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Dapsone toxicity: Some current perspectives
Pergamon
0306-3623(95)00029-1
Gen. Pharmac. Vol. 26, No. 7, pp. 1461-1467, 1995 Copyright © 1995 Elsevier ScienceInc. Printed in Great Britain. All rights reserved. 0306-3623/95 $29.00 + 0.00
REVIEW
Dapsone Toxicity: Some Current Perspectives* MICHAEL
D. C O L E M A N
Mechanisms o f Drug Toxicity Group, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K. [TEL"0121-359-3611 ext. 4204; Fax: 021-359-0733] (Received 12 December 1994)
Abstract-1. Dapsone is a potent anti-inflammatory and anti-parasitic compound, which is metabolised by cytochrome P-450 to hydroxylamines, which in turn cause methaemoglobinaemia and haemolysis. However, during the process of methaemoglobin formation, erythrocytes are capable of detoxifying the hydroxylamine to the parent drug, which may either reach the tissues to exert a therapeutic effect or return to the liver and be r¢-oxidised in a form of systemic cycling. This glutathione-dependent effect, combined with the un-ionised state of the drug at physiological pH, may contribute to its efficacy. 2. Paradoxically, other aspects of the glutathione-dependent cycling of the hydroxylamine metabolite may contribute to the major adverse reaction of the drug, agranulocytosis. Erythrocytes exposed to the metabolite and repeatedly washed may still release the hydroxylamine in sufficient concentration to kill mononuclear leucocytes in vitro. Thus, erythrocytes may be a conduit for the hydroxylamine to reach the bone marrow to covalently bind to granulocyte precursors, which may trigger an immune response in certain individuals and may lead to the potentially fatal eradication of granulocytes from the circulation. 3. Attempts to increase patient tolerance to dapsone have been most successful using a metabolic inhibitor to reduce hepatic oxidation of the drug to the hydroxylamine. Methaemoglobin formation in the presence of cimetidine was maintained at 30°70 below control levels for almost 3 mo, and patients' reported side effects such as headache and lethargy were significantly reduced. 4. As clinical application of new and safer dapsone analogues is years away, the use of cimetidine provides an immediate route to increasing patient compliance during dapsone therapy, especially in those maintained on dapsone dosages in excess of 200 rag/day. Key Words: Dapsone, toxicity, erythrocyte, organulocytosis
INTRODUCTION Although dapsone was first synthesised in the early part of the century, it was not established as an anti-leprous agent until the late 1940s and early 1950s. Its antiinflammatory and anti-malarial properties were discovered soon after and are now well documented (for a review, see Zuidema et al., 1986). In the last few years, dapsone has been applied to conditions as diverse as asthma, Kaposi's sarcoma and rheumatoid arthritis (for reviews, see Coleman, 1993; Coleman and Tingle, 1992; Zuidema et al., 1986). It has shown recent promise as a second-line therapy for Pneumocystis carinii pneum o n i a either alone as a prophylactic or in combination with trimethoprim (for a review, see Gallant et al., 1994). A l t h o u g h it is no more effective than the first-
*This review is dedicated to the memory of Mark J. Winn.
line approach aerosolized pentamidine, it is cheaper, easier to administer and provides added protection against toxoplasmosis in combination with pyrimethamine (Torres et al., 1993). It also demonstrates fewer adverse reactions in combination with trimethoprim compared with sulphmethoxazole and is better tolerated (Pertel and Hirschtick, 1994). The drug is so effective in the control of dermatitis herpetiformis that it is virtually diagnostic o f that disease, and it is generally effective in other conditions involving activated polymorphonuclear leucocytes such as Linear IgA disease (Zone, 1991).
HEPATIC METABOLISM OF DAPSONE Dapsone is responsible for a variety of adverse reactions ranging from methaemoglobinaemia, which occurs in all subjects, to occasional fatal agranulocytosis. With the exception o f psychosis (Gawkrodger, 1461
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Michael D. Coleman
1989), which is probably due to the parent drug, all side effects of dapsone are linked to its hepatic Nhydroxylation by cytochrome P-450 (Coleman and Tingle, 1992). Before studies on its oxidative metabolism, the acetylation of the drug was initially thought to be its major route of elimination. Dapsone acetylation is polymorphic and is mediated by cytosolic liver N-acetyltransferases (NAT) (Gelber et al., 1971), which exists in two forms (NAT-I and NAT-2) (Grant et al., 1990). Erythrocytes are also capable of acetylating the drug (Irshaid et al., 1993). Paradoxically, acetylation is not a true elimination pathway, as although plasma levels may be high, it is found only in trace amounts in urine (Coleman and Tingle, 1992). Acetylated dapsone is not linked with toxicity, unless its remaining amino group has been hydroxylated (Coleman and Tingle, 1992). The N-hydroxylation of dapsone and its acetylated derivative was recently ascribed to the 3A family of isoenzymes of cytochrome P-450 (3A3, 3A4, and 3A5) and has been proposed as a marker for this group of enzymes (for a review, see Watkins, 1994). However, a number of studies indicated that other enzymes are also capable of metabolising the drug, especially 2El but also 2C9 (Mitra et al., 1993, 1994). In addition, there was no correlation between dapsone and two other putative "probe" compounds for the same isoenzyme, 3A4 (Kinirons et al., 1993). Hence, it appears that dapsone is not a particularly "clean" experimental probe drug for this isoenzyme. Although sulphation does occur, the hydroxylamines mostly undergo glucuronidation, and the major metabolite, approximately 30070 of the dose, is dapsone hydroxylamine N-glucuronide (Israili et al., 1973; Coleman and Tingle, 1992). Some of the hydroxylamine enters the blood and is taken up extremely rapidly by erythrocytes (Coleman and Jacobus, 1993a) so that it cannot be directly detected in human plasma (Zuidema et al., 1986). The hydroxylamine causes the characteristic methaemoglobinaemia that occurs within 30-60 min of administration. It is clear that a substantial proportion of the hydroxylamine escapes phase II glucuronidation. Close structural analogues of dapsone also undergo oxidative metabolism, but with little systemic toxicological manifestation of this, that is, methaemoglobin levels are low to non-existent (Coleman and Tingle, 1992). The analogues are thought to undergo extensive phase II metabolism, and it is possible that dapsone itself and its hydroxylamine are both relatively poor substrates for glucuronyl transferases compared with those compounds. Small quantities (~ 8°70) of the hydroxylamines are found unchanged in urine (Israili et al., 1973; May et al., 1990), and this observation has been used as the basis for the calculation of a ratio of hydroxylamine (stabilised by ascorbate
added to the urine) excreted in relation to parent drug. This ratio has been used to correlate cytochrome P-450 3A4 activity in patients with susceptibility to bladder cancer (Fleming et al., 1994). However, because such low levels of hydroxylamine are eliminated unchanged and the major metabolite is an N-glucuronide of the hydroxylamine, it would appear to be more logical, accurate and reproducible to hydrolyse the conjugate, free the hydroxylamine and then use this much larger quantity as a measure of metabolism. Although the conjugate is reasonably stable and requires several hours of acid hydrolysis to fully degrade it (Coleman et al., 1990), Israili et aL (1973) suggested that it could be labile, as are many glucuronides. These conjugates can degrade in urine in the bladder because of instability or pH changes caused by change in diet constituents. If this occurred with dapsone, correlations made with parent drug and such small free hydroxylamine quantities might be unreliable, as only a small amount of degradation would greatly increase the hydroxylamine recovery in relation to dapsone. As the parent drug is also glucuronidated (Coleman et al., 1990), no studies have been carried out to the author's knowledge comparing the relative stabilities of the two conjugates. This, again, could be a complicating factor that may not have been envisaged in the studies of May et al. (1990). EXTRA-HEPATIC DAPSONE METABOLISM A number of studies showed that dapsone and its metabolites may undergo oxidation and reduction in erythrocytes (Coleman and Jacobus, 1993a,b) and neutrophils (Uetrecht et aL, 1988) as well as bone marrow (Weetman et al., 1980). The methaemoglobinaemia caused by the drug is normally reasonably well tolerated at low to moderate dapsone doses but may become a serious problem at dosages required to control dermatitis herpetiformis in some patients, which can exceed 200 mg/day (Coleman, 1993). The basic process by which hydroxylamines in general cause haemoglobin oxidation has long been known (Kiese et aL, 1950). Within the erythrocyte, aromatic amine hydroxy derivatives derived from dapsone and the carcinogen 4-aminobiphenyl bind to the superoxo-ferrihaem complex (Fe3*O2_) of oxyhaemoglobin leading to methaemoglobin (Fe 3÷) formation, as well as hydrogen peroxide. The hydroxylamines are converted to unstable nitrosoarenes in this process (Heilmair et al., 1991; Kramer et al., 1972). The reducing power of erythrocytic glutathione regenerates the nitrosoarenes to hydroxylamines that in their turn oxidise other haemoglobin molecules, thus continuing the redox cycle. The hydroxylamines deplete glutathione during this process within minutes (Coleman and Jacobus 1993a).
Dapsone toxicity Although dapsone hydroxylamine may spontaneously oxidise to the nitrosoarene in the presence of oxygen, nitroso derivatives cannot react with haemoglobin themselves but must be reduced to the hydroxylamine (Karreth and Lenk, 1991). As methaemoglobin may be reduced to haemoglobin by erythrocytic NADHdependent reductase, this is not really a "toxic" reaction in the strictest sense, as once methaemoglobin is reduced, the erythrocyte is once more available to carry oxygen. If glutathione is fully depleted and reduction of the hydroxylamine ceases, then the nitroso derivative is free to react with other intracellular thiol groups, such as those of haemoglobin itself, as well as various membrane structures in the erythrocyte. Early reports indicated that hydroxylamine-derived material was strongly associated with erythrocyte membranes (Israili et al., 1973), and more recently, it has been shown that the hydroxylamines mediate the formation of disulphide linked adducts between haemoglobin and erythrocyte skeletal proteins (Grossman et aL, 1992). This process in turn may account for the characteristic acceleration of red cell destruction seen in normal patients on dapsone therapy (Zuidema et aL, 1986), as senescent antigens may become exposed on the cell surface that promote premature sequestration by spleen macrophages (Kay, 1985). Glucose-6-phosphate dehydrogenase deficient subjects cannot generate enough N A D P H to maintain sufficient glutathione to prevent nitroso-mediated membrane damage, and acute intravascular haemolytic anaemia may result in a short period of time (Zuidema et al., 1986). However, recent evidence has emerged that the process of methaemoglobinaemia can be viewed as an elaborate and highly effective detoxification process. In a study using an experimental three-compartment system, it was shown that erythrocytes would preferentially take up dapsone hydroxylamine and protect mononuclear leucocytes from what would have been a lethal level of hydroxylamine in the absence of the red cells (Tingle et al., 1992). Subsequent studies showed that like 4-aminobiphenyl (Heilmair et al., 1991), dapsone hydroxylamine could be detoxified by erythrocytes by reduction to the parent drug (Coleman and Jacobus, 1993a). This process of reduction of hydroxylamine to dapsone was in direct proportion to the level of methaemoglobin formed. It was found that hydroxylamine-dependent methaemoglobin production could be accelerated by diethyl dithiocarbamate, and this process also accelerated hydroxylamine reduction. This theme was further explored using a two-compartment in vitro method to evaluate the ability of the dapsone thus formed to leave the red cells where reduction took place and diffuse to other red cells (Coleman and Jacobus, 1993b). It is conceivable that erythrocytes may act
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as a delivery system, whereby hydroxylamine produced in the liver is taken up by erythrocytes, rapidly reduced to the parent drug and then released into tissue compartments. This process might contribute to the overall process of diffusion of parent drug from plasma to sites of therapeutic action (Fig. 1). In the studies with the two-compartment method, it was also clear that methaemoglobin occurred in drugfree erythrocytes exposed to erythrocytes that contained the hydroxylamine. Because all excess hydroxylamine had been washed off the ceils before their insertion in the compartments, it appeared that the methaemoglobin forming hydroxylamine, as well as the parent drug, escaped the erythrocytes into other drug free cells (Coleman and Jacobus, 1993b). This theme was pursued, and it was found that hydroxylamine-exposed washed erythrocytes were capable of liberating enough hydroxylamine to kill significant numbers of mononucleocytes in the two-compartment method (Coleman et al., 1994). Hence, although Tingle et al. (1992) showed the protective effect of erythrocytes when a high concentration of the hydroxylamine was avidly taken up by the red cells, after metabolite exposure, metabolite could be released from the cells as well as parent drug over a longer period of time. Exactly how the hydroxylamine is released is debatable, although glutathione is necessary for this process to occur (Coleman et al., 1994). It is also likely that dapsone "produced" by red cells from the metabolite would then be re-oxidised by the
H\N/H
.
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O\ N/ H
~e
p~r'~ntd, ll's~es
"~1' l°\"/"
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Erythrocyte ~ " f Q ~ ' ~ t Erythrocyte O~S ~0
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Fig. 1. A scheme describing the possible "systemic cycling" of dapson¢ and its hydroxylamine in humans.
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Michael D. Coleman
liver to the hydroxylamine, and thus a "systemic cycling" process might occur where the drug would be alternately metabofised and detoxified, possibly retarding the elimination of the drug still further (Fig. 1). The acetylated hydroxylamine can also be reduced by erythrocytes to monoacetyl dapsone (Coleman et al., in press). It has also been shown that monoacetyl dapsone hydroxylamine is equipotent with dapsone hydroxylamine as a methaemglobin former (Vage et al., 1994); however, the initial rate (<10 min) of haemoglobin oxidation mediated by the acetylated hydroxylamine is more rapid compared with dapsone hydroxylamine, probably due to the increased lipid solubility of the acetylated derivative that leads to increased penetration of the erythrocytic membrane (Coleman et al., in press). DAPSONE AND AGRANULOCYTOSIS Agranulocytosis is almost exclusively a drug-mediated condition and is as poorly understood as it is unpredictable (Uetrecht, 1992). Recent studies indicate that the usual observation that agranulocytosis occurs suddenly at 2-3 mo after commencement of dapsone therapy is simplistic and that loss of cells is rather more gradual and starts soon after therapy begins (Duhra and Charles-Holmes, 1991). Considering the possible causes of the condition, the parent drug itself is not directly toxic to white or red cells (Coleman and Jacobus, 1993b). Exogenous hydroxylamine metabolite of dapsone is certainly cytotoxic to mononucleocytes and bone marrow in vitro (Coleman and Tingle, 1992; Weetman et al., 1980). It has been proposed, based on the metabolizing ability of granulocytes and their precursors, that the neutrophil myeloperoxidase oxidises dapsone to its hydroxylamine, which then inhibits the enzyme and kills the cell (Uetrecht et al., 1988). However, most recent work has indicated that dapsone inhibits compound I of myeloperoxidase by converting it to the inactive compound II (Kettle and Winterbourn, 1991; Kettle et al., 1993). In addition, van Zyl et al. (1991) found that the myeloperoxidase system did not oxidise dapsone. If any dapsone oxidation does occur in neutrophils during respiratory burst, the actual oxidant is likely to be hydrogen peroxide rather than myeloperoxidase (Kettle et al., 1993). Other sources of oxidation might include isoenzymes of cytochrome P-450, which might be capable of making the hydroxylamine and are present in various bone marrow systems (Schnier et al., 1989). In addition, myeloperoxidase has been shown to be capable of N-chlorinating sulphonamides, leading to the formation of reactive metabolites that could be involved in granulocyte destruction (Uetrecht et al., 1993). However, in a healthy individual, it seems unlikely that neutrophils would undergo respiritory
burst in bone marrow and liberate large quantities of oxidants without some clear manifestation of patient symptoms; generally, the first signs of agranulocytosis are well after cell numbers have already fallen below critical levels. Overall, the possibility that a gradual apparently symptomless and highly cell selective condition such as agranulocytosis could be caused by such explosive and indiscriminate tissue destructive processes as those involved in cellular oxidant production appears to be remote. Alternatively, it may be postulated that a link may exist between agranulocytosis and erythrocytic metabolism of dapsone hydroxylamine originally derived from the liver. If the hydroxylamine "leeches" out of erythrocytes as they circulate through bone marrow, it is possible that small sub-lethal quantities of the metabolite may enter granulocyte precursor cells and auto-oxidise to the tissue reactive (Coleman and Tingle, 1992) nitrosoarene. This sequence of events, if it does indeed occur, would happen in all patients exposed to the drug, and clearly only a small fraction of patients develop agranulocytosis. Therefore, it has been suggested that some form of immune reaction is involved. The evidence for this is sketchy, but it is apparent that during the condition of agranulocytosis, if dapsone is immediately stopped and the patient survives the infections, the granulocyte population recovers in a matter of days, suggesting that the marrow is intact and undamaged (Uetrecht, 1992). This is at odds with conditions such as aplastic anaemia, where the marrow is badly damaged and recovers far more slowly. Therefore, whichever process is happening, it is highly specific. In the case of dapsone-mediated agranulocytosis, either dapsone itself is acting as an "off switch" by mimicking an endogenous regulator of granulocyte production or the hydroxylamine itself may be killing the cells progressively, but only in certain susceptible individuals or some form of highly specific immune response is triggered that selectively eliminates the cells concerned. One way an immune response might be involved could be through the hapten hypothesis (Park et al., 1988), where binding of drug related material, such as the nitrosoarene, would be followed by some form of antigen processing and ultimately expression of the "hapten" on the cell surface. The granulocyte would then be recognised as foreign and destroyed. In the case of dapsone, once hydroxylamine production is stopped and "haptenation" ceases, then in the absence of the stimulus, the immune system ceases to recognise and destroy granulocytes. This theory does not explain why other populations of white cells are not apparently exposed to this risk. However, if the parent drug is not acting in some way to switch off granulocyte produc-
Dapsone toxicity tion, then the hydroxylamine metabolite seems to be the most likely cause of agranulocytosis. It is also plausible that erythrocytes provide a conduit for hydroxylamine penetration into the bone marrow, which may complement any possible in situ activation of dapsone to its metabolite by white cell precursors. In view of dapsone's inhibitory action on myeloperoxidase metabolism, it appears more likely that hydroxylamine delivered by erythrocytes may be the major contributor to nitrosoarene binding to bone marrow (Fig. 1). Among complex risk factors such as age, sex and ethnic origin, immune reactivity may be the greatest parameter: agranulocytosis is unknown in partially immunosuppressed leprosy patients, whereas in conditions characterised by immune hyper-responsiveness such as dermatitis herpetiformis, the frequency is an alarming 0.3%, compared with the much lower figure (> 1:10,000) for normal individuals (Hornsten et al., 1990; Ognibene, 1970). The possibility of dapsone itself acting as an off switch for granulocyte production has not, to the author's knowledge, been previously discussed. How this interaction might occur is open to speculation. In the case of drug metabolism, low molecular weight compounds are capable of indirectly modulating or inducing the expression of enzyme systems such as cytochrome P-450. This can be achieved by binding of the compound to cytoplasmic receptors that then communicate with DNA (for a review, see Gonzalez, 1989). Apoptosis, or programmed cell death, is a process in which cytoplasmic regulation is thought to occur (Stewart, 1994). It is conceivable that a low molecular weight compound such as dapsone in certain individuals with defective cytoplasmic receptors might perhaps have their granulocytes artificially programmed to die by dapsone. Once the drug is withdrawn, binding to the receptor would cease and cell survival would return to normal. In any case, recent reports showed an increasing number of specific aspects of neutrophil function to be compromised by dapsone, including inhibition of adherance and capillary extravasation (Booth et al., 1992), lysosomal enzyme activity, possible binding to IgA and attenuation of chemotaxis (Thuong-Nguyen et al., 1993). In light of these interactions, the potential for dapsone to somehow regulate development and maturation o f granulocytes might not be far-fetched.
INCREASING PATIENT TOLERANCE TO DAPSONE A number of attempts have been made to increase the tolerance of patients to dapsone, including the co-
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administration with dapsone of vitamins E and C either alone or in combination (Prussick et al., 1992). These antioxidant studies demonstrated little impact on the methaemoglobinaemia caused by the drug. An alternative approach was first explored in rats where a number of metabolic inhibitors of cytochrome P-450 were evaluated in their ability to attenuate the methaemoglobin due to single dose dapsone administration (Coleman, 1993). Cimetidine was selected as the most practical inhibitor for use in humans. Single-dose studies using cimetidine (3 x 400 mg/day) in human volunteers showed a significant reduction in methaemoglobin formation, as well as a fall in hydroxylamine excretion as its N-glucuronide (Coleman, 1993). Meanwhile, chronic studies in rats indicated that methaemoglobin formation could be suppressed for several days (Coleman and Tingle, 1991), and a study in patients on dapsone suffering from dermatitis herpetiformis was organised. This work involved the establishment of baseline methaemoglobin formation and dapsone plasma concentrations for 14 days. The range of dapsone dosage was 50-350 mg/day. After commencement of cimetidine (3 × 400 mg/day), methaemoglobin fell by 27%, peak dapsone plasma levels were unchanged but trough levels rose significantly. Most importantly, four of the six patients reported a reduction in lethargy and headaches and suppression of their disease was unchanged (Coleman, 1993). Follow up studies using a lower cimetidine dose were not successful in reducting methaemoglobin levels in patients (Rhodes et al., 1992). A longer duration study of 3 mo using 1600 mg/day of cimetidine as inhibitor showed that dapsone levels were elevated significantly for the full 12 wk, whereas methaemoglobin levels were depressed by 30% for 11 of 12 wk0 Visual analogue scores for headache also fell significantly (Rhodes et al., 1995). Thus, this approach demonstrated that higher plasma levels of dapsone may be achieved without a concomitant increase in toxicity. This therapeutic approach may be applied with minimal risk to the patient, as cimetidine is now considered safe enough to be available over the counter at pharmacies, net hydroxylamine formation is reduced and control of disease is maintained. It is possible that the reduction in hydroxylamine formation may in turn reduce the risk of agranulocytosis, which would be especially welcome in view of the stated risks to dermatitis herpatiformis patients (Duhra and Charles-Holmes 1991; Hornsten et al., 1990). More potent and less toxic derivatives of dapsone have been synthesised and tested and are very promising (Seydel, 1993). However, they may take many years to reach patients as the market for these compounds is relatively small. In the meantime, the use of a metabolic inhibitor during dapsone administration is currently available.
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Michael D. Coleman CONCLUSIONS
D a p s o n e is a useful b u t rather toxic drug t h a t has found wider therapeutic application in recent years. Often it is not the m o s t p o t e n t drug in any given experimental system, b u t because of its un-ionised state at physiological p H ( C o l e m a n a n d Tingle 1992), it can penetrate tissues in sufficient c o n c e n t r a t i o n to exert a p o t e n t therapeutic action. As more is u n d e r s t o o d o f its modes of action a n d toxicology, it is hoped that these wider applications may benefit patients while reducing the risks associated with the drug. It is now recomm e n d e d t h a t d u r i n g the first 3 m o o f therapy, white cell counts should be m o n i t o r e d every 2 wk ( C o c k b u r n et al., 1993). A l t h o u g h p a t i e n t tolerance to d a p s o n e varies widely, those o n high dosage or those with genetic predispositions to high m e t h a e m o g l o b i n p r o b l e m s would surely benefit f r o m c o n c u r r e n t cimetidine therapy. Physicians may not always see control o f side effects as a problem, as patients experiencing t h e m often d o n o t c o m p l a i n o f s y m p t o m s such as h e a d a c h e or lethargy. In the case o f dapsone, in view o f the potential severity o f its adverse reactions, it would be very desirable for i n f o r m a t i o n o n patients' s y m p t o m s to be m o s t actively sought. Acknowledgement-Preparation of this review was aided by
fruitful discussions with Dr. N. A. Helsby.
REFERENCES Booth S., Moody C., Dahl M., Herron M. and Nelson R. (1992) Dapsone suppresses integrin-mediated neutrophil adherance function. J. Invest. Dermatol. 98, 135-140. Cockburn E. M., Wood S. M., Waller P. C. and Bleehan S. S. (1993) Dapsone induced agranulocytosis: spontaneous reporting data. Br. J. Dermatol. 128, 702-703. Coleman M. D. (1993) Dapsone: modes of action, toxicity and possible strategies for increasing patient tolerance. Br. J. Dermatol. 129, 507-513. Coleman M. D. and Jacobus D. P. (1993a) Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro. Biochem. Pharmac. 45, 1027-1033. Coleman M. D. and Jacobus D. P. (1993b) Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro. II. Movement of dapsone across a semipermeable membrane. Biochem. Pharmac. 46, 1363-1368. Coleman M. D., Pahal K. K. and Gardiner J. M. (in press). The effect of acetylation and deacetylation on the disposition of dapsone and monoacetyl dapsone hydroxylamines in human erythrocytes in vitro. J. Pharm. Pharmacol. Coleman M. D., Simpson J. and Jacobus D. P. (1994) Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro. IV. The role of the erythrocyte in the development of dapsonedependent agranulocytosis. Biochem. Pharmac. 48, 13491354. Coleman M. D. and Tingle M. D. (1992) The use of a metabolic inhibitor to reduce dapsone-dependent haematological toxicity. Drug Dev. Res. 25, 1-16. Duhra P. and Charles-Holmes R. (1991) Linear lgA disease
with haemorrhagic pompholyx and dapsone-induced neutropenia. Br. J. Dermatol. 125, 172-174. Fleming C. M., Persad R., Kaisary A., Smith P., Adedoyin A., Porter J., Wilkinson G. R. and Branch R. A. (1994) Low activity of dapsone N-hydroxylation as a susceptibility risk factor in aggressive bladder cancer. Pharmacogenetics 4, 199-207. Gallant J. E., Moore R. D. and Chaisson R. E. (1994) Prophylaxis for opportunistic infections in patients with HIV infection. Ann. Intern. Med. 120, 932-944. Gawkrodger D. (1989) Manic depression induced by dapsone in patient with dermatitis herpetiformis. Br. Med. J. 299, 860. Gelber R., Peters., J. H., Ross-Gordon M. S., Glazko A. J., and Levy L. (1971) The polymorphic acetylation of dapsone in man. Clin. Pharmac. Ther. 12, 225-238. Gonzalez E (1989) The molecular biology of cytochrome P-450s. Pharmac. Rev. 40, 243-288. Grant D. M., Morike K., Eichelbaum M. and Meyer U. A. (1990) Acetylation Pharmacogenetics. J. Clin. Invest. 85, 968-972. Grossman S. J., Simson J. and Jollow D. J. (1992) Dapsoneinduced hemolytic anemia: effect of N-hydroxydapsone on the sulfhydryl status and membrane proteins of rat erythrocytes. Toxicol. Appl. Pharmac. 117, 208-217. Heilmair R., Karreth S. and Lenk W. (1991) The metabolism of 4-aminobiphenyl in the rat II. Reaction of 4-hydroxy-4aminobiphenyl with rat blood in vitro. Xenobiotica 21, 805-815. Hornsten P., Keisu M. and Wiholm B. E. (1990) The incidence of agranulocytosis during treatment of dermatitis herpetiformis with dapsone as reported in Sweden, 1972 through 1988. Arch. Dermatol. 126, 919-922. Irshaid Y. M., A1-Hadidi H. F., Abuirjeie M. A., Rawashdeh N. M., and Gharaibeh N. S. (1993) Acetylation of dapsone by human whole blood. Int. J. Clin. Pharmac. Ther. Toxicol. 31, 18-22. Israili Z. H., Cucinell S. A., Vaught J., Davis B., Zesser J. M. and Dayton P. G. (1973) Studies of the metabolism of dapsone in man and experimental animals. Formation of N-hydroxy metabolites. J. Pharmac. Exp. Ther 187, 138-151. Karreth S. and Lenk W. (1991) The metabolism of 4-aminobiphenyl in the rat. IV. Ferrihaemoglobin formation by 4aminobiphenyl metabolites. Xenobiotica 21, 971-977. Kay M. M. (1985) Aging of cell membrane molecules leads to an appearance of aging antigen and removal of senescent cells. Gerontology 31, 215-235. Kettle A. J. and Winterbourn C. C. (1991) Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs. Biochem. Pharmac. 41, 1485-1492. Kettle A. J., Gedye C. A. and Winterbourn C. C. (1993) Superoxide is an antagonist of antiiinflammatory drugs that inhibit hypochlorous acid production by myeloperoxidase. Biochem. Pharmac. 45, 2003-2010. Kiese M., Reinwein D. and Waller H. D. (1950) Kinetik der Hamiglobinbildung. IV Mitteilung. Die Hamiglobinbildung durch Phenylhydroxylamin und Nitrosobenzol in roten Zellen in vitro. Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmac. 210, 393-398. Kinirons M. T., O'Shea D., Downing T. E., Fitzwilliam A. T., Joelenbeck L., Groopman J., Wilkinson G. R. and Wood A. J. (1993) Absence of correlations among three putative in vivo probes of human cytochrome P4503A activity in young healthy men. Clin. Pharmac. Ther. 54, 621-629. Kramer P. A., Glader B. E. and Li T. K. (1972) Mechanism of methaemoglobin formation by diphenylsulphones. Biochem. Pharmac. 21, 1265-1274. May D. G., Porter J. A., Wilkinson G. R. and Branch R. A. (1990) The contribution of N-hydroxylation and acetylation to dapsone pharmacokinetics in normal subjects. Clin. Pharmac. Ther. 48, 619-627.
Dapsone toxicity Mitra A. K., Kalhorn T. F., Thummel K. E., Unadkat J. A. and Slattery J. T. (1993) Metabolism of arylamines by human liver microsomal cytochrome P-450(S). Proc. ISSX, Fifth North American Meeting, Tucson, AZ. October 17-21. Mitra A. K., Thummel K. E., Unadkat J. A. and Slattery J. T. (1994) Metabolism of dapsone in human liver by cytochrome P-450 2El. Proc. Sixth North American Meeting, Raleigh, NC, October 23-27. Ognibene A. J. (1970) Agranulocytosis due to dapsone. Ann. Intern. Med. 72, 521-524. Park B. K., Coleman J. W. and Kitteringham N. R. (1988) Drug disposition and drug hypersensitivity. Biochem. Pharmac. 36, 581-590. Pertel P. and Hirschtick R. (1994) Adverse reactions to dapsone in persons infected with human immunodeficiency virus. Clin. Infect. Dis. 18, 630-632. Prussick R., Ali M. A. M., Rosenthal D. and Guyatt G. (1992) The protective effect of vitamin E on the haemolysis associated with dapsone treatment in patients with dermatitis herpetiformis. Arch. Dermatol. 128, 210-213. Rhodes L. E., Coleman M. D., Tingle M. D., Verbov J. L., Park B. K. and Friedmann P. S. (1992) Cimetidine reduces dapsone-induced haematological toxicity in dermatitis herpetiformis (DH). Br. J. Dermatol. 127, 426-427. Rhodes L. E., Tingle M. D., Verbov J. L., Park B. K. and Friedmann P. S. (1995) Cimetidine improves the therapeutic/toxic ratio of dapsone in patients on chronic dapsone therapy. Br. J. Dermatol. 132, 257-262. Schnier G. G., Laethem C. L. and Koop D. R. (1989) Identification and induction of cytochromes P450, 450 E1 and P450 IAl in Rabbit bone marrow. £ Pharmac. Exp. Ther. 251, 790-796. Seydel J. K. (1993) In vitro and in vivo results of brodimoprim and analogues alone and in combination against E. coli and mycobacteria. Stewart B. W. (1994) Mechanisms of apoptosis: integration of genetic, biochemical and cellular indicators. JNCI 86, 1286-1296. Tingle M. D., Blee M. A. B., Coleman M. D. and Park B. K. (1992) The use of a novel three compartment system to investigate cell selective drug toxicity in vitro. Br. J. Clin. Pharmac. 33, 216P-217P.
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Thuong-Nguyen V., Kadunce D. P., Hendrix J. D., Gammon W. R. and Zone J. J. (1993) Inhibition of neutrophil adherance to antibody by dapsone: a possible therapeutic mechanism of dapsone in the treatment of IgA dermatoses. J. Invest. Dermatol. 10O, 349-355. Tortes R. A., Barr M., Thorn M., Gregory G., Kiely S., Chanin E., Carlo C., Martin M. and Thornton J. (1993) Randomized trial of dapsone and aerosolized pentamidine for the prophylaxis of Pneumocystis carinii pneumonia and toxoplasmic encephalitis. Am. J. Med. 95, 573-583. Uetrecht J. P., Zahid N., Shear N. H. and Rubin R. (1988) Metabolism of dapsone to a hydroxylamine by human neutrophils and mononuclear cells. J. Pharmac. Exp. Ther. 245, 274-279. Uetrecht J. P. (1992) The Role of leukocyte-generated reactive metabolites in the pathogenesis of idiosyncratic drug reactions. Drug Metab. Rev. 24, 299-366. Uetrecht J. P., Shear N. H. and Zahid N. (1993) N-chlorination of sulfamethazole and dapsone by the myeloperoxidase system. Drug Metab. Disp. 21, 830-834. Vage C., Saab N., Woster P. M. and Svensson C. K. (1994) Dapsone-induced hematologic toxicity: comparison of the methemoglobin-formingability of hydroxylamine metabolites of dapsone in rat and human blood. Toxicol. Appl. Pharmacol. 129, 309-316. Watkins P. B. (1994) Noninvasive tests of CYP3A enzymes. Pharmacogenetics 4, 171-184. Weetman R. M., Boxer L. A., Brown M. P., Mantich N. H. and Baehner R. L. (1980)In vitro inhibition of granulopoiesis by 4 amino 4' hydroxylamino phenyl sulphone. Br. J. Hematol. 45, 361-370. Zone J. J. (1991) Dermatitis herpetiformis. Curt. Prob. Dermatol. 3, 4-41. Zuidema J., Hilbers-Modderman E. S. M. and Markus F. W. H. M. (1986) Clinical pharmacokinetics of dapsone. Clin. Pharmac. l l , 299-325. van Zyl J. M., Basson K., Kriegler A. and van der Walt (1991) Mechanisms by which clofazimine and dapsone inhibit the myeloperoxidase system. Biochem. Pharmac. 42, 599-608.
0306-3623(95)00029-1
Gen. Pharmac. Vol. 26, No. 7, pp. 1461-1467, 1995 Copyright © 1995 Elsevier ScienceInc. Printed in Great Britain. All rights reserved. 0306-3623/95 $29.00 + 0.00
REVIEW
Dapsone Toxicity: Some Current Perspectives* MICHAEL
D. C O L E M A N
Mechanisms o f Drug Toxicity Group, Department of Pharmaceutical Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, U.K. [TEL"0121-359-3611 ext. 4204; Fax: 021-359-0733] (Received 12 December 1994)
Abstract-1. Dapsone is a potent anti-inflammatory and anti-parasitic compound, which is metabolised by cytochrome P-450 to hydroxylamines, which in turn cause methaemoglobinaemia and haemolysis. However, during the process of methaemoglobin formation, erythrocytes are capable of detoxifying the hydroxylamine to the parent drug, which may either reach the tissues to exert a therapeutic effect or return to the liver and be r¢-oxidised in a form of systemic cycling. This glutathione-dependent effect, combined with the un-ionised state of the drug at physiological pH, may contribute to its efficacy. 2. Paradoxically, other aspects of the glutathione-dependent cycling of the hydroxylamine metabolite may contribute to the major adverse reaction of the drug, agranulocytosis. Erythrocytes exposed to the metabolite and repeatedly washed may still release the hydroxylamine in sufficient concentration to kill mononuclear leucocytes in vitro. Thus, erythrocytes may be a conduit for the hydroxylamine to reach the bone marrow to covalently bind to granulocyte precursors, which may trigger an immune response in certain individuals and may lead to the potentially fatal eradication of granulocytes from the circulation. 3. Attempts to increase patient tolerance to dapsone have been most successful using a metabolic inhibitor to reduce hepatic oxidation of the drug to the hydroxylamine. Methaemoglobin formation in the presence of cimetidine was maintained at 30°70 below control levels for almost 3 mo, and patients' reported side effects such as headache and lethargy were significantly reduced. 4. As clinical application of new and safer dapsone analogues is years away, the use of cimetidine provides an immediate route to increasing patient compliance during dapsone therapy, especially in those maintained on dapsone dosages in excess of 200 rag/day. Key Words: Dapsone, toxicity, erythrocyte, organulocytosis
INTRODUCTION Although dapsone was first synthesised in the early part of the century, it was not established as an anti-leprous agent until the late 1940s and early 1950s. Its antiinflammatory and anti-malarial properties were discovered soon after and are now well documented (for a review, see Zuidema et al., 1986). In the last few years, dapsone has been applied to conditions as diverse as asthma, Kaposi's sarcoma and rheumatoid arthritis (for reviews, see Coleman, 1993; Coleman and Tingle, 1992; Zuidema et al., 1986). It has shown recent promise as a second-line therapy for Pneumocystis carinii pneum o n i a either alone as a prophylactic or in combination with trimethoprim (for a review, see Gallant et al., 1994). A l t h o u g h it is no more effective than the first-
*This review is dedicated to the memory of Mark J. Winn.
line approach aerosolized pentamidine, it is cheaper, easier to administer and provides added protection against toxoplasmosis in combination with pyrimethamine (Torres et al., 1993). It also demonstrates fewer adverse reactions in combination with trimethoprim compared with sulphmethoxazole and is better tolerated (Pertel and Hirschtick, 1994). The drug is so effective in the control of dermatitis herpetiformis that it is virtually diagnostic o f that disease, and it is generally effective in other conditions involving activated polymorphonuclear leucocytes such as Linear IgA disease (Zone, 1991).
HEPATIC METABOLISM OF DAPSONE Dapsone is responsible for a variety of adverse reactions ranging from methaemoglobinaemia, which occurs in all subjects, to occasional fatal agranulocytosis. With the exception o f psychosis (Gawkrodger, 1461
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Michael D. Coleman
1989), which is probably due to the parent drug, all side effects of dapsone are linked to its hepatic Nhydroxylation by cytochrome P-450 (Coleman and Tingle, 1992). Before studies on its oxidative metabolism, the acetylation of the drug was initially thought to be its major route of elimination. Dapsone acetylation is polymorphic and is mediated by cytosolic liver N-acetyltransferases (NAT) (Gelber et al., 1971), which exists in two forms (NAT-I and NAT-2) (Grant et al., 1990). Erythrocytes are also capable of acetylating the drug (Irshaid et al., 1993). Paradoxically, acetylation is not a true elimination pathway, as although plasma levels may be high, it is found only in trace amounts in urine (Coleman and Tingle, 1992). Acetylated dapsone is not linked with toxicity, unless its remaining amino group has been hydroxylated (Coleman and Tingle, 1992). The N-hydroxylation of dapsone and its acetylated derivative was recently ascribed to the 3A family of isoenzymes of cytochrome P-450 (3A3, 3A4, and 3A5) and has been proposed as a marker for this group of enzymes (for a review, see Watkins, 1994). However, a number of studies indicated that other enzymes are also capable of metabolising the drug, especially 2El but also 2C9 (Mitra et al., 1993, 1994). In addition, there was no correlation between dapsone and two other putative "probe" compounds for the same isoenzyme, 3A4 (Kinirons et al., 1993). Hence, it appears that dapsone is not a particularly "clean" experimental probe drug for this isoenzyme. Although sulphation does occur, the hydroxylamines mostly undergo glucuronidation, and the major metabolite, approximately 30070 of the dose, is dapsone hydroxylamine N-glucuronide (Israili et al., 1973; Coleman and Tingle, 1992). Some of the hydroxylamine enters the blood and is taken up extremely rapidly by erythrocytes (Coleman and Jacobus, 1993a) so that it cannot be directly detected in human plasma (Zuidema et al., 1986). The hydroxylamine causes the characteristic methaemoglobinaemia that occurs within 30-60 min of administration. It is clear that a substantial proportion of the hydroxylamine escapes phase II glucuronidation. Close structural analogues of dapsone also undergo oxidative metabolism, but with little systemic toxicological manifestation of this, that is, methaemoglobin levels are low to non-existent (Coleman and Tingle, 1992). The analogues are thought to undergo extensive phase II metabolism, and it is possible that dapsone itself and its hydroxylamine are both relatively poor substrates for glucuronyl transferases compared with those compounds. Small quantities (~ 8°70) of the hydroxylamines are found unchanged in urine (Israili et al., 1973; May et al., 1990), and this observation has been used as the basis for the calculation of a ratio of hydroxylamine (stabilised by ascorbate
added to the urine) excreted in relation to parent drug. This ratio has been used to correlate cytochrome P-450 3A4 activity in patients with susceptibility to bladder cancer (Fleming et al., 1994). However, because such low levels of hydroxylamine are eliminated unchanged and the major metabolite is an N-glucuronide of the hydroxylamine, it would appear to be more logical, accurate and reproducible to hydrolyse the conjugate, free the hydroxylamine and then use this much larger quantity as a measure of metabolism. Although the conjugate is reasonably stable and requires several hours of acid hydrolysis to fully degrade it (Coleman et al., 1990), Israili et aL (1973) suggested that it could be labile, as are many glucuronides. These conjugates can degrade in urine in the bladder because of instability or pH changes caused by change in diet constituents. If this occurred with dapsone, correlations made with parent drug and such small free hydroxylamine quantities might be unreliable, as only a small amount of degradation would greatly increase the hydroxylamine recovery in relation to dapsone. As the parent drug is also glucuronidated (Coleman et al., 1990), no studies have been carried out to the author's knowledge comparing the relative stabilities of the two conjugates. This, again, could be a complicating factor that may not have been envisaged in the studies of May et al. (1990). EXTRA-HEPATIC DAPSONE METABOLISM A number of studies showed that dapsone and its metabolites may undergo oxidation and reduction in erythrocytes (Coleman and Jacobus, 1993a,b) and neutrophils (Uetrecht et aL, 1988) as well as bone marrow (Weetman et al., 1980). The methaemoglobinaemia caused by the drug is normally reasonably well tolerated at low to moderate dapsone doses but may become a serious problem at dosages required to control dermatitis herpetiformis in some patients, which can exceed 200 mg/day (Coleman, 1993). The basic process by which hydroxylamines in general cause haemoglobin oxidation has long been known (Kiese et aL, 1950). Within the erythrocyte, aromatic amine hydroxy derivatives derived from dapsone and the carcinogen 4-aminobiphenyl bind to the superoxo-ferrihaem complex (Fe3*O2_) of oxyhaemoglobin leading to methaemoglobin (Fe 3÷) formation, as well as hydrogen peroxide. The hydroxylamines are converted to unstable nitrosoarenes in this process (Heilmair et al., 1991; Kramer et al., 1972). The reducing power of erythrocytic glutathione regenerates the nitrosoarenes to hydroxylamines that in their turn oxidise other haemoglobin molecules, thus continuing the redox cycle. The hydroxylamines deplete glutathione during this process within minutes (Coleman and Jacobus 1993a).
Dapsone toxicity Although dapsone hydroxylamine may spontaneously oxidise to the nitrosoarene in the presence of oxygen, nitroso derivatives cannot react with haemoglobin themselves but must be reduced to the hydroxylamine (Karreth and Lenk, 1991). As methaemoglobin may be reduced to haemoglobin by erythrocytic NADHdependent reductase, this is not really a "toxic" reaction in the strictest sense, as once methaemoglobin is reduced, the erythrocyte is once more available to carry oxygen. If glutathione is fully depleted and reduction of the hydroxylamine ceases, then the nitroso derivative is free to react with other intracellular thiol groups, such as those of haemoglobin itself, as well as various membrane structures in the erythrocyte. Early reports indicated that hydroxylamine-derived material was strongly associated with erythrocyte membranes (Israili et al., 1973), and more recently, it has been shown that the hydroxylamines mediate the formation of disulphide linked adducts between haemoglobin and erythrocyte skeletal proteins (Grossman et aL, 1992). This process in turn may account for the characteristic acceleration of red cell destruction seen in normal patients on dapsone therapy (Zuidema et aL, 1986), as senescent antigens may become exposed on the cell surface that promote premature sequestration by spleen macrophages (Kay, 1985). Glucose-6-phosphate dehydrogenase deficient subjects cannot generate enough N A D P H to maintain sufficient glutathione to prevent nitroso-mediated membrane damage, and acute intravascular haemolytic anaemia may result in a short period of time (Zuidema et al., 1986). However, recent evidence has emerged that the process of methaemoglobinaemia can be viewed as an elaborate and highly effective detoxification process. In a study using an experimental three-compartment system, it was shown that erythrocytes would preferentially take up dapsone hydroxylamine and protect mononuclear leucocytes from what would have been a lethal level of hydroxylamine in the absence of the red cells (Tingle et al., 1992). Subsequent studies showed that like 4-aminobiphenyl (Heilmair et al., 1991), dapsone hydroxylamine could be detoxified by erythrocytes by reduction to the parent drug (Coleman and Jacobus, 1993a). This process of reduction of hydroxylamine to dapsone was in direct proportion to the level of methaemoglobin formed. It was found that hydroxylamine-dependent methaemoglobin production could be accelerated by diethyl dithiocarbamate, and this process also accelerated hydroxylamine reduction. This theme was further explored using a two-compartment in vitro method to evaluate the ability of the dapsone thus formed to leave the red cells where reduction took place and diffuse to other red cells (Coleman and Jacobus, 1993b). It is conceivable that erythrocytes may act
1463
as a delivery system, whereby hydroxylamine produced in the liver is taken up by erythrocytes, rapidly reduced to the parent drug and then released into tissue compartments. This process might contribute to the overall process of diffusion of parent drug from plasma to sites of therapeutic action (Fig. 1). In the studies with the two-compartment method, it was also clear that methaemoglobin occurred in drugfree erythrocytes exposed to erythrocytes that contained the hydroxylamine. Because all excess hydroxylamine had been washed off the ceils before their insertion in the compartments, it appeared that the methaemoglobin forming hydroxylamine, as well as the parent drug, escaped the erythrocytes into other drug free cells (Coleman and Jacobus, 1993b). This theme was pursued, and it was found that hydroxylamine-exposed washed erythrocytes were capable of liberating enough hydroxylamine to kill significant numbers of mononucleocytes in the two-compartment method (Coleman et al., 1994). Hence, although Tingle et al. (1992) showed the protective effect of erythrocytes when a high concentration of the hydroxylamine was avidly taken up by the red cells, after metabolite exposure, metabolite could be released from the cells as well as parent drug over a longer period of time. Exactly how the hydroxylamine is released is debatable, although glutathione is necessary for this process to occur (Coleman et al., 1994). It is also likely that dapsone "produced" by red cells from the metabolite would then be re-oxidised by the
H\N/H
.
J
O\ N/ H
~e
p~r'~ntd, ll's~es
"~1' l°\"/"
I
Erythrocyte ~ " f Q ~ ' ~ t Erythrocyte O~S ~0
H/N\H Hydroxylarnlne
granulo~y[~
via erythrocytes
Fig. 1. A scheme describing the possible "systemic cycling" of dapson¢ and its hydroxylamine in humans.
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Michael D. Coleman
liver to the hydroxylamine, and thus a "systemic cycling" process might occur where the drug would be alternately metabofised and detoxified, possibly retarding the elimination of the drug still further (Fig. 1). The acetylated hydroxylamine can also be reduced by erythrocytes to monoacetyl dapsone (Coleman et al., in press). It has also been shown that monoacetyl dapsone hydroxylamine is equipotent with dapsone hydroxylamine as a methaemglobin former (Vage et al., 1994); however, the initial rate (<10 min) of haemoglobin oxidation mediated by the acetylated hydroxylamine is more rapid compared with dapsone hydroxylamine, probably due to the increased lipid solubility of the acetylated derivative that leads to increased penetration of the erythrocytic membrane (Coleman et al., in press). DAPSONE AND AGRANULOCYTOSIS Agranulocytosis is almost exclusively a drug-mediated condition and is as poorly understood as it is unpredictable (Uetrecht, 1992). Recent studies indicate that the usual observation that agranulocytosis occurs suddenly at 2-3 mo after commencement of dapsone therapy is simplistic and that loss of cells is rather more gradual and starts soon after therapy begins (Duhra and Charles-Holmes, 1991). Considering the possible causes of the condition, the parent drug itself is not directly toxic to white or red cells (Coleman and Jacobus, 1993b). Exogenous hydroxylamine metabolite of dapsone is certainly cytotoxic to mononucleocytes and bone marrow in vitro (Coleman and Tingle, 1992; Weetman et al., 1980). It has been proposed, based on the metabolizing ability of granulocytes and their precursors, that the neutrophil myeloperoxidase oxidises dapsone to its hydroxylamine, which then inhibits the enzyme and kills the cell (Uetrecht et al., 1988). However, most recent work has indicated that dapsone inhibits compound I of myeloperoxidase by converting it to the inactive compound II (Kettle and Winterbourn, 1991; Kettle et al., 1993). In addition, van Zyl et al. (1991) found that the myeloperoxidase system did not oxidise dapsone. If any dapsone oxidation does occur in neutrophils during respiratory burst, the actual oxidant is likely to be hydrogen peroxide rather than myeloperoxidase (Kettle et al., 1993). Other sources of oxidation might include isoenzymes of cytochrome P-450, which might be capable of making the hydroxylamine and are present in various bone marrow systems (Schnier et al., 1989). In addition, myeloperoxidase has been shown to be capable of N-chlorinating sulphonamides, leading to the formation of reactive metabolites that could be involved in granulocyte destruction (Uetrecht et al., 1993). However, in a healthy individual, it seems unlikely that neutrophils would undergo respiritory
burst in bone marrow and liberate large quantities of oxidants without some clear manifestation of patient symptoms; generally, the first signs of agranulocytosis are well after cell numbers have already fallen below critical levels. Overall, the possibility that a gradual apparently symptomless and highly cell selective condition such as agranulocytosis could be caused by such explosive and indiscriminate tissue destructive processes as those involved in cellular oxidant production appears to be remote. Alternatively, it may be postulated that a link may exist between agranulocytosis and erythrocytic metabolism of dapsone hydroxylamine originally derived from the liver. If the hydroxylamine "leeches" out of erythrocytes as they circulate through bone marrow, it is possible that small sub-lethal quantities of the metabolite may enter granulocyte precursor cells and auto-oxidise to the tissue reactive (Coleman and Tingle, 1992) nitrosoarene. This sequence of events, if it does indeed occur, would happen in all patients exposed to the drug, and clearly only a small fraction of patients develop agranulocytosis. Therefore, it has been suggested that some form of immune reaction is involved. The evidence for this is sketchy, but it is apparent that during the condition of agranulocytosis, if dapsone is immediately stopped and the patient survives the infections, the granulocyte population recovers in a matter of days, suggesting that the marrow is intact and undamaged (Uetrecht, 1992). This is at odds with conditions such as aplastic anaemia, where the marrow is badly damaged and recovers far more slowly. Therefore, whichever process is happening, it is highly specific. In the case of dapsone-mediated agranulocytosis, either dapsone itself is acting as an "off switch" by mimicking an endogenous regulator of granulocyte production or the hydroxylamine itself may be killing the cells progressively, but only in certain susceptible individuals or some form of highly specific immune response is triggered that selectively eliminates the cells concerned. One way an immune response might be involved could be through the hapten hypothesis (Park et al., 1988), where binding of drug related material, such as the nitrosoarene, would be followed by some form of antigen processing and ultimately expression of the "hapten" on the cell surface. The granulocyte would then be recognised as foreign and destroyed. In the case of dapsone, once hydroxylamine production is stopped and "haptenation" ceases, then in the absence of the stimulus, the immune system ceases to recognise and destroy granulocytes. This theory does not explain why other populations of white cells are not apparently exposed to this risk. However, if the parent drug is not acting in some way to switch off granulocyte produc-
Dapsone toxicity tion, then the hydroxylamine metabolite seems to be the most likely cause of agranulocytosis. It is also plausible that erythrocytes provide a conduit for hydroxylamine penetration into the bone marrow, which may complement any possible in situ activation of dapsone to its metabolite by white cell precursors. In view of dapsone's inhibitory action on myeloperoxidase metabolism, it appears more likely that hydroxylamine delivered by erythrocytes may be the major contributor to nitrosoarene binding to bone marrow (Fig. 1). Among complex risk factors such as age, sex and ethnic origin, immune reactivity may be the greatest parameter: agranulocytosis is unknown in partially immunosuppressed leprosy patients, whereas in conditions characterised by immune hyper-responsiveness such as dermatitis herpetiformis, the frequency is an alarming 0.3%, compared with the much lower figure (> 1:10,000) for normal individuals (Hornsten et al., 1990; Ognibene, 1970). The possibility of dapsone itself acting as an off switch for granulocyte production has not, to the author's knowledge, been previously discussed. How this interaction might occur is open to speculation. In the case of drug metabolism, low molecular weight compounds are capable of indirectly modulating or inducing the expression of enzyme systems such as cytochrome P-450. This can be achieved by binding of the compound to cytoplasmic receptors that then communicate with DNA (for a review, see Gonzalez, 1989). Apoptosis, or programmed cell death, is a process in which cytoplasmic regulation is thought to occur (Stewart, 1994). It is conceivable that a low molecular weight compound such as dapsone in certain individuals with defective cytoplasmic receptors might perhaps have their granulocytes artificially programmed to die by dapsone. Once the drug is withdrawn, binding to the receptor would cease and cell survival would return to normal. In any case, recent reports showed an increasing number of specific aspects of neutrophil function to be compromised by dapsone, including inhibition of adherance and capillary extravasation (Booth et al., 1992), lysosomal enzyme activity, possible binding to IgA and attenuation of chemotaxis (Thuong-Nguyen et al., 1993). In light of these interactions, the potential for dapsone to somehow regulate development and maturation o f granulocytes might not be far-fetched.
INCREASING PATIENT TOLERANCE TO DAPSONE A number of attempts have been made to increase the tolerance of patients to dapsone, including the co-
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administration with dapsone of vitamins E and C either alone or in combination (Prussick et al., 1992). These antioxidant studies demonstrated little impact on the methaemoglobinaemia caused by the drug. An alternative approach was first explored in rats where a number of metabolic inhibitors of cytochrome P-450 were evaluated in their ability to attenuate the methaemoglobin due to single dose dapsone administration (Coleman, 1993). Cimetidine was selected as the most practical inhibitor for use in humans. Single-dose studies using cimetidine (3 x 400 mg/day) in human volunteers showed a significant reduction in methaemoglobin formation, as well as a fall in hydroxylamine excretion as its N-glucuronide (Coleman, 1993). Meanwhile, chronic studies in rats indicated that methaemoglobin formation could be suppressed for several days (Coleman and Tingle, 1991), and a study in patients on dapsone suffering from dermatitis herpetiformis was organised. This work involved the establishment of baseline methaemoglobin formation and dapsone plasma concentrations for 14 days. The range of dapsone dosage was 50-350 mg/day. After commencement of cimetidine (3 × 400 mg/day), methaemoglobin fell by 27%, peak dapsone plasma levels were unchanged but trough levels rose significantly. Most importantly, four of the six patients reported a reduction in lethargy and headaches and suppression of their disease was unchanged (Coleman, 1993). Follow up studies using a lower cimetidine dose were not successful in reducting methaemoglobin levels in patients (Rhodes et al., 1992). A longer duration study of 3 mo using 1600 mg/day of cimetidine as inhibitor showed that dapsone levels were elevated significantly for the full 12 wk, whereas methaemoglobin levels were depressed by 30% for 11 of 12 wk0 Visual analogue scores for headache also fell significantly (Rhodes et al., 1995). Thus, this approach demonstrated that higher plasma levels of dapsone may be achieved without a concomitant increase in toxicity. This therapeutic approach may be applied with minimal risk to the patient, as cimetidine is now considered safe enough to be available over the counter at pharmacies, net hydroxylamine formation is reduced and control of disease is maintained. It is possible that the reduction in hydroxylamine formation may in turn reduce the risk of agranulocytosis, which would be especially welcome in view of the stated risks to dermatitis herpatiformis patients (Duhra and Charles-Holmes 1991; Hornsten et al., 1990). More potent and less toxic derivatives of dapsone have been synthesised and tested and are very promising (Seydel, 1993). However, they may take many years to reach patients as the market for these compounds is relatively small. In the meantime, the use of a metabolic inhibitor during dapsone administration is currently available.
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Michael D. Coleman CONCLUSIONS
D a p s o n e is a useful b u t rather toxic drug t h a t has found wider therapeutic application in recent years. Often it is not the m o s t p o t e n t drug in any given experimental system, b u t because of its un-ionised state at physiological p H ( C o l e m a n a n d Tingle 1992), it can penetrate tissues in sufficient c o n c e n t r a t i o n to exert a p o t e n t therapeutic action. As more is u n d e r s t o o d o f its modes of action a n d toxicology, it is hoped that these wider applications may benefit patients while reducing the risks associated with the drug. It is now recomm e n d e d t h a t d u r i n g the first 3 m o o f therapy, white cell counts should be m o n i t o r e d every 2 wk ( C o c k b u r n et al., 1993). A l t h o u g h p a t i e n t tolerance to d a p s o n e varies widely, those o n high dosage or those with genetic predispositions to high m e t h a e m o g l o b i n p r o b l e m s would surely benefit f r o m c o n c u r r e n t cimetidine therapy. Physicians may not always see control o f side effects as a problem, as patients experiencing t h e m often d o n o t c o m p l a i n o f s y m p t o m s such as h e a d a c h e or lethargy. In the case o f dapsone, in view o f the potential severity o f its adverse reactions, it would be very desirable for i n f o r m a t i o n o n patients' s y m p t o m s to be m o s t actively sought. Acknowledgement-Preparation of this review was aided by
fruitful discussions with Dr. N. A. Helsby.
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