Dapsone Hydroxylamine Research Articles

Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro III: Effect of diabetes

The fate of dapsone hydroxylamine has been investigated in diabetic and normal human erythrocytes. In erythrocytes from four type 1 (insulin dependent) diabetic subjects, there was a significant decrease in dapsone hydroxylamine-mediated methaemoglobin formation compared with cells drawn from normal individuals (P < 0.01). However, the ability of the diabetic cells to detoxify the hydroxylamine to dapsone was not correspondingly reduced and was not different to normal cells. The initial rate of the accelerating effect of diethyl dithiocarbamate (DDC) on hydroxylamine-mediated methaemoglobin and dapsone formation was significantly reduced in diabetic compared with normal cells. There was no significant difference in hydroxylamine-dependent methaemoglobin formation between diabetic erythrocytes pretreated with either statil or sorbinil and untreated diabetic cells. Dapsone recovery in diabetic erythrocytes incubated with statil was not significantly different from statil-free incubations. However, in the presence of sorbinil, there was a marked reduction in dapsone formation at all four time points, ( P < 0.001 at 15 min). Mean measured levels of glutathione did not differ significantly between the normal (380 ± 30.9 mg/L; N = 8) and diabetic (349 ± 58.7 mg/L; N = 8) volunteers. In summary, although diabetic erythrocytes were less sensitive to the effect of dapsone hydroxylamine-mediated methaemoglobin formation in comparison with normal cells, glutathione-dependent hydroxylamine reduction to dapsone was unaffected.

Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro—II : Movement of dapsone across a semipermeable membrane into erythrocytes and plasma

We have used an in vitro two-compartment model, to investigate the ability of dapsone, formed by erythrocyte-mediated detoxification of its hydroxylamine metabolite, to escape the cells and cross a semi-permeable membrane into both plasma and other erythrocytes. Both diethyl dithiocarbamate (DDC) treated and untreated erythrocytes were incubated with dapsone hydroxylamine and dialysed against either fresh cells or plasma. Methaemoglobin was predominantly detectable in compartment A although the presence of low levels of methaemoglobin in compartment B indicated that the hydroxylamine itself had crossed the membrane. In contrast to methaemoglobin disposition, recovery of dapsone was higher (P < 0.05) in compartment B compared with A for all three treatment groups at 30 and 60 min, but not at the remaining time points. Regression analysis of the cumulative recovery of dapsone over 150 min in all three treatment groups for both compartments A and B showed correlation coefficients close to unity. In compartment A, analysis of the mean slopes of the regression lines indicated that, overall, significantly more dapsone was recovered from group 1 (erythrocytes, hydroxylamine and DDC dialysed against untreated red cells) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma) (0.22 ± 0.05 vs 0.09 ± 0.005; P < 0.025). Also in compartment A, significantly more dapsone was recovered from group 2 (erythrocytes and hydroxylamine dialysed against untreated red cells) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma: 0.16 ± 0.02 vs 0.09 ± 0.005). In compartment B, dapsone recovery was significantly greater in group 1 (erythrocytes, hydroxylamine and DDC dialysed against untreated red cells; slope of regression line: 0.59 ± 0.05) compared with group 2 (erythrocytes and hydroxylamine dialysed against untreated red cells; slope of line: 0.28 ± 0.02, P < 0.005). In addition, dapsone recovery was significantly greater in group 1 (0.59 ± 0.05) compared with group 3 (erythrocytes and hydroxylamine dialysed against plasma; 0.21 ± 0.02, P < 0.005). Dialysis of erythrocytes with dapsone itself over 120 min caused no detectable methaemoglobin formation. The process of erythrocyte-mediated dapsone formation from its hydroxylamine may feasibly occur in vivo and contribute to the systemic persistence and therapeutic effect of dapsone.

The use of a three compartment in vitro model to investigate the role of hepatic drug metabolism in drug-induced blood dyscrasias.

1. N-hydroxylation is thought to be an essential step in the haemotoxicity of dapsone (DDS). To investigate both metabolism-dependent and cell-selective drug toxicity in vitro we have developed a three-compartment system in which an hepatic drug metabolizing system is contained within a central compartment separated by semipermeable membranes from compartments containing mononuclear leucocytes (MNL) and red blood cells (RBC). 2. Metabolism of dapsone (100 microM) by rat liver microsomes resulted in toxicity to RBC cells (47.3 +/- 2.1% methaemoglobin), but there was no significant toxicity toward MNL (3.7 +/- 1.3% cell death) compared with control values (1.6 +/- 0.9%). However, when RBC were replaced with buffer in the third compartment there was significantly greater (P < 0.001) white cell toxicity (17.6 +/- 0.6% cell death), demonstrating the protection of MNL by RBC. Metabolism of dapsone by human liver microsomes again resulted in RBC toxicity (12.5 +/- 3.3% methaemoglobin) but no significant MNL toxicity (2.9 +/- 0.8% cell death). Replacement of RBC resulted in a significant (P < 0.001) increase in MNL toxicity (6.5 +/- 0.7% cell death). Addition of synthetic dapsone hydroxylamine (30 microM) in the absence of a metabolizing system and with no RBC in the third compartment resulted in significant (P < 0.001) toxicity toward MNL (43.36 +/- 5.82% cell death) compared with control (1.8 +/- 1.1%). The presence of RBC in the third compartment resulted in a significant (P < 0.001) decrease in MNL toxicity (17.6 +/- 2.2% cell death), with 40.1 +/- 3.7% methaemoglobin in the RBC.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of dapsone hydroxylamine to dapsone during methaemoglobin formation in human erythrocytes in vitro

The fate of the toxic metabolite of dapsone, dapsone hydroxylamine, has been studied in the human red cell. Twice-washed red cells were incubated at 37 with dapsone hydroxylamine: at 3 and 5 min, 27.0 ± 2.2 and 33.2 ± 2.7% of the haemoglobin had been converted to methaemoglobin, leading to a maximum at 45 min (45 ± 1.8%). HPLC analysis revealed that parent amine was produced from dapsone hydroxylamine during methaemoglobin formation in the red cells. At 3 min, conversion of dapsone hydroxylamine to dapsone reached 7.0 ± 3.9% leading to a maximum at 30 min (18.1 ± 3.7%). There was a linear relationship between hydroxylamine-dependent methaemoglobin formation and conversion of hydroxylamine to dapsone ( r = 0.97). At 4, methaemoglobin and dapsone formation was greatly retarded, and did not exceed 10%. Co-incubation of diethyl dithiocarbamate (DDC) with dapsone hydroxylamine and red cells led to a marked increase in methaemoglobin formation (61.4 ± 3.4%) compared with hydroxylamine and red cells alone (45.0 ± 1.8%, P < 0.001) at 45 min, and conversion of dapsone hydroxylamine to dapsone was almost doubled at 45 min (35.7 ± 5.3%) compared with hydroxylamine and red cells (18.1 ± 2.5%). A linear relationship between methaemoglobin formation and dapsone formation ( r = 0.96) was also shown to occur in the presence of DDC. Incubation of red cells with DDC and dapsone hydroxylamine caused a significantly greater reduction in glutathione levels (98.3 ± 1.6%) compared with red cells and dapsone hydroxylamine alone (84.8 ± 2.7%) at 5 min (P < 0.001), although there was no significant difference between the groups at 15 min (96.9 ± 2.6 vs 98.1 ± 2.2%). Intra-erythrocytic glutathione was then depleted by 75 ± 3.4%, by pretreatment with diethyl maleate (6 mM), and these cells in the presence of the hydroxylamine showed a significant fall in both methaemoglobin generation (29.7 ± 1.2 vs 35.0 ± 1.7%) and parent amine formation (11.1 ± 0.2 vs 16.5 ± 1.1%) compared with untreated red cells at 45 min. It is possible that a cycle exists between hepatic oxidation of dapsone to its hydroxylamine and reduction to the amine within the red cell, which may lead to re-oxidation by hepatic cytochrome P450. This process may contribute to the persistence of the drug in vivo.

Dapsone-induced hemolytic anemia: Effect of N-hydroxy dapsone on the sulfhydryl status and membrane proteins of rat erythrocytes

Dapsone hydroxylamine (DDS-NOH), a known metabolite of dapsone, has recently been shown to be a direct-acting hemotoxin responsible in part for dapsone-induced hemolytic anemia in the rat. The effect of DDS-NOH on the morphology, sulfhydryl status, and membrane skeletal proteins of the rat red cell has been investigated. Exposure of rat red cells to a TC 50 of DDS-NOH induced transformation of about 50% of the cells to an extreme echinocyte morphology. Reduced glutathione content of the cells was rapidly lost with concomitant increase in the formation of mixed disulfide between glutathione and the soluble protein of the cell. Oxidized glutathione content of the cells did not increase at any time during exposure to DDS-NOH. Examination of the skeletal membrane proteins by SDS-PAGE indicated that DDS-NOH caused the apparent loss of band 4.2, decrease in peaks 1,2.1, and 3, and the appearance of new bands at about 16, 27, 40, and 54 kDa. Bands 4.1 and 7 appeared unchanged. Treatment of DDS-NOH altered proteins with dithiothreitol, reversed the protein changes, and indicated that the observed alterations were due to the formation of disulfide-linked adducts between hemoglobin and the various skeletal proteins as well as between hemoglobin monomers. The possible significance of the parallel changes in cell morphology and in membrane skeletal proteins for the premature splenic sequestration of the injured rat red cells is discussed.

The use of cimetidine to reduce dapsone-dependent methaemoglobinaemia in dermatitis herpetiformis patients.

1. We have attempted to reduce dapsone-dependent methaemoglobinaemia formation in six dermatitis herpetiformis patients stabilised on dapsone by the co-administration of cimetidine. 2. In comparison with control, i.e. dapsone alone, methaemoglobinaemia due to dapsone fell by 27.3 +/- 6.7% and 26.6 +/- 5.6% the first and second weeks after commencement of cimetidine administration. The normally cyanotic appearance of the patient on the highest dose of dapsone (350 mg day-1), underwent marked improvement. 3. There was a significant increase in the trough plasma concentration of dapsone (2.8 +/- 0.8 x 10(-5)% dose ml-1) at day 21 in the presence of cimetidine compared with control (day 7, 1.9 +/- 0.6 x 10(-5)% dose ml-1, P less than 0.01). During the period of the study, dapsone-mediated control of the dermatitis herpetiformis in all six patients was unchanged. 4. Trough plasma concentrations of monoacetyl dapsone were significantly increased (P less than 0.05) at day 21 (1.9 +/- 1.0 x 10(-5)% dose ml-1) compared with day 7 (1.6 +/- 0.9 x 10(-5)% dose ml-1:control). 5. Over a 12 h period, 20.6 +/- 8.9% (day 0) of a dose of dapsone was detectable in urine as dapsone hydroxylamine. Significantly less dapsone hydroxylamine was recovered from urine at day 14 (15.0 +/- 8.4) in the presence of cimetidine, compared with day 0 (control: P less than 0.05). 6. The co-administration of cimetidine may be of value in increasing patient tolerance to dapsone, a widely used, effective, but comparatively toxic drug.

Use of a metabolic inhibitor to reduce dapsone‐dependent haematological toxicity

AbstractAside from its established use as an antileprotic and anti‐inflammatory drug, dapsone is also effective in the therapy of Pneurnocystis carinii pneumonia. Unfortunately, its use is often limited by its dose‐dependent toxicity, such as methaemoglobinaemia and haemolysis; the latter condition occurs most frequently in gIucose‐6‐dehydrogenase deficient individuals. It is also responsible for occasional life‐threatening disorders such as agranulocytosis. Dapsone may undergo acetylation, but its toxicity is due to the product of its oxidative metabolism, dapsone hydroxylamine. This is generated in man by the constitutive hepatic cytochrome P450 enzyme IIIA4. Studies in the rat revealed that dapsone‐dependent methaemoglobinaemia could be greatly diminished by the co‐administration of metabolic inhibitors. In the isolated perfused rat liver, dapsone hydroxylamine levels and hence methaemoglobin formation fell significantly in the presence of cimetidine. In addition, the clearance of the parent drug was retarded, and perfusate concentrations of monoacetyl dapsone increased. The protective effect of cimetidine also reduced methaemoglobin formation in the whole rat during the chronic administration of dapsone. Incubation of dapsone in a two‐compartment in vitro system using human tissues in the presence of cimetidine or ketoconazole resulted in a decrease in methaemoglobin formation in all the human livers tested. Although cimetidine was only effective if incubated with microsomes and NADPH prior to the addition of dapsone. Administration of cimetidine (3 × 400 mg daily) to volunteers 3 days prior to and 4 days post administration of a single dose of 100 mg dapsone caused drug concentrations to increase by almost 30%. There was a marked fall in peak methaemoglobin levels and the percentage of the dose excreted in urine as dapsone hydroxylamine N‐glucuronide was reduced by almost one third. During high dose dapsone therapy it may be possible that the co‐administration of cimetidine might reduce toxicity while maintaining drug efficacy.

The effect of preincubation with cimetidine on the N‐hydroxylation of dapsone by human liver microsomes.

We have examined the ability of cimetidine to inhibit the oxidative metabolism and hence haemotoxicity of dapsone in vitro, using a two compartment system in which two Teflon chambers are separated by a semi-permeable membrane. Compartment A contained a drug metabolizing system (microsomes prepared from human or rat liver +/- NADPH), whilst compartment B contained human red cells. Preincubation (30 min) of human liver microsomes with cimetidine (0-1000 microM) and NADPH prior to the addition of dapsone (100 microM) and NADPH (1 mM) resulted in a concentration-dependent decrease in the concentrations of dapsone hydroxylamine (from 179 +/- 47 to 40 +/- 6 ng) in compartment B. This reduction of hydroxylamine metabolite was reflected in the concentration-dependent reduction in methaemoglobin measured (from 7.1 +/- 0.7 to 3.5 +/- 1.5%) in parallel experiments. Preincubation of microsomes with cimetidine in the absence of NADPH had no effect. The effect of cimetidine pretreatment on dapsone-dependent methaemoglobin was confirmed using microsomes prepared from a further three sources of human liver, as well as from rat liver.

The use of cimetidine as a selective inhibitor of dapsone N‐ hydroxylation in man.

1. The N-hydroxylation of dapsone is thought to be responsible for the methaemoglobinaemia and haemolysis associated with this drug. We wished to investigate the effect of concurrent administration of cimetidine (400 mg three times per day) on the disposition of a single dose (100 mg) of dapsone in seven healthy volunteers in order to inhibit selectively N-hydroxylation. 2. The AUC of dapsone (31.0 +/- 7.2 micrograms ml-1 h) was significantly increased (P less than 0.001) in the presence of cimetidine (43.3 +/- 8.8 micrograms ml-1 h). 3. Peak methaemoglobin levels observed after dapsone administration (2.5 +/- 0.6%) were significantly (P less than 0.05) reduced in the presence of cimetidine (0.98 +/- 0.35%). 4. The percentage of the dose excreted in urine as the glucuronide of dapsone hydroxylamine was significantly (P less than 0.05) reduced in the presence of cimetidine (34.2 +/- 9.3 vs 23.1 +/- 4.2%). 5. Concurrent cimetidine therapy might reduce some of the haematological side-effects of dapsone.

Bioactivation of dapsone to a cytotoxic metabolite: in vitro use of a novel two compartment system which contains human tissues.

1. A two compartment system, comprising two adjacent teflon chambers separated by a semi-permeable membrane, has been devised with which to investigate the generation of drug metabolites that are toxic to human cells in vitro. 2. Compartment A contained a drug-metabolising system (human liver microsomes +/- NADPH) and compartment B contained target cells (human mononuclear leucocytes). The semi-permeable membrane retained protein (m.w. greater than 10,000) but allowed equilibration (within 1 h) of drug and drug metabolites, during which time cells remained viable. 3. Incubation of dapsone (100 microM) with human microsomal protein (2 mg ml-1) and NADPH (1 mM) in compartment A caused cell death (8.7 +/- 1.8%) in compartment B, which was reduced significantly (P less than 0.05) by the addition of glutathione (500 microM). Dapsone in the absence of NADPH was not cytotoxic. 4. Chemical analysis showed the presence of dapsone hydroxylamine as the only stable metabolite in both compartment A (5.2 +/- 0.4% incubated drug) and compartment B (3.5 +/- 0.5%). 5. Irreversible binding of dapsone to cells was significantly (P less than 0.05) reduced by omission of NADPH (85 +/- 13 pmol/10(6) cells) or addition of glutathione (103 +/- 9) compared with control values (153 +/- 51).

Stimulation of K-C1 cotransport in rat red cells by a hemolytic anemia-producing metabolite of dapsone.

Dapsone, a sulfone compound used in the treatment of leprosy and, more recently, Pneumocystis carinii pneumonia, produces as a major side effect a hemolytic anemia. This anemia is characterized by oxidation of hemoglobin to methemoglobin and increased splenic uptake of red blood cells. Using a rat model, Grossman and Jollow (J. Pharmacol. Exp. Ther. 244: 118-125, 1988) found that dapsone hydroxylamine (DDS-NOH), a dapsone metabolite, is responsible for its hemolytic effect in vivo. DDS-NOH also promotes hemoglobin binding to SH groups on rat red cell membrane proteins (Budinsky et al., FASEB J. 2: A801, 1988). Since the binding of hemoglobin and other reagents (e.g., N-ethylmaleimide) to membrane SH groups has been associated with increased K transport in red blood cells, we examined the effect of DDS-NOH on K efflux from rat red blood cells in vitro. Cells shrink when exposed to DDS-NOH (100 microM) in media with plasma-like ionic composition. This shrinkage is prevented if extracellular K is raised to 110 mM or if intra- and extracellular Cl are replaced by methylsulfate (MeSO4), suggesting involvement of a K-Cl cotransport pathway. Indeed, 100 microM DDS-NOH produces a 4- to 5-fold increase in K efflux in cells containing Cl but less than a 2-fold increase in cells containing MeSO4. This stimulatory effect is specific for K; Na efflux is slightly inhibited by 100 microM DDS-NOH. The concentrations of DDS-NOH required for half-maximal stimulation of Cl-dependent K efflux (53 microM) is similar to its half-maximal hemolytic concentration in rats (approximately 100 microM). Furthermore, the stimulation of Cl-dependent K efflux by DDS-NOH is greater than 80% reversed by subsequent treatment of the cells with dithiothreitol, suggesting involvement of SH groups. Our results indicate that DDS-NOH exposure stimulates an apparent K-Cl cotransport in rat red blood cells, resulting in cell shrinkage under physiological ionic conditions. Since shrinkage of red blood cells renders them less deformable (Mohandas et al., J. Clin. Invest. 66: 563-573, 1980), this suggests a pathophysiological mechanism whereby DDS-NOH exposure in vivo could promote increased splenic uptake of red blood cells and hemolytic anemia.