Int. J. Immunopharmac., Vol. 13, No. 4, pp. 419-428, 1991. Printed in Great Britain.

0192-0561/91 $3.00 + .DO Pergamon Press plc.

International Society for lmmunopharmacology.

EFFECT OF A N T I - L E P R O S Y D R U G S ON S U P E R O X I D E A N I O N P R O D U C T I O N BY RAT P E R I T O N E A L M A C R O P H A G E WITH SPECIAL

REFERENCE TO LIGHT E X P O S E D CLOFAZIMINE

ARVIND SAHU,* KUNAL SAHA, *+ N. R. BANERJEE,* V. N. SEHGAL § and C. R. JAGGA rl

*Department of Immunology, Vallabhbhai Patel Chest Institute; *Department of Chemistry, University of Delhi, Delhi-110007, India; ~Department of Dermatology, Maulana Azad Medical College, New Delhi-110002, India;

~qTMMEC, Indian Institute of Technology, New Delhi, India

(Received 25 June 1990 and in final form 24 October 1990)

Abstract - - The present study describes the in vitro effect of anti-leprosy drugs on superoxide anion (O2) production by rat resident peritoneal macrophages. Of the three drugs tested i.e. clofazimine, rifampicin and dapsone, the first was most effective in increasing 02 production in a dose dependent manner, while rifampicin had some stimulatory effect and dapsone exhibited minimal action. Furthermore, when clofazimine and dapsone were added together it was observed that the increase of 02 production by macrophages due to clofazimine was not significantly altered by the addition of dapsone. Moreover, it was found that killed Mycobacterium leprae could induce a lesser amount of 02 production in comparison to that of Staphylococcus aureus and the enhancement of 02 release due to clofazimine was stimulus dependent. This increase of O~ release after addition of clofazimine was inhibited by the addition of p- bromophenacyl bromide. Another interesting finding was that the enhancement of O~ production by clofazimine gradually decreased as clofazimine was exposed to light for days. On further investigation it was found that ultraviolet, NMR, infrared and mass spectra of the light unexposed and exposed drug were similar, but the diffusion current of the polarogram of light exposed drug was remarkably more than that observed in light unexposed drug, indicating, thereby, a possible increase in the electron accepting capacity of the light reacted molecule. As far as we know this is the first report describing the effect of light exposed clofazimine on the respiratory burst activity of macrophages.

Multidrug therapy, consisting of dapsone, clofazimine and r ifampicin is considered to be a useful tool in the t reatment of leprosy (Hastings & Franzblau, 1988). These drugs are known to affect various arms of the immune system, such as humoral , cellular (Humber , Nsanzumuhire, Aluoch, Webster, Aber, Mitchinson, Giring & Nunn, 1980), phagocytic (Anderson, Gatner, van Rensburg, Grabow, Imkamp, Kak & van Rensburg, 1981) and complement (Sahu, Saha, Kashyap & Chakrabarty, 1988).

Macrophages play a key role in our immune system (Adams & Hamil ton , 1984). It endocytoses the drugs in solution as particles or as a coating on other particles (Lewis & Adams, 1985). Furthermore, these cells are known to have oxygen dependent and oxygen independent killing mechanisms (Roitt, Bros tof f & Male, 1985). The superoxide anion is one

of the important radicals in oxidative killing. Its product ion has been correlated with the intracellular killing of several microorganisms including Mycobacterium tuberculosis (Jackett, Aber & Lowrie, 1978). In lepromatous leprosy patients, macrophages contain an enormous number of Mycobacterium leprae without inducing superoxide anion production (Niwa, Sakane, Miyachi & Ozaki, 1984). The concentrat ion of dapsone, clofazimine and rifampicin achieved after taking standard doses o f these drugs was reported to be 1 0 - 1 5 / a g / m l (Mandell & Sande, 1985), 1 /ag/ml (Yawalker & Vischer, 1979) and 7 t~g/ml (Mandell & Sande, 1985), respectively. Anti-leprosy drugs, especially clofazimine are known to accumulate in macrophages in high concentrations and the accumulation of the drug is proportional to the number of macrophages present in the organ or tissue (Conalty & Jackson,

"tAuthor to whom correspondence should be addressed.

419

420 A. SAHU et at.

1962). In humans, clofazimine is deposited in the tissues of leprosy patients, where its concentration reaches as high as 3.3 mg/g tissue (Desikan & Balakrishnan, 1976). Moreover, clofazimine and dapsone are thought to act in lepromatous and non- leprotic diseases by affecting polymorphonuclear leukocyte (PMNL) function (Winkelmann & Roth, 1960; Molin, 1975; Stendahl, Molin & Aahlgren, 1978; Mac-Dougall, Horsfall, Hede & Chaplin, 1980). Previous studies described the effect of clofazimine (Niwa et al., 1984) and dapsone (Miyachi & Niwa, 1982; Harvath, Yancey & Katz, 1986) on superoxide anion production by PMNLs. It was reported that there was no spontaneous generation of 02 from resting cells incubated with clofazimine alone (Zeis, Savage, O'Sullivan & Anderson, 1990). However, the mechanism of superoxide anion production by PMNLs and macrophages is not the same (Kitagawa, Takaku & Sakamoto, 1980). This report describes the in vitro effect of anti-leprosy drugs on the 02 release by rat peritoneal macrophages containing S. aureus or M. leprae with special reference to the effects of light on clofazimine's respiratory burst augmenting activity.

EXPERIMENTAL PROCEDURES

Anti-leprosy drugs. Solutions were prepared as described earlier (Sahu et al., 1988). Twenty milligrams of dapsone (WeUcome, India) were dissolved in 1 ml of 50O7o dimethyl sulphoxide; 100 mg clofazimine (a kind gift from Ciba Geigy, Switzerland) was dissolved in 50 ml ethyl alcohol in dark. At this concentration clofazimine was not completely solubilized and some of the drug remained as microparticles (Cline, 1970). One half was kept in dark while the other half was kept in a Corning glass beaker which was exposed to a fluorescent electric lamp (40 W), from a distance of 30 cm at room temperature. It was exposed to light for a period varying from 0 to 15 days. The solution was stirred occasionally to achieve uniform exposure of the sample to light. Aliquots were withdrawn at varying times during light exposure, stored in the dark and then all samples were tested simultaneously for their ability to increase 02 production using the same preparation of macrophages. It was further diluted in Hank's balanced salt solution (HBSS); 20 mg/ml rifampicin (Lupin Laboratories, India) solution was made in normal saline to which 10 IA of 4 N NaOH was added to achieve a clear solution.

Isolation o f rat peritoneal macrophages. Fifteen millilitres of sterile HBSS (without phenol red) was injected intra-peritoneally into a male Wistar rat weighing 200 g. After 1 h, peritoneal fluid was collected from the animal under light ether anaesthesia. One millilitre of peritoneal exudate, containing 1 × 10 6 cells, was poured into a plastic Petri-dish of 3.5 cm diameter, kept for 1 h at 37°C and thereafter the non-adherent cells were removed by rinsing the plates three times with HBSS. A monolayer of adherent cells was then obtained. Viability of the cells always exceeded 95% as determined by trypan blue exclusion.

Superoxide anion estimation. Superoxide anions released by rat peritoneal macrophages monolayers were estimated according to the method of Holzer et al. (Holzer, Nelson, Schauf, Crispen & Anderson, 1986). In brief, each dish containing macrophages was covered with 1 ml of sterile HBSS and 0.2 ml of 200 pM ferricytochrome c (Type VI, Sigma, U.S.A.) in HBSS, incubated for 5 rain with or without various concentrations of drugs/or vehicles at 37°C and thereafter stimulated with 1 × 107 live S. aureus or 1 × 106 and 1 × 107 killed M. leprae (WHO, armadillo derived lepromin). After addition of stimuli the total reaction mixture was adjusted to 2.2 ml by addition of HBSS and the plates were incubated at 37°C (test) and 0°C (control) for 1 h. The respective supernatants were centrifuged at 1500 g for 15 rain at 4°C, and their optical densities were scanned between 660 and 500 nm in a Shimadzu UV-240 Spectrophotometer. The superoxide anions liberated were calculated by subtracting the optical density of the control (0°C) at 550 nm from that of the test (37°C) at 550 rim; the difference of optical densities was then converted into nanomoles of cytochrome c reduced (for 2.2ml) using an extinction coefficient of reduced cytochrome c (21 × 103 mol ' /cm ~). To quantify the adherent macrophages, their cell protein was estimated by the following method: 1 ml of 1 N NaOH was added to each Petri-dish containing unstimulated adherent cells, allowed to stand overnight and thereafter the protein was estimated by employing the method of Lowry et al. (Lowry, Rosebrough, Farr & Randall, 1951). The results were expressed as nanomoles of cytochrome c reduced/60 min/mg protein.

4-bromophenacyl bromide mediated inhibition o f 02 release induced by S. aureus in the presence o f clofazimine. Macrophages were pre-incubated for 5 rain with 5/~M p-bromophenacyl bromide, an

Anti-leprosy Drugs and Superoxide Anion Production 421

Table 1. Release of superoxide anion by rat peritoneal macrophage stimulated by S. aureus in the presence of clofazimine alone and in combination with dapsone

Superoxide Amount of the drugs anion release

(Pg) Number of (nmols/60 min/mg protein) Statistical

Group Clofazimine Dapsone experiments mean + S.D. analysis

I. Control 0 0 3 56.3 _+ 17.7 II. Clofazimine 30 0 3 210.3 _+ 49.9

III. (a) Clof. + dapsone

(b) Clof. + dapsone

(c) Clof. + dapsone

II vs I, P<0.05

30 10 3 219.6 + 54.3 IIIa vs II, NS

30 100 3 197.8 + 24.8 IIIb vs II, NS

30 1000 3 153.3 _+ 46.7 IIIc vs II, NS

1 x 10 6 macrophages were mixed with the above drugs in HBSS and stimulated with 1 x 107 S. aureus. Maximum increase of O£ release was found when dapsone (10 ~g) was used with clofazimine 30 pg. NS: not significant.

inhibitor of phospholipase A2 (Sigma, U.S.A.) in HBSS according to the method of Bromberg & Pick (1983) and thereafter incubated with clofazimine and stimulated with S. aureus as described previously.

Physico - chemical methods used to study the light unexposed and exposed clofazimine.

(a) The u.v. and visible spectra of light unexposed and exposed (15 days) clofazimine solution in alcohol (2 x 10 5M) was studied from 700 to 190 nm in a recording spectrophotometer (Gilford, U.S.A.).

(b) Infrared absorption spectra of the above two species of the drug were studied in a Beckman 620 spectrophotometer.

(c) The 90 MHz ~H NMR spectra of both the species of clofazimine were taken in a Perkin Elmer R-32 instrument, with TMS as an internal standard.

(d) Polarograms of clofazimine were taken in a modified H type cell under nitrogen atmosphere. A 2.1 x 10 4M solution of clofazimine was made by dissolving 1 mg of the drug in 9.0 ml methanol and 1.0 ml of 0.2 M phosphate buffer (pH 7.0). The final concentration of Triton-X 100 (suppressor) added to the cell was 0.002°7o. The polarograms were taken in a cell containing fresh drug in the dark and also after exposing the solution of the drug to an electric light for 15 min. Half wave potential and diffusion current were recorded.

(e) Thin layer chromatograms of the light unexposed and exposed (15 days) clofazimine were carried out on a silica gel plate using to luene- acetic

ac id -wa te r solvent system (50 :50 :4 ) and studied under u.v. light (Lanyl & Dubois, 1982).

(f) Mass spectra of the light exposed and unexposed clofazimine were recorded in a mass spectrometer, JEOL (Japan) D-300 under electron impact (EI) 70 eV.

Statistical analysis. Results were grouped and statistically evaluated using Student's t-test.

RESULTS

Effect o f clofazimine, rifampicin and dapsone on superoxide anion release. Clofazimine significantly increased the 02 production by the rat resident peritoneal macrophages after engulfing S. aureus as a stimulus (Table 1), in a dose dependent manner (Fig. 1). Rifampicin marginally enhanced Oz release and dapsone was found to be the least effective in stimulating O2 production (Fig. 1). Higher concen- trations of rifampicin (30 tag) alone were able to reduce cytochrome c directly by a considerable amount and therefore this method was not suitable for studying the effect of higher concentrations of rifampicin on 02 release. Thirty micrograms of clofazimine and rifampicin enhanced maximum release of O2 (199.7 and 28.8 nmols, respectively). Dapsone also showed a dose dependent activity. Thus, 100 tag of dapsone could induce 25.2 nmols 02. Its production was thereafter impaired by addition of a higher amount of the drug.

A. SAHU et al.

Io9/Jg dopsone odded 1.0 E.O 3.0

i i i

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o ,0o,o2o,.. .o.20o.20 Fig. 1. Effect of clofazimine, rifampicin and dapsone on OF release by rat resident peritoneal macrophages after engulfing S. aureus. 1 × 106 rat peritoneal macrophages were pre-incubated with cytochrome c in HBSS and a different amount of the above anti-leprosy drugs for 5 rain, thereafter challenged with 1 x 107 live S. aureus and incubated for 1 h. Maximum release of 02 was found after addition of 30/~g clofazimine, 30/ag rifampicin and 100/ag dapsone. Each point represents the mean of three different

experiments.

Effect o f S. aureus and M. leprae on the superoxide anion release by rat peritoneal macrophages and its modulation by addition o f clofazimine. Figure 2 depicts tha t S. aureus could release larger amoun t s of 0 2 f rom macrophages than the same n u m b e r of M. leprae when added to an equal n u m b e r of macrophages , showing thereby that M. leprae was a poor inducer of O: product ion . Interest ingly, 30/ag of c lofazimine p ro found ly increased 0 2 release f rom 84.1 +_ 31.5(a) to 266.1 _+ 48.8(b) n m o l s / 6 0 m i n / m g pro te in in the presence of S. aureus (P<0.01) , while the same a m o u n t of c lofazimine increased the release of 0 2 f rom 58.8 _+ 13.6(c) to 189.4 __ 32.7(d) n m o l s / 60 m i n / m g prote in in the presence of M. leprae (P<0 .01) (Fig. 2). Fu r the rmore , the difference in the increase of O~ release induced by S. aureus and M. leprae in the presence of the same a m o u n t of c lofazimine (30 t~g) was signif icant (i.e. d - c<b - a, P < 0 . 0 5 , Fig. 2). W h e n 30 ~g of c lofazimine was added to the test system in the presence of 1 × 106 M. leprae, enhancemen t of O7 release also decreased (174.5 + 19.6 n m o l s / 6 0 m i n / m g prote in) in com- par ison with tha t released by same a m o u n t of

3 0 0

I

2 4 0

c g 18o

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~ ] S oureu5

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m Fig. 2. Effect of different stimuli, on the Oz release by rat peritoneal macrophages, vis-&vis the modulation of O_~ release by addition of clofazimine. 1 x 106 macrophages were incubated with (a)1 x 107 S. aureus; (b)1 × 107 S. aureus + 30 /ag clofazimine; (c) 1 x 107 M. leprae; (d) 1 × 107 M. leprae + 30 /~g clofazimine; (e) 1 × 106 M. leprae and (f) 1 × 106 M. leprae + 30 ~g clofazimine. O2 production was decreased by the addition of M. leprae in comparison with that released by S. aureus. The increase in 02 production due to clofazimine was significantly less in the presence of M. leprae compared with S. aureus (d-c<b-a , P<0.05). Each bar is the mean of three

experiments and a straight line shows S,D.

c lofazimine in the presence of 1 x 107 M. leprae (189.4 _+ 32.7 n m o l s / 6 0 m i n / m g protein) , however, the difference was not significant.

Effect o f the combination o f clofazimine and dapsone on superoxide anion release by macrophages stimulated by S. aureus. Table 1 shows tha t c lofazimine alone significantly increased O: release in compar i son with the cont ro l (wi thout drug, P<0 .05 ) . It was found that when 30 pg of c lofazimine and varying doses of dapsone were used in combina t ion , there was a progressive fall of 02 release with the increasing dose of dapsone. However , the differences were not significant.

4-Bromophenacyl bromide (BPB) mediated inhibition o f superoxide anion release. Table 2 shows signif icant inhib i t ion of 02 release by macrophages induced by S. aureus in the presence of clofazimine and prior to incuba t ion with BPB (P<0.05) .

Anti-leprosy Drugs and Superoxide Anion Production 423

Top

,Origin

A B Fig. 4. Thin layer chromatogram of light unexposed (A) and light exposed (B) clofazimine on silica gel. Both the species

exhibited same mobility.

Anti-leprosy Drugs and Superoxide Anion Production

Table 2.4-Bromophenacyl bromide (BPB) mediated inhibition of superoxide release

425

Group Number of Oz release Statistical experiments (nmols/60 min/mg protein) analysis

I. Control 4 66.0 + 18.1 II. Control + BPB 4 37.2 __ 20.6 II vs I, NS

III. Clof. 4 228.2 + 64.7 III vs I, P<0.01 IV. Clof. + BPB 4 139.1 _+ 28.5 IV vs III, P<0.05

1 × 106 macrophages were challenged with 1 x 10 7 S. aureus and 30 Mg clofazimine. Significant inhibition of clofazimine induced 02 release was observed after addition of BPB (5/aM). Results are expressed as mean _+ S.D. NS: not significant.

5

A- Clof" (light' exposed): E / = - 0 . 4 3 v l ~ ~ j ~ A

,4 / 1 l

3

id ,rio 2

I B

0

0.0 -0,1 -0 .2 - 3 - .4 - 0 5 -0 ,6 -0.7

Ed,e. Fig. 3. Polarogram of light unexposed and exposed clofazimine. Polarogram was taken with the light unexposed drug, thereafter the drug in the cell of the polarogram was exposed to strong light for 15 min and the polarogram was repeated. The rate of fall of mercury was 1 drop/2 s; bias time was 1.7; intensity of current was 10/aA; sweep rate was 10 mV/s. The diffusion current was remarkably increased in light exposed drug and the half wave potential remained unchanged.

Decrease in the induction o f superoxide anion production by light exposed clofazimine. Superoxide an ion p roduc t ion by ra t per i toneal macrophages progressively decreased, as the drug was exposed to light. Thus , there was 43 .3% decrease in O~ p roduc t ion at day 1. This decrease was fu r ther increased to 77 .7% at day 2 and 94 .7% at day 15.

The Rf values remained unal te red (Rf values of light unexposed and exposed samples were 0.73 and 0.72, respectively). The molecular weights of b o t h the light unexposed and exposed drugs were 473 (Fig. 5) which was similar to the expected values. The ad jacent peaks were due to the presence of an isotopic chlor ine a tom in the molecule.

Physico - chemical studies o f light unexposed and exposed clofazimine. No remarkab le changes in the u.v. - visible light, in f ra red and N M R spectra of the two species of c lofazimine were found . Figure 3 shows a r emarkab le increase of the d i f fus ion current f rom 1.4 to 4 .3/aA, however , the ha l f wave potent ia l remained unal tered . Figure 4 depicts the th in layer c h r o m a t o g r a m of b o t h the species of clofazimine.

DISCUSSION

The superoxide an ion is the p r imary p roduc t of the phagocytosis-associated burs t of oxidat ive metab- olism ( Johns ton , 1981). However , role of 0 2 in the intracel lular killing of M. leprae has not yet been repor ted. The 02 produced by pa t ien ts ' monocytes

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Fig. 5. Mass spectra of light unexposed and exposed clofazimine molecule. There was no change in the atomic

weights of the two species.

containing M. leprae has been shown to be inactivated by the superoxide dismutase (SOD) of M. leprae (Niwa et al., 1984). Furthermore, Neill & Klebanoff (1988) have explained the intracellular survival of M. leprae by the fact that PGL-1 could scavenge the reactive oxygen species and thereby prevent the killing of M. leprae within phagocytes. It has been proposed previously that clofazimine besides having direct anti-microbial activity also possesses indirect anti-microbial mechanisms. It was observed that under aerobic conditions the addition of clofazimine potentiated the killing of Listeria by macrophages (Cline, 1970) and also enhanced the spontaneous production of H202 (Wadee, Anderson & Rabson, 1988). Importantly, clofazimine could partially reverse the inhibitory effect of mycobacterial components on HzO2 production by phagocytes (Wadee et al., 1988). These observations have stimulated us to undertake the present study in which we have attempted to document the effect of

A. SAHU et al.

clofazimine, rifampicin and dapsone alone as well as in combinations after stimulating the rat peritoneal macrophages with bacteria, especially M. leprae.

We have shown that heat killed M. leprae, despite lacking SOD, was still a poor inducer of 02 by rat peritoneal macrophages in comparison with live S. aureus (Fig. 2). Another striking observation was that out of the three anti-leprosy drugs tested, clofazimine could enhance a significant amount of O~ release (Table 1). We have also compared the amount of 02 production by rat peritoneal macrophages in the presence of clofazimine after induction by live S. aureus with that induced by killed M. leprae. The production of 02 by macrophages in the presence of M. leprae and clofazimine minus that produced in the presence of M. leprae alone ( d - c ) was found to be significantly less than that produced by S. aureus and clofazimine minus S. aureus alone ( b - a ) (P<0.05, Fig. 2), showing thereby that two signals (i.e. signal I by bacteria and signal II by drug) are dependent on each other. These findings get some support from the study of Kitagawa et al. (1980), who stimulated human PMNLs by both N-formylmethionyl phenylalanine and Con A. However, unlike clofazimine which by itself is, apparently, incapable of eliciting O~ production above the basal level (Zeis et al., 1990), both N-formylmethionyl phenylalanine and Con A are inducers of an oxidative burst. This impairment of 02 production by M. leprae might throw some light on intracellular persistence of M. leprae in lepromatous leprosy patients even after administration of multidrug therapy (Suite & Edinborough, 1989).

Until now, there is no information regarding the therapeutic efficacy of the light exposed drug. Earlier Sahu et al. (1988) have shown that this drug retains its anti-complement activity even after light exposure. Surprisingly, on the other hand, the light reacted drug looses its property to enhance 02 release. To investigate this dichotomy of the above two properties of the clofazimine molecule we undertook a physico-chemical study of the light exposed and unexposed drug. We could not differentiate these two species on the basis of u . v . - visible, infrared and NMR spectra, thin layer chromatogram and mass spectrogram. However, the light exposed drug showed a marked increase in the diffusion current (three times) in polarogram suggesting, thereby, an increased electron accepting capacity of the clofazimine molecule after exposure to light.

Recently, Zeis & Anderson (1986) have demonstrated that clofazimine activates the

Anti-leprosy Drugs and Superoxide Anion Production

a rachidonic acid cascade of P M N L s . They have also shown tha t c lofazimine can mobi l ize a rachidonic acid f rom the m e m b r a n e phosphol ip ids of h u m a n P M N L s (Anderson , Beyers, Savage & Nel, 1988). It is k n o w n tha t a rach idonic acid acts as a second messenger of superoxide an ion p roduc t ion elicited by various stimuli and as an inducer by itself (Bromberg & Pick, 1983) via ac t iva t ion of prote in kinase C (Lambe th , 1988). This s tudy showed tha t an increase in O~ release due to c lofazimine was b locked by BPB (Table 2) suggesting tha t there might be enhanced release of a rach idonic acid due to the addi t ion of c lofazimine which increases the 02 p roduc t ion by rat

427

per i toneal macrophages . Exposure of c lofazimine to light impairs the respi ra tory burs t augment ing proper ty of the drug. A l though , the mechan i sm of this ac t ion is not clear, it may be pos tu la ted tha t the increased electron accepting capaci ty of the drug (Fig. 3) might result in the impa i rmen t of respi ra tory burs t activity. This issue needs fur ther explora t ion.

Acknowledgements - - The authors express their gratefulness to the Council of Scientific and Industrial Research, New Delhi for financial support to the senior author (A.S.). The kind gift of clofazimine from Ciba Geigy, Switzerland is also acknowledged.

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