Dapsone Hydroxylamine Research Articles

Hemolysis and Metabolic Lesion of G6PD Deficient RBCs in Response to Dapsone Hydroxylamine in a Humanized Mouse Model.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans (~5% of all individuals). G6PD deficiency (G6PDd) is caused by an unstable enzyme and manifests most strongly in red blood cells (RBCs) that cannot synthesize new protein. G6PDd RBCs have decreased ability to mitigate oxidative stress due to lower levels of NADPH, as a result of a defective pentose phosphate pathway. Accordingly, oxidative drugs can result in hemolysis and potentially life-threatening anemia in G6PDd patients. Dapsone is a highly useful drug for treating a variety of pathologies but oral dapsone is contraindicated in patients with G6PDd due to oxidative stress-induced anemia. Dapsone must be metabolized to become hemolytic. Dapsone hydroxylamine (DDS-NOH) has been implicated as the major hemolytic dapsone metabolite, but this has never been tested on G6PDd RBCs with in vivo circulation as a metric. Moreover, the metabolic lesion caused by DDS-NOH is unknown. We report that RBCs from a novel humanized mouse expressing the human Mediterranean G6PD-deficient variant have increased sensitivity to DDS-NOH. In addition, we show that DDS-NOH damaged RBCs can either undergo sequestration (with subsequent return to circulation) or permanent removal in a dose-dependent manner, with G6PD-sufficient RBCs mostly being sequestered, and G6PDd RBCs mostly being permanently removed. Finally, we characterize the metabolic lesion caused by DDS-NOH in G6PDd RBCs and report a blockage in terminal glycolysis resulting in a cellular accumulation of pyruvate. These findings confirm DDS-NOH as a hemolytic metabolite and elucidate metabolic effects of DDS-NOH on G6PDd RBCs. Significance Statement These findings confirm that dapsone hydroxylamine, an active metabolite of dapsone, causes in vivo clearance of murine RBCs expressing a human variant of deficient G6PD, an enzymopathy that affects half a billion individuals (G6PD deficiency). Both cellular mechanisms of clearance (sequestration vs. destruction) and specific metabolic disturbances caused by dapsone hydroxylamine are elucidated, providing novel mechanistic understanding.

Alpha-Lipoic Acid and Its Enantiomers Prevent Methemoglobin Formation and DNA Damage Induced by Dapsone Hydroxylamine: Molecular Mechanism and Antioxidant Action

Dapsone (DDS) therapy can frequently lead to hematological side effects, such as methemoglobinemia and DNA damage. In this study, we aim to evaluate the protective effect of racemic alpha lipoic acid (ALA) and its enantiomers on methemoglobin induction. The pre- and post-treatment of erythrocytes with ALA, ALA isomers, or MB (methylene blue), and treatment with DDS-NOH (apsone hydroxylamine) was performed to assess the protective and inhibiting effect on methemoglobin (MetHb) formation. Methemoglobin percentage and DNA damage caused by dapsone and its metabolites were also determined by the comet assay. We also evaluated oxidative parameters such as SOD, GSH, TEAC (Trolox equivalent antioxidant capacity) and MDA (malondialdehyde). In pretreatment, ALA showed the best protector effect in 2.5 µg/mL of DDS-NOH. ALA (1000 µM) was able to inhibit the induced MetHb formation even at the highest concentrations of DDS-NOH. All ALA tested concentrations (100 and 1000 µM) were able to inhibit ROS and CAT activity, and induced increases in GSH production. ALA also showed an effect on DNA damage induced by DDS-NOH (2.5 µg/mL). Both isomers were able to inhibit MetHb formation and the S-ALA was able to elevate GSH levels by stimulating the production of this antioxidant. In post-treatment with the R-ALA, this enantiomer inhibited MetHb formation and increased GSH levels. The pretreatment with R-ALA or S-ALA prevented the increase in SOD and decrease in TEAC, while R-ALA decreased the levels of MDA; and this pretreatment with R-ALA or S-ALA showed the effect of ALA enantiomers on DNA damage. These data show that ALA can be used in future therapies in patients who use dapsone chronically, including leprosy patients.

Development and Characterization of topical microemulsion as novel drug delivery system for Dapsone

Dapsone is a Biopharmaceutical Classification System class II drug with anti inflammatory, immunosuppressive, antibacterial, and antibiotic properties and is used as an antileprotic. The purpose of the present study was to investigate the potential of a microemulsion formulation for topical delivery of dapsone to enhance permeation and to avoid systemic side effects. When administered orally, dapsone undergoes hepatic metabolism. Its hepatic metabolite, dapsone hydroxylamine, shows systemic side effects such as hemolytic anaemia peripheral neuropathy, nausea, and headache. A novel drug delivery system in the form of a microemulsion was developed for dapsone. This is the first attempt that dapsone has been combined with chaulmoogra oil in a topical microemulsion. The primary drugs used for the treatment of leprosy are found in chaulmoogra seeds. Considering its good solubilizing capacity and its use in the treatment of leprosy, chaulmoogra oil was chosen as the oil phase. Based on emulsification ability, Cremophor RH40 and PEG 400 were selected as surfactant and co-surfactant, respectively. A pseudo-ternary phase diagram was constructed to identify the microemulsion region. Smix (Cremophor RH40: PEG-400 in the ratio of 1:2) was most effective in imparting stability to the formulation. The selected formulation exhibited appropriate diffusion behavior (in vitro). The developed dapsone containing microemulsion formulation exhibited the optimal homogeneity, clarity, pH, type of microemulsion, viscosity, percent drug content, and percent transmittance to qualify as a topical drug delivery system for local treatment of leprosy.

Endometriosis Susceptibility to Dapsone-Hydroxylamine-Induced Alterations Can Be Prevented by Licorice Intake: In Vivo and In Vitro Study.

Endometriosis, an estrogen-dependent chronic gynecological disease, is characterized by a systemic inflammation that affects circulating red blood cells (RBC), by reducing anti-oxidant defenses. The aim of this study was to investigate the potential beneficial effects of licorice intake to protect RBCs from dapsone hydroxylamine (DDS-NHOH), a harmful metabolite of dapsone, commonly used in the treatment of many diseases. A control group (CG, n = 12) and a patient group (PG, n = 18) were treated with licorice extract (25 mg/day), for a week. Blood samples before (T0) and after (T1) treatment were analyzed for: i) band 3 tyrosine phosphorylation and high molecular weight aggregates; and ii) glutathionylation and carbonic anhydrase activity, in the presence or absence of adjunctive oxidative stress induced by DDS-NHOH. Results were correlated with plasma glycyrrhetinic acid (GA) concentrations, measured by HPLC–MS. Results showed that licorice intake decreased the level of DDS-NHOH-related oxidative alterations in RBCs, and the reduction was directly correlated with plasma GA concentration. In conclusion, in PG, the inability to counteract oxidative stress is a serious concern in the evaluation of therapeutic approaches. GA, by protecting RBC from oxidative assault, as in dapsone therapy, might be considered as a new potential tool for preventing further switching into severe endometriosis.

HLA-B*13 :01 Is a Predictive Marker of Dapsone-Induced Severe Cutaneous Adverse Reactions in Thai Patients.

HLA-B*13:01 allele has been identified as the genetic determinant of dapsone hypersensitivity syndrome (DHS) among leprosy and non-leprosy patients in several studies. Dapsone hydroxylamine (DDS-NHOH), an active metabolite of dapsone, has been believed to be responsible for DHS. However, studies have not highlighted the importance of other genetic polymorphisms in dapsone-induced severe cutaneous adverse reactions (SCAR). We investigated the association of HLA alleles and cytochrome P450 (CYP) alleles with dapsone-induced SCAR in Thai non-leprosy patients. A prospective cohort study, 16 Thai patients of dapsone-induced SCARs (5 SJS-TEN and 11 DRESS) and 9 Taiwanese patients of dapsone-induced SCARs (2 SJS-TEN and 7 DRESS), 40 dapsone-tolerant controls, and 470 general Thai population were enrolled. HLA class I and II alleles were genotyped using polymerase chain reaction-sequence specific oligonucleotides (PCR-SSOs). CYP2C9, CYP2C19, and CYP3A4 genotypes were determined by the TaqMan real-time PCR assay. We performed computational analyses of dapsone and DDS-NHOH interacting with HLA-B*13:01 and HLA-B*13:02 alleles by the molecular docking approach. Among all the HLA alleles, only HLA-B*13:01 allele was found to be significantly associated with dapsone-induced SCARs (OR = 39.00, 95% CI = 7.67–198.21, p = 5.3447 × 10−7), SJS-TEN (OR = 36.00, 95% CI = 3.19–405.89, p = 2.1657 × 10−3), and DRESS (OR = 40.50, 95% CI = 6.38–257.03, p = 1.0784 × 10−5) as compared to dapsone-tolerant controls. Also, HLA-B*13:01 allele was strongly associated with dapsone-induced SCARs in Asians (OR = 36.00, 95% CI = 8.67–149.52, p = 2.8068 × 10−7) and Taiwanese (OR = 31.50, 95% CI = 4.80–206.56, p = 2.5519 × 10−3). Furthermore, dapsone and DDS-NHOH fit within the extra-deep sub pocket of the antigen-binding site of the HLA-B*13:01 allele and change the antigen-recognition site. However, there was no significant association between genetic polymorphism of cytochrome P450 (CYP2C9, CYP2C19, and CYP3A4) and dapsone-induced SCARs (SJS-TEN and DRESS). The results of this study support the specific genotyping of the HLA-B*13:01 allele to avoid dapsone-induced SCARs including SJS-TEN and DRESS before initiating dapsone therapy in the Asian population.

Dapsone Hydroxylamine, an Active Metabolite of Dapsone, Can Promote the Procoagulant Activity of Red Blood Cells and Thrombosis.

Dapsone hydroxylamine (DDS-NHOH), N-hydroxylated metabolite of a sulfonamide antibiotic, dapsone, is responsible for various adverse effects of dapsone that include methemoglobinemia, hemolytic anemia, and thrombosis. However, the mechanism underlying DDS-NHOH-induced thrombosis remains unclear. Here, we demonstrated that DDS-NHOH, but not dapsone, could increase prothrombotic risks through inducing the procoagulant activity of red blood cells (RBCs). In freshly isolated human RBCs in vitro, sub-hemolytic concentrations of DDS-NHOH (10-50 μM) increased phosphatidylserine (PS) exposure and augmented the formation of PS-bearing microvesicles (MV). Reactive oxygen species (ROS) generation and the subsequent dysregulation of enzymes maintaining membrane phospholipid asymmetry were found to induce the procoagulant activity of DDS-NHOH. Dapsone hydroxylamine also accelerated thrombin generation and enhanced RBC self-aggregation and adherence of RBCs to endothelial cells in vitro. Most importantly, both the single dose of 50 or 100 mg/kg (i.p.) DDS-NHOH and repeated doses of 10 mg/kg per day (i.p.) for 4 days increased thrombus formation in rats (six rats per dose) in vivo, substantiating a potential prothrombotic risk of DDS-NHOH. Collectively, these results demonstrated the central role of RBC procoagulant activity induced by DDS-NHOH in the thrombotic risk of dapsone.

Dapsone hydroxylamine-mediated alterations in human red blood cells from endometriotic patients

Endometriosis, an estrogen-dependent chronic gynecological disease in women of reproductive age, is characterized by a systemic inflammation status involving also red blood cells (RBCs). In this study, we evaluated how the protein oxidative status could be involved in the worsening of RBC conditions due to dapsone intake in endometriotic women in potential treatment for skin or infection diseases. Blood samples from two groups of volunteers, control group (CG) and endometriosis patient group (PG), were analyzed for their content of band 3 tyrosine phosphorylation (Tyr-P) and high molecular weight aggregate (HMWA) in membranes, and glutathione (GSH) content and carbonic anhydrase (CA) activity in cytosol. In endometriotic patients, RBC showed the highest level of oxidative-related alterations both in membrane and cytosol. More interestingly, the addition of dapsone hydroxylamine (DDS-NHOH) could induce further increase of both membranes and cytosol markers, with an enhancement of CA activity reaching about 66% of the total cell enzyme amount. In conclusion, in PG the systemic inflammatory status leads to the inability of counteracting adjunctive oxidative stress, with a potential involvement of CA-related pathologies, such as glaucoma. Hence, the importance of the evaluation of therapeutic approaches worsening oxidative imbalance present in PG RBC is underlined.

Quercetin Potentiates Toxicity Of Hemolytic Drugs In G6PD Deficient Human Erythrocytes

Quercetin, a flavonoid, is widely distributed in plants and a prominent constituent of dietary supplements. Quercetin-based supplements are recommended for prevention of cancer, improvement of cardiovascular functions and mental health. However, these effects have not been validated clinically. Except for limited interactions with some drugs (fluoroquinolone antibiotics and taxol/paclitaxel), there is little evidence of safety concerns, and with oral use, quercetin-containing preparations have been generally well tolerated. Quercetin inhibits NRH: quinone oxidoreductase 2 (NQO2), an enzyme involved in quinone metabolism and detoxification. Individuals with genetic deficiency of glucose 6-phosphate dehydrogenase (G6PD) are highly susceptible to hemolytic toxicity of certain oxidant drugs. Treatment of G6PD deficient human erythrocytes in vitro with quercetin caused significant increase in methemoglobin formation and selective depletion of reduced glutathione (GSH) levels. Further, when tested in combination with 5, 6-orthoquinone PQ [6] and dapsone hydroxylamine [3], the hemotoxic metabolites of primaquine [4] and dapsone [2] respectively, caused significant potentiation of their hemotoxic effect as determined through an increase in methemoglobin formation and depletion of GSH. Quercetin also augmented the hemotoxic effect of 2-amino-4-chlorophenol in vitro. The results warrant further investigation of quercetin regarding its toxic interaction with drugs clinically known to cause hemolytic toxicity.

In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells.

Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy.

Dapsone‐Associated Methemoglobinemia in a Patient With Slow NAT2*5B Haplotype and Impaired Cytochrome b5 Reductase Activity

Dapsone (4,4′-diaminodiphenylsulfone; DDS) is a sulfone antimicrobial used to treat leprosy, malaria, and inflammatory dermatoses and to prevent and treat opportunistic infections in immunocompromised patients, particularly those who cannot tolerate sulfonamide antimicrobials.1 However, dapsone can lead to dose-dependent hematologic toxicity and is the most commonly recognized cause of acquired methemoglobinemia in human patients.2 The hydroxylamine metabolite of dapsone oxidizes the iron moiety of hemoglobin,3 which leads to increased levels of methemoglobin, impaired oxygen delivery, and cyanosis. Dapsone hydroxylamine is generated by CYP2C9 and CYP2C19, with minor contributions from other P450s.4,5 A rapid activity variant for CYP2C19 (CYP2C19*17) has been reported6 but not for CYP2C9. Dapsone hydroxylamine is detoxified by cytochrome b5 (b5) and its electron donor, cytochrome b5 reductase (b5R),7 which are expressed in blood, liver, and other tissues. This reduction pathway converts the hydroxylamine back to the parent dapsone. Dapsone itself is inactivated and eliminated following N-acetylation by the enzyme NAT2 in the liver,8 and impaired N-acetylation activity is a risk factor for hematologic and neurologic toxicity from dapsone.9,10 However, genotypic analysis of the CYP2C19, NAT2, and b5/b5R pathways has not been performed in the setting of dapsone-associated methemoglobinemia. This report describes a patient who developed methemoglobinemia at prophylactic dosages of dapsone, for which we characterized wild-type alleles at the CYP2C19*17 loci, but low activity of the b5/b5R pathway along with low expression of the CYB5A gene encoding b5, and a homozygous slow NAT2*5B haplotype, both of which may have contributed to defective dapsone detoxification.

O1-2.2 Sparse instrumental variables: an integrative approach to biomarker validation

Dapsone activates CYP2C9-mediated metabolism in various expression systems and is itself metabolized by CYP2C9 to its hydroxylamine metabolite. Studies were conducted with expressed CYP2C9 to characterize the kinetic effects of dapsone (0–100 μM) on (<i>S</i>)-flurbiprofen (2–300 μM), (<i>S</i>)-naproxen (10–1800 μM), and piroxicam (5–900 μM) metabolism in 6 × 6 matrix design experiments. The influence of (<i>S</i>)-flurbiprofen on dapsone hydroxylamine formation was also studied. Dapsone increased the Michaelis-Menten-derived <i>V</i>_max of flurbiprofen 4′-hydroxylation from 12.6 to 20.6 pmol/min/pmol P450, and lowered its <i>K</i>_m from 28.9 to 10.0 μM, suggesting that dapsone activates CYP2C9-mediated flurbiprofen metabolism without displacing flurbiprofen from the active site, supporting a two-site model describing activation. Similar results were observed with piroxicam 5′-hydroxylation, as<i>V</i>_max was increased from 0.08 to 0.20 pmol/min/pmol P450 and <i>K</i>_m was decreased from 183 to 50 μM in the presence of dapsone. In addition, the kinetic profile for naproxen was converted from biphasic to hyperbolic in the presence of dapsone, while exhibiting similar decreases in<i>K</i>_m and increases in<i>V</i>_max. Kinetic parameters were also estimated using the two-site binding equation, with α values &lt;1 and β values &gt;1, indicative of activation. Additionally, dapsone hydroxylamine formation was measured from incubations containing flurbiprofen, exhibiting a kinetic profile that was minimally affected by the presence of flurbiprofen. Overall, these results suggest that dapsone activates the metabolism of multiple substrates of CYP2C9 by binding within the active site and causing positive cooperativity, thus lending further support to a two-site binding model of P450-mediated metabolism.

Effect of Diethyldithiocarbamate on Dapsone Hydroxylamine‐dependent Methaemoglobin Formation in Human Erythrocytes in Static and Rotating Systems In‐vitro*

The potentiating effect of DDC (diethyldithiocarbamate) on dapsone hydroxylamine-mediated methaemoglobin formation was investigated in human erythrocytes in static and rotating in-vitro models. The potentiating effect shown in the static model was retained in the non-rotating two-compartment model. Rotation of the two-compartment model caused the effect to be first lost, and finally reversed as rotation speed increased. Rotation was associated with increased oxygenation of the erythrocytes in this system compared with the static system. Although both DDC and dapsone hydroxylamine individually oxidize haemoglobin by similar redox reactions, these processes are clearly different in velocity. Depending on oxygen availability and speed of motion of the incubation mixture, DDC both potentiates the redox cycling of the hydroxylamine in lower oxygen tensions and directly reverses methaemoglobin formation in higher oxygen levels. It is unlikely that DDC or its parent compound, disulfiram would have any methaemoglobin-promoting effect during dapsone therapy in-vivo.

Dapsone hydroxylamine induces premature removal of human erythrocytes by membrane reorganization and antibody binding

N-hydroxylation of dapsone leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with dapsone therapy. Dapsone has been associated with decreased lifespan of erythrocytes, with consequences such as anaemia and morbidity in patients treated with dapsone for malaria. Here, we investigated how dapsone and/or its hydroxylamine derivative (DDS-NHOH) induced erythrocyte membrane alterations that could lead to premature cell removal. Erythrocytes from healthy donors were subjected to incubation with dapsone and DDS-NHOH for varying times and the band 3 protein tyrosine-phosphorylation process, band 3 aggregation, membrane alteration and IgG binding were all examined and compared with erythrocytes from two patients receiving dapsone therapy. The hydroxylamine derivative, but not dapsone (the parent sulphone) altered membrane protein interactions, leading both to aggregation of band 3 protein and to circulating autologous antibody binding, shown in erythrocytes from patients receiving dapsone therapy. The band 3 tyrosine-phosphorylation process can be used as a diagnostic system to monitor membrane alterations both in vitro, assessing concentration and time-dependent effects of DDS-NHOH treatment, and in vivo, evaluating erythrocytes from dapsone-treated patients, in resting or oxidatively stimulated conditions. DDS-NHOH-induced alterations of human erythrocytes can be directly monitored in vitro by tyrosine-phosphorylation level and formation of band 3 protein aggregates. The latter, together with antibody-mediated labelling of erythrocytes, also observed after clinical use of dapsone, may lead to shortening of erythrocyte lifespan.

Very late relapse of dapsone-induced methaemoglobinemia

Dapsone, a sulfone active against a wide range of bacteria, is mainly used for Mycobacterium leprae. Moreover, because of its immunosuppressive effect, it is used for many auto-immune diseases [1]. Methaemoglobinemia is an expected adverse drug reaction of dapsone, seldom symptomatic. Long-term administration of dapsone at standard doses (100 mg/day) results in methaemoglobinemia in about 15% of patients [2]. When symptomatic (dyspnea, cyanosis, neurological disorders), methaemoglobinemia is treated with methylene blue (1–2 mg/kg) [3]. Rebounds have been described after dapsone withdrawal, with a maximum delay of 64 h [4]. We report a case of dapsone-induced methaemoglobinemia which relapsed 10 days after drug withdrawal. A 25-year-old woman was diagnosed with systemic lupus erythematosus in January 2009. Organs involved were skin and joints, and hydroxychloroquine alone was prescribed. In October 2009, she developed a bullous dermatitis and dapsone was added to hydroxychloroquine on 26 October 2009. About 1 month later, on 24 November, she was admitted to the hospital for severe dyspnea. Physical examination was normal except for cutaneous cyanosis. Chest computed tomography and electrocardiogram were normal. Arterial blood gas analysis showed pH: 7.63, PaO2: 110 mmHg, PaCO2: 15 mmHg, haemoglobin: 11 g/dL, SaO2: 99.1%; methaemoglobin: 17%. Methylene blue (2 mg/kg) was injected intravenously. Twenty minutes later, the patient was asymptomatic and arterial methaemoglobin was 4%. Dapsone was discontinued and the patient left the hospital on 26 November. Ten days later, she consulted for relapse of dyspnea. Arterial blood gas analysis showed methaemoglobin at 14%. No symptomatic treatment was done and methaemoglobin rate decreased spontaneously to 5% the next day. Hydroxychloroquine was stopped. The compliance of the patient was not doubtful, and no other cause of intoxication causing methaemoglobinemia was found. Methaemoglobinemia in dapsone intoxication is due to the ability of dapsone hydroxylamine, a metabolite of the drug, to react with haemoglobin (Fe) to form methaemoglobin (Fe) [5]. Methaemoglobin is unable to bind dioxygen, which results in functional anaemia. The clinical G. Moulis Service de Medecine Interne, Centre Hospitalier Universitaire de Toulouse, Universite Paul-Sabatier, Toulouse, France e-mail: gmoulis@hotmail.com

The effect of pregnenolone 16alpha-carbonitrile on the pharmacokinetics and metabolism of dapsone in rats.

The purpose of this study was to evaluate the effect of pregnenolone 16 alpha-carbonitrile (PCN) on the interconversion pharmacokinetics and metabolism of dapsone. To determine microsomal CYP3A activity and protein, eight rats (4 PCN, 4 corn oil) received a 1 mg kg(-1) intravenous bolus dose of dapsone, followed by blood and urine sampling. The formation clearance of dapsone hydroxylamine (CLf DDS-NOH) was calculated from the obtained samples. Interconversion pharmacokinetics estimates were obtained after 10 rats (5 PCN, 5 control) received 1 mg kg(-1) dapsone or 1.17 mg kg(-1) monoacetyldapsone, with a 24-h wash-out. Results from the interconversion analysis demonstrated that PCN significantly increased systemic clearance (CLs) of dapsone, but not its interconversion. The in-vivo/in-vitro correlation study demonstrated that PCN significantly increased CLs of dapsone (8.55 to 16.39mLmin(-1); P<0.01) and CLf DDS-NOH (0.13 to 0.18mLmin(-1); P<0.01). PCN treatment produced a 69% increase in CYP3A protein, and increased 6beta- and 2beta-hydroxytestosterone formation rates. Significant correlations were found between CLf DDS-NOH and either 6beta- (r2 = 0.925), 2beta-hydroxytestosterone (r2 = 0.92), or CYP3A1/2 protein (r2= 0.60). We conclude that PCN treatment produces significant increases in CLs (dapsone) and CLf (DDS-NOH) in rats. These changes were not due to changes in the reversible metabolism of dapsone. These results suggest that the formation clearance of dapsone hydroxylamine reflects alterations in CYP3A activity, despite the fact that it accounted for a small part of the systemic clearance of dapsone.

Dapsone induces oxidative stress and impairs antioxidant defenses in rat liver

Dapsone (DDS) is currently used in the treatment of leprosy, malaria and in infections with Pneumocystis jirovecii and Toxoplasma gondii in AIDS patients. Adverse effects of DDS involve methemoglobinemia and hemolysis and, to a lower extent, liver damage, though the mechanism is poorly characterized. We evaluated the effect of DDS administration to male and female rats (30 mg/kg body wt, twice a day, for 4 days) on liver oxidative stress through assessment of biliary output and liver content of reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and expression/activities of the main antioxidant enzymes glutathione peroxidase, superoxide dismutase, catalase and glutathione S-transferase. The influence of DDS treatment on expression/activity of the main DDS phase-II-metabolizing system, UDP-glucuronosyltransferase (UGT), was additionally evaluated. The involvement of dapsone hydroxylamine (DDS-NHOH) generation in these processes was estimated by comparing the data in male and female rats since N-hydroxylation of DDS mainly occurs in males. Our studies revealed an increase in the GSSG/GSH biliary output ratio, a sensitive indicator of oxidative stress, and in lipid peroxidation, in male but not in female rats treated with DDS. The activity of all antioxidant enzymes was significantly impaired by DDS treatment also in male rats, whereas UGT activity was not affected in any sex. Taken together, the evidence indicates that DDS induces oxidative stress in rat liver and that N-hydroxylation of DDS was the likely mediator. Impairment in the activity of enzymatic antioxidant systems, also associated with DDS-NHOH formation, constituted a key aggravating factor.

Cimetidine and dapsone‐mediated methaemoglobinaemia

I read with interest the case report from Choi et al. [1] relating to dapsone-mediated methaemoglobinaemia. The role of cimetidine in partially attenuating the haematological side-effects of dapsone does not appear to have been discussed in the anaesthesia literature. I wish to suggest the possible therapeutic benefit of peri-operative administration of cimetidine in patients receiving dapsone. The oxidative metabolism of dapsone leads to its toxicity. The majority of dapsone undergoes reversible acetylation in liver followed by elimination as N-hydroxylamine and to a lesser degree monoacetylated hydroxylamine. Dapsone N- hydroxylation is responsible for the haematological side-effects such as methaemoglobinaemia and haemolysis. Dapsone hydroxylamine depletes glutathione within glucose-6-phosphate dehydrogenase (G6PD)-deficient cells. The nitroso derivative then causes peroxidation reactions, leading to rapid haemolysis. Doses of 100 mg or less in normal healthy persons and 50 mg or less in G6PD-deficient individuals do not cause haemolysis [2]. A maximum dose of 150–300 mg a day, in divided doses, is recommended to minimise the risk of methaemoglobinaemia [3]. Cimetidine, an H2-recector antagonist, competes with dapsone for cytochrome P 450 enzymes. Thus cimetidine may be used as a selective inhibitor of N-hydroxylation and decrease the quantity of methaemoglobinaemia produced by dapsone [3, 4]. Cimetidine 400 mg three times daily has been shown to lead to a significant (> 25%) and sustained fall in methaemoglobin in patients on chronic dapsone therapy [5]. This dose appears to be well tolerated and not associated with adverse effects. The peri-operative use of cimetidine is likely to benefit patients by increasing the clinical safety margin, minimising the incidence and severity of methaemoglobinaemia and, possibly, avoiding the complications associated with treatment modalities such as methylene blue or ascorbic acid [6]. The mean elimination half-life of dapsone is about 20–30 h. Sulphones are retained in the circulation for long periods because of intestinal re-absorption from bile, requiring periodic interruption of treatment in dermatological diseases [2]. It is imperative to discontinue dapsone at least 4–7 days pre-operatively in patients listed for elective surgery. Furthermore, the G6PD level should be checked in all patients receiving dapsone therapy to identify patients at risk of peri-operative methaemoglobinaemia.

Detection of Haptenated Proteins in Organotypic Human Skin Explant Cultures Exposed to Dapsone

Bioactivation of parent drug to reactive metabolite(s) followed by protein haptenation has been suggested to be a critical step in the elicitation of cutaneous drug reactions. Although liver is believed to be the primary organ of drug bioactivation quantitatively, other organs including skin may also metabolize drugs. Cultured human epidermal keratinocytes and dermal fibroblasts have been shown to be capable of bioactivating sulfonamides and sulfones, giving rise to haptenated proteins. It is, however, unclear whether metabolic events in these isolated cells reflect bioactivation in vivo. Hence, split-thickness human skin explants were exposed to dapsone (DDS) or its arylhydroxylamine metabolite (dapsone hydroxylamine, D-NOH) and probed for protein haptenation. DDS and D-NOH were applied either epicutaneously or mixed in the medium (to mimic its entry into skin from the systemic circulation). DDS-protein adducts were readily detected in skin explants exposed to either DDS or D-NOH. Adducts were detected mainly in the upper epidermal region in response to epicutaneous application, whereas adducts were formed all over the explants when DDS/D-NOH were mixed in the culture medium. In addition, adducts were visible in HLA-DR+ cells, indicating their presence in the dendritic cell population in the skin. Our results demonstrate the ability of intact human skin to bioactivate DDS leading to protein haptenation.

Formation and Uptake of Arylhydroxylamine-Haptenated Proteins in Human Dendritic Cells

Bioactivation of sulfonamides and the subsequent formation of haptenated proteins is believed to be a critical step in the development of hypersensitivity reactions to these drugs. Numerous lines of evidence suggest that the presence of such adducts in dendritic cells (DCs) migrating to draining lymph nodes is essential for the development of cutaneous reactions to xenobiotics. Our objective was to determine the ability of human DCs to form drug-protein covalent adducts when exposed to sulfamethoxazole (SMX), dapsone (DDS), or their arylhydroxylamine metabolites [sulfamethoxazole hydroxylamine (S-NOH) and dapsone hydroxylamine (D-NOH)] and to take up preformed adduct. Naive and immature CD34+ KG-1 cells were incubated with SMX, DDS, or metabolites. Formation of haptenated proteins was probed using confocal microscopy and ELISA. Cells were also incubated with preformed adduct (drug-bovine serum albumin conjugate), and uptake was determined using confocal microscopy. Both naive and immature KG-1 cells were able to bioactivate DDS, forming drug-protein adducts, whereas cells showed very little protein haptenation when exposed to SMX. Exposure to S-NOH or D-NOH resulted in protein haptenation in both cell types. Both immature and naive KG-1 cells were able to take up preformed haptenated proteins. Thus, DCs may acquire haptenated proteins associated with drugs via intracellular bioactivation, uptake of reactive metabolites, or uptake of adduct formed and released by adjacent cells (e.g., keratinocytes).

Pharmacokinetics of Dapsone Gel, 5% for the Treatment of Acne Vulgaris

Oral dapsone has been available for over 60 years and has been used to treat severe acne vulgaris; however, the oral formulation is known to cause dose-dependent haematological reactions and is currently indicated only for diseases such as dermatitis herpetiformis and Hansen's disease. A gel formulation of dapsone was recently developed to treat acne vulgaris. As dapsone is administered topically, it was expected that systemic absorption would be considerably lower than that observed with oral dapsone therapy, thereby avoiding any adverse haematological effects. To report the pharmacokinetic profile of topically applied dapsone gel, 5% in the treatment of acne vulgaris. Three prospective, open-label studies enrolled a total of 548 subjects with acne vulgaris: two phase I pharmacokinetic studies (crossover and drug interaction) and one phase III long-term safety study. In the crossover study (n = 18), topical dapsone gel applied twice daily for a total of 14 days to 22.5% of the body surface area was compared with a single dose of oral dapsone 100mg (the typical clinical dose). In the drug-interaction study (n = 24), oral trimethoprim/sulfamethoxazole monotherapy, topical dapsone gel monotherapy and the two in combination were used twice daily for 7, 21 and 7 days, respectively. In the long-term safety study (n = 506), topical dapsone gel was applied twice daily to acne-affected areas for up to 12 months. Blood samples were drawn at various timepoints in each study to assess drug and metabolite concentrations. Systemic concentrations of dapsone, N-acetyl dapsone, dapsone hydroxylamine, trimethoprim and sulfamethoxazole were determined, according to the study design. In the crossover study, the mean area under the plasma concentration-time curve (AUC) from 0 to 24 hours for dapsone was 417.5 ng x h/mL after 2 weeks of dapsone gel therapy (n = 10), compared with an AUC from time zero to infinity of 52,641 ng x h/mL after a single dose of oral dapsone; this represents a 126-fold lower systemic exposure for dapsone gel at typical therapeutic doses. In the drug-interaction study, the AUC from 0 to 12 hours for dapsone was 221.52 ng x h/mL after 3 weeks of dapsone gel monotherapy compared with 320.3 ng x h/mL after 1 week of coadministration with trimethoprim/sulfamethoxazole. In the long-term safety study, the mean plasma dapsone concentrations ranged from 7.5 to 11 ng/mL over 12 months. Overall, total systemic exposures to dapsone and its metabolites were approximately 100-fold less for dapsone gel than for oral dapsone, even in the presence of trimethoprim/sulfamethoxazole. There were no reports of any haematological adverse events. Topical application of dapsone gel in various settings ranging from 2 weeks to 12 months resulted in systemic exposures to dapsone and its metabolites that were approximately 100-fold less than those after oral dapsone at a therapeutic dose level. The concentrations of dapsone and its metabolites reached steady state and did not increase during prolonged treatment.