed the passive Arthus reaction.£5] Anderson et aU4] have shown both in patients with leprosy and in healthy volunteers that dapsone exposure can cause an ex vivo increase in lymphocyte responsiveness after stimulation with phytohaemaglutinin (PHA). Neutrophil migration was also stimulated ex vivo in the patients with leprosy, and this was ascribed to inhibition of the H 20 2halide system. However, in another in vitro study
no effect of dapsone on PHA-stimulated lymphocytes could be demonstrated, but inhibition of proliferation was shown when PPD (purified protein derivative of tuberculin) was used as a stimulant. [8] Dapsone has also been implicated in the cure of a Kaposi's sarcoma in a patient with AIDS,f 9 ] and this could indicate immunostimulation. Because of these conflicting findings, the aim of the present study in healthy volunteers was to investigate the in vivo effects of a therapeutic dose of dapsone on a known delayed hypersensitivity reaction, i.e. PPD-reaction.£IO-12] The tuberculin skin test (Montoux) is a reliable test frequently used to determine a patient's cellular immune response to the tubercule bacilli.[11 ,12] Participants and Methods
This open study included 13 healthy volunteers (7 males and 6 females aged from 20 to 23 years)
who had previously been inoculated with bacille Calmette-Guerin (BCG) vaccine, Ethical approval was obtained from the Ethics Committee of the University of Pretoria, and informed consent was obtained from all participants. O.lml of Japan freeze-dried PPD (Japan BCG Laboratory, Tokyo) was injected intradermally on
180
Snyman et al.
Table I. Area (mm2) of induration 48 hours after intradermal PPD administration Baseline
Dapsone
1309.94" SO 1221.37 Range 211.69 - 4755.09 a Significantly larger compared with baseline; p < 0.001 . Mean
405.89 275.77 132.44 - 933.08
Abbreviation: PPD = purified protein derivative of tuberculin.
the volar aspect of the volunteers' forearms. The injection consisted of 5 TU (tuberculin units) PPD in Tween-80 as antigen l II) and was administered on two occasions, 7 days apart, using alternate forearms, as it is known that no booster phenomenon occurs during this time-period. lID) The reaction after intradermal administration of PPD on day 1, taken as baseline and read 48 hours later, was followed by the administration of dapsone (Lennon Medicines Pty Ltd, Randburg, South Africa) 3 mgt kg/day for 7 consecutive days (i.e. days 3 to 9). The reaction to the day 7 injection was read on day 9. The outlines of the indurations were drawn on transparent plastic film and measured by the same blinded coworker using computerised planimetry. A 3mm punch biopsy was taken from the centre of every reaction site 48 hours after intradermal antigen administration and immediately frozen in liquid nitrogen for histological evaluation by another coworker blinded to the protocol. The biopsies were stained with haematoxylin and eosin, and the inflammatory cell infiltration was evaluated. Peripheral blood samples were taken for full blood and differential counts on days I and 9 of the study and blood for dapsone plasma levels on day 9.
Results As seen in table I, dapsone significantly enhanced the cell-mediated reaction in all volunteers. The mean dapsone plasma level on day 9 was 6.33 mg/L, and ranged from 3.33 to 12.57 mg/L. There was no correlation between the size of the reactions and the dapsone plasma levels. Histologically there was a pronounced increase in inflammatory celIs in all biopsies taken from the reaction sites when on dapsone treatment compared with baseline biopsies. Examples of this increase in inflammatory cell infiltration are depicted in figures I and 2. The only significant change in the peripheral blood counts was an increase in circulating basophils, i.e. an increase from a mean of 0.0 I (range 0 to 0.5) x 109/L to 0.06 (range 0 to 1.8) x 109/L. Six volunteers had no detectable basophils in their
Statistical Analysis Each individual acted as hislher own control, and the Wilcoxon signed ranks test was used to evaluate differences between paired observations. Values were regarded as significant at the 5% level throughout. © Adis International Limited. All rights reserved.
Fig. 1. Baseline histological examination of a biopsy from a purified protein derivative of tuberculin reaction site. (A) 100 x magnification of cryostat section stained with haematoxylin and eosin; h hair follicle. (8) 400 x magnification of a part of the same section as in (A).
=
Clin. Drug Invest. 10 (3) 1995
Immunomodulatory Effects of Dapsone
Fig. 2. Histological examination of a biopsy from a purified protein derivative of tuberculin reaction site after dapsone treatment. (A) 100 x magnification of cryostat section stained with haematoxylin and eosin; h hair follicle. (8) 400 x magnification of a part of the same section as in (A). Compared with baseline (fig. 1), there is a clear increase in inflammatory cells in the connective tissue.
=
blood at baseline, and only one of them still had no basophils after dapsone therapy. There were no significant changes in the total white-cell count or in other leucocyte populations.
Discussion The main finding of this study was that dapsone enhanced the delayed cell-mediated reaction to the POO skin test. This is in contrast to findings by others who demonstrated in vivo and in vitro reduction of lymphocyte function,[1.3,S.SI but corroborates findings by Anderson et al.,[4] who showed an increase in PHA-stimulated lymphocyte transformation and stimulation of polymorphonuclear leucocyte motility after the oral administration of dapsone (lOOmg) in both healthy controls and © Adis International Limited. All rights reserved.
181
patients with leprosy. In the latter study, it was also shown that the level of dapsone necessary to stimulate neutrophil function in vitro was IOO-fold greater than that obtained in vivo. In a previous studyllO] with similar time periods to those in the present study, we were unable to demonstrate a booster effect with PPO. Although there was no placebo group in the present study, it is unlikely that sequential PPO stimulation influenced the results. It is, however, not uncommon for nonspecific immunomodulators such as levamisole to have either stimulatory or suppressant properties, depending on the type of study, species studied and antigenic stimulations used.!13] With in vivo studies, the antigen presentation to the immune system differs from the in vitro situation in that a cascade of cytokines eventually causes effector cell stimulation. Along this pathway many aspects of the immune function may be influenced by a drug such as dapsone. It is, therefore, not meaningful to speculate on the immunomodulatory mechanism of action of dapsone on the basis of the present results. Follow-up in vivo studies in this regard should be undertaken.
Acknowledgements The authors would like to thank Lennon Medicines Pty Ltd, South Africa, for providing the test drug, Mrs J. Bekker for secretarial and Miss N. Lourens for technical assistance. This research was funded by the Department of Pharmacology, University of Pretoria, Pretoria, South Africa.
References I. McDougall AC. Dapsone. Clin Exp Dermatol 1979; 4: 139-42 2. Coleman MD. Dapsone: modes of action, toxicity and possible strategies for increasing patient tolerance. Br J Dermatol 1993; 229: 507-13 3. Beiguelman B, Pisani RCB. Effects of DDS on phytohemaglutinin-induced lymphocyte transformation. Int J Lepr 1974; 42: 412-5 4. Anderson R, Gatner EMS, Van Rensburg CE, et al. In vitro and in vivo effects of dapsone on neutrophil and lymphocyte functions in normal individuals and patients with lepromatous leprosy. Antimicrob Agents Chemother 1981; 19: 495-503 5. Ruzicka T, Bauer A, GlUck S, et al. Effects of dapsone on passive Arthus reaction and chemotaxis and phagocytosis of polymorphonuclear leucocytes. Arch Dermatol Res 1981 ; 270: 347-51
Clin. Drug Invest. 10 (3) 1995
182
6. Williams K. Capstick RB. Lewis DA. et al. Anti-inflammatory actions of dapsone and its related biochemistry. J Pharm Pharmacol 1976; 28: 555-8 7. Barranco VP. Inhibition of lysosomal enzymes by dapsone. Arch Dermatol 1974; 110: 563-6 8. Chen M. Kappel M. Lemnge M. et al. Tn vitro effects of artesun ate and other antimalarial agents on the function of human lymphocytes and neutrophils. Transplant Proc 1994; 26: 3172-4 9. Poulsen A. Hultberg B. Thomsen K. et al. Regression of Kaposi 's sarcoma in AIDS after treatment with dapsone. Lancet 1984; I: 560 10. Snyman JR. Meyer EC. Schoeman HS. Cimetidine as modulator of the cell-mediated immune response in vivo using the
© Adis Internaflonal Limited. All rights reserved.
Snymall ct at.
tuberculin skin test as parameter. Br J C1in Pharmacol 1990; 29: 257-60 I \. Hale KA. Tuberculin skin test. proper technique and interpretation. Postgrad Med J 1985; 77: 252-3 12. Snider DE. Bacille Cal mette-Guerin vaccination and tuberculin skin test. JAMA 1985; 253: 3438-9 13. Van Wauwe J. Janssen PAJ. On the biochemical mode of action of levamisole: an update. Int J Immunopharmacol 1991 : 13: 3-9
Correspondence and reprints: Dr f.R. Snyman, Department of Pharmacology, Faculty of Medicine, PO Box 2034, 0001 Pretoria, South Africa.
Clin. Drug Invest. 10 (3) 1995
Clin. Drug Inve st . 10 (3): 179-1 82, 1995
1 173-2563/95/0009-{) 179/ S02.rfJ/O
© Adls International Limited. All rights reserved.
Dapsone as an Immunomodulator In Vivo Using the Tuberculin Skin Test as a Parameter 1.R. Snyman,1 WK. lacyk,2 D.J. Lizamore 3 and De K. Sommers1 1 Department of Pharmacology, University of Pretoria, Pretoria, South Africa 2 Department of Dermatology, University of Pretoria, Pretoria, South Africa 3 Department of Anatomy, University of Pretoria, Pretoria, South Africa
Dapsone has been in extensive clinical use for infectious diseases such as leprosy and the prophylaxis of malaria for many years.[l] This agent also possesses anti-inflammatory properties that have been employed successfully in the treatment ofbullous pemphigoid and dermatitis herpetiformis.[l] Dapsone a~ts as an anti-infective agent by inhibiting the synthesis of dihydrofolic acid through competition with para-arninobenzoate for the active site on the enzyme dihydropteroate synthetase pi Its anti-inflammatory action is, however, still not clear despite numerous animal and in vitro studiesp-7] Reports on its seemingly dose-related[4,8] immunomodulatory effects are conflicting. In one study, dapsone in doses of 100 to 200 mg/kg suppressed experimental arthritis in laboratory animals to the same extent as prednisolone;[6] however, in another study 5 mg/kg but not 2 mg/kg suppressed the passive Arthus reaction.£5] Anderson et aU4] have shown both in patients with leprosy and in healthy volunteers that dapsone exposure can cause an ex vivo increase in lymphocyte responsiveness after stimulation with phytohaemaglutinin (PHA). Neutrophil migration was also stimulated ex vivo in the patients with leprosy, and this was ascribed to inhibition of the H 20 2halide system. However, in another in vitro study
no effect of dapsone on PHA-stimulated lymphocytes could be demonstrated, but inhibition of proliferation was shown when PPD (purified protein derivative of tuberculin) was used as a stimulant. [8] Dapsone has also been implicated in the cure of a Kaposi's sarcoma in a patient with AIDS,f 9 ] and this could indicate immunostimulation. Because of these conflicting findings, the aim of the present study in healthy volunteers was to investigate the in vivo effects of a therapeutic dose of dapsone on a known delayed hypersensitivity reaction, i.e. PPD-reaction.£IO-12] The tuberculin skin test (Montoux) is a reliable test frequently used to determine a patient's cellular immune response to the tubercule bacilli.[11 ,12] Participants and Methods
This open study included 13 healthy volunteers (7 males and 6 females aged from 20 to 23 years)
who had previously been inoculated with bacille Calmette-Guerin (BCG) vaccine, Ethical approval was obtained from the Ethics Committee of the University of Pretoria, and informed consent was obtained from all participants. O.lml of Japan freeze-dried PPD (Japan BCG Laboratory, Tokyo) was injected intradermally on
180
Snyman et al.
Table I. Area (mm2) of induration 48 hours after intradermal PPD administration Baseline
Dapsone
1309.94" SO 1221.37 Range 211.69 - 4755.09 a Significantly larger compared with baseline; p < 0.001 . Mean
405.89 275.77 132.44 - 933.08
Abbreviation: PPD = purified protein derivative of tuberculin.
the volar aspect of the volunteers' forearms. The injection consisted of 5 TU (tuberculin units) PPD in Tween-80 as antigen l II) and was administered on two occasions, 7 days apart, using alternate forearms, as it is known that no booster phenomenon occurs during this time-period. lID) The reaction after intradermal administration of PPD on day 1, taken as baseline and read 48 hours later, was followed by the administration of dapsone (Lennon Medicines Pty Ltd, Randburg, South Africa) 3 mgt kg/day for 7 consecutive days (i.e. days 3 to 9). The reaction to the day 7 injection was read on day 9. The outlines of the indurations were drawn on transparent plastic film and measured by the same blinded coworker using computerised planimetry. A 3mm punch biopsy was taken from the centre of every reaction site 48 hours after intradermal antigen administration and immediately frozen in liquid nitrogen for histological evaluation by another coworker blinded to the protocol. The biopsies were stained with haematoxylin and eosin, and the inflammatory cell infiltration was evaluated. Peripheral blood samples were taken for full blood and differential counts on days I and 9 of the study and blood for dapsone plasma levels on day 9.
Results As seen in table I, dapsone significantly enhanced the cell-mediated reaction in all volunteers. The mean dapsone plasma level on day 9 was 6.33 mg/L, and ranged from 3.33 to 12.57 mg/L. There was no correlation between the size of the reactions and the dapsone plasma levels. Histologically there was a pronounced increase in inflammatory celIs in all biopsies taken from the reaction sites when on dapsone treatment compared with baseline biopsies. Examples of this increase in inflammatory cell infiltration are depicted in figures I and 2. The only significant change in the peripheral blood counts was an increase in circulating basophils, i.e. an increase from a mean of 0.0 I (range 0 to 0.5) x 109/L to 0.06 (range 0 to 1.8) x 109/L. Six volunteers had no detectable basophils in their
Statistical Analysis Each individual acted as hislher own control, and the Wilcoxon signed ranks test was used to evaluate differences between paired observations. Values were regarded as significant at the 5% level throughout. © Adis International Limited. All rights reserved.
Fig. 1. Baseline histological examination of a biopsy from a purified protein derivative of tuberculin reaction site. (A) 100 x magnification of cryostat section stained with haematoxylin and eosin; h hair follicle. (8) 400 x magnification of a part of the same section as in (A).
=
Clin. Drug Invest. 10 (3) 1995
Immunomodulatory Effects of Dapsone
Fig. 2. Histological examination of a biopsy from a purified protein derivative of tuberculin reaction site after dapsone treatment. (A) 100 x magnification of cryostat section stained with haematoxylin and eosin; h hair follicle. (8) 400 x magnification of a part of the same section as in (A). Compared with baseline (fig. 1), there is a clear increase in inflammatory cells in the connective tissue.
=
blood at baseline, and only one of them still had no basophils after dapsone therapy. There were no significant changes in the total white-cell count or in other leucocyte populations.
Discussion The main finding of this study was that dapsone enhanced the delayed cell-mediated reaction to the POO skin test. This is in contrast to findings by others who demonstrated in vivo and in vitro reduction of lymphocyte function,[1.3,S.SI but corroborates findings by Anderson et al.,[4] who showed an increase in PHA-stimulated lymphocyte transformation and stimulation of polymorphonuclear leucocyte motility after the oral administration of dapsone (lOOmg) in both healthy controls and © Adis International Limited. All rights reserved.
181
patients with leprosy. In the latter study, it was also shown that the level of dapsone necessary to stimulate neutrophil function in vitro was IOO-fold greater than that obtained in vivo. In a previous studyllO] with similar time periods to those in the present study, we were unable to demonstrate a booster effect with PPO. Although there was no placebo group in the present study, it is unlikely that sequential PPO stimulation influenced the results. It is, however, not uncommon for nonspecific immunomodulators such as levamisole to have either stimulatory or suppressant properties, depending on the type of study, species studied and antigenic stimulations used.!13] With in vivo studies, the antigen presentation to the immune system differs from the in vitro situation in that a cascade of cytokines eventually causes effector cell stimulation. Along this pathway many aspects of the immune function may be influenced by a drug such as dapsone. It is, therefore, not meaningful to speculate on the immunomodulatory mechanism of action of dapsone on the basis of the present results. Follow-up in vivo studies in this regard should be undertaken.
Acknowledgements The authors would like to thank Lennon Medicines Pty Ltd, South Africa, for providing the test drug, Mrs J. Bekker for secretarial and Miss N. Lourens for technical assistance. This research was funded by the Department of Pharmacology, University of Pretoria, Pretoria, South Africa.
References I. McDougall AC. Dapsone. Clin Exp Dermatol 1979; 4: 139-42 2. Coleman MD. Dapsone: modes of action, toxicity and possible strategies for increasing patient tolerance. Br J Dermatol 1993; 229: 507-13 3. Beiguelman B, Pisani RCB. Effects of DDS on phytohemaglutinin-induced lymphocyte transformation. Int J Lepr 1974; 42: 412-5 4. Anderson R, Gatner EMS, Van Rensburg CE, et al. In vitro and in vivo effects of dapsone on neutrophil and lymphocyte functions in normal individuals and patients with lepromatous leprosy. Antimicrob Agents Chemother 1981; 19: 495-503 5. Ruzicka T, Bauer A, GlUck S, et al. Effects of dapsone on passive Arthus reaction and chemotaxis and phagocytosis of polymorphonuclear leucocytes. Arch Dermatol Res 1981 ; 270: 347-51
Clin. Drug Invest. 10 (3) 1995
182
6. Williams K. Capstick RB. Lewis DA. et al. Anti-inflammatory actions of dapsone and its related biochemistry. J Pharm Pharmacol 1976; 28: 555-8 7. Barranco VP. Inhibition of lysosomal enzymes by dapsone. Arch Dermatol 1974; 110: 563-6 8. Chen M. Kappel M. Lemnge M. et al. Tn vitro effects of artesun ate and other antimalarial agents on the function of human lymphocytes and neutrophils. Transplant Proc 1994; 26: 3172-4 9. Poulsen A. Hultberg B. Thomsen K. et al. Regression of Kaposi 's sarcoma in AIDS after treatment with dapsone. Lancet 1984; I: 560 10. Snyman JR. Meyer EC. Schoeman HS. Cimetidine as modulator of the cell-mediated immune response in vivo using the
© Adis Internaflonal Limited. All rights reserved.
Snymall ct at.
tuberculin skin test as parameter. Br J C1in Pharmacol 1990; 29: 257-60 I \. Hale KA. Tuberculin skin test. proper technique and interpretation. Postgrad Med J 1985; 77: 252-3 12. Snider DE. Bacille Cal mette-Guerin vaccination and tuberculin skin test. JAMA 1985; 253: 3438-9 13. Van Wauwe J. Janssen PAJ. On the biochemical mode of action of levamisole: an update. Int J Immunopharmacol 1991 : 13: 3-9
Correspondence and reprints: Dr f.R. Snyman, Department of Pharmacology, Faculty of Medicine, PO Box 2034, 0001 Pretoria, South Africa.
Clin. Drug Invest. 10 (3) 1995
