High frequency of ofloxacin resistance patterns of Mycobacterium leprae from India: An indication to revisit second line anti-leprosy treatment regimen.

OBJECTIVES: Drug resistance in leprosy is an emerging concern, leading to treatment failures, recurrences, and potential spread of resistant Mycobacterium leprae in the community. In this study, we aimed to assess drug resistance prevalence and patterns amongst leprosy patients at a tertiary care referral hospital in India. METHODS: Mutations in drug resistance determining regions for dapsone, rifampicin, and ofloxacin of the M. leprae genome in DNA extracted from skin biopsies of 136 leprosy patients (treatment-naive = 67, with persistent skin lesions = 35, with recurrence = 34) were analysed by polymerase chain reaction followed by Sanger sequencing. Wild-type strain (Thai-53) was used as a reference strain. RESULTS: Resistance mutations were identified in a total of 23 patients, constituting 16.9% of the cohort. Within this subset of 23 cases, resistance to ofloxacin was observed in 17 individuals (12.5%), while resistance to both dapsone and rifampicin was detected in three patients each (2.2% for both). The occurrence of ofloxacin resistance showed minimal disparity between recurrent and treatment-naive cases, at 17.6% and 16.4%, respectively. Dapsone resistance emerged in two treatment-naive cases and one case with persistent skin lesions. Notably, none of the treatment-naive cases or those with recurrence/relapse exhibited rifampicin resistance. Subsequently, no statistically significant correlation was identified between other clinical variables and the presence of antimicrobial resistance. CONCLUSIONS: The occurrence of resistance to the current multidrug therapy regimen (specifically dapsone and rifampicin) and to ofloxacin, a secondary antileprosy medication in M. leprae, represents a concerning scenario. This calls for an expansion towards bactericidal drug options and the establishment of robust surveillance for drug resistance in countries burdened with high leprosy rates. Moreover, the introduction of stringent antimicrobial stewardship initiatives is imperative. As a single centre study, it represents a limited, cross-sectional view of the real situation in the field.

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 red blood cells expressing a human variant of deficient glucose 6-phosphate dehydrogenase (G6PD), an enzymopathy that affects half a billion individuals (G6PD deficiency). Both cellular mechanisms of clearance (sequestration versus destruction) and specific metabolic disturbances caused by dapsone hydroxylamine are elucidated, providing novel mechanistic understanding.

Inhibitory effect of natural compounds on Dihydropteroate synthase of Mycobacterium leprae: Molecular dynamic study.

Leprosy is a chronic infectious disease caused by a bacillus, Mycobacterium leprae. According to official data from 139 countries in the 6 WHO Regions, there were 127558 new leprosy cases worldwide in 2020. Leprosy mainly affects the skin, the peripheral nerves, mucosa of the upper respiratory tract, and the eyes. If this disease is left untreated, can harm the skin, nerves, limbs, eyes, and skin permanently. The disease is curable with multidrug therapy. Over a period of time Mycobacterium leprae has become resistant to these drugs. Therefore, new therapeutic molecules are warranted. This study was aimed to carry out the in-silico analysis to determine the inhibitory effect of natural compounds on Dihydropteroate synthase (DHPS) of Mycobacterium leprae. The DHPS is a key enzyme in the folate biosynthesis pathway in M. leprae and acts as a competitive inhibitor of PABA. The 3D structure of DHPS protein was modeled using homology modeling and was validated. Molecular docking and simulation along with other in-silico methods were employed to determine the inhibitory effect of ligand molecules towards DHPS target protein. Results revealed ZINC03830554 molecule as a potential inhibitor of DHPS. Binding experiments and bioassays utilizing this strong inhibitor molecule against purified DHPS protein are necessary to validate these early findings.Communicated by Ramaswamy H. Sarma.

Detection of Hepatic Drug Metabolite-Specific T-Cell Responses Using a Human Hepatocyte, Immune Cell Coculture System.

Drug-responsive T-cells are activated with the parent compound or metabolites, often via different pathways (pharmacological interaction and hapten). An obstacle to the investigation of drug hypersensitivity is the scarcity of reactive metabolites for functional studies and the absence of coculture systems to generate metabolites in situ. Thus, the aim of this study was to utilize dapsone metabolite-responsive T-cells from hypersensitive patients, alongside primary human hepatocytes to drive metabolite formation, and subsequent drug-specific T-cell responses. Nitroso dapsone-responsive T-cell clones were generated from hypersensitive patients and characterized in terms of cross-reactivity and pathways of T-cell activation. Primary human hepatocytes, antigen-presenting cells, and T-cell cocultures were established in various formats with the liver and immune cells separated to avoid cell contact. Cultures were exposed to dapsone, and metabolite formation and T-cell activation were measured by LC-MS and proliferation assessment, respectively. Nitroso dapsone-responsive CD4+ T-cell clones from hypersensitive patients were found to proliferate and secrete cytokines in a dose-dependent manner when exposed to the drug metabolite. Clones were activated with nitroso dapsone-pulsed antigen-presenting cells, while fixation of antigen-presenting cells or omission of antigen-presenting cells from the assay abrogated the nitroso dapsone-specific T-cell response. Importantly, clones displayed no cross-reactivity with the parent drug. Nitroso dapsone glutathione conjugates were detected in the supernatant of hepatocyte immune cell cocultures, indicating that hepatocyte-derived metabolites are formed and transferred to the immune cell compartment. Similarly, nitroso dapsone-responsive clones were stimulated to proliferate with dapsone, when hepatocytes were added to the coculture system. Collectively, our study demonstrates the use of hepatocyte immune cell coculture systems to detect in situ metabolite formation and metabolite-specific T-cell responses. Similar systems should be used in future diagnostic and predictive assays to detect metabolite-specific T-cell responses when synthetic metabolites are not available.

Characterization of the anticonvulsant effect of dapsone on metabolic activity assessed by [18F]FDG -PET after kainic acid-induced status epilepticus in rats.

BACKGROUND: Development of effective drugs for epilepsy are needed, as nearly 30 % of epileptic patients, are resistant to current treatments. This study is aimed to characterize the anticonvulsant effect of dapsone (DDS), in the kainic acid (KA)-induced Status Epilepticus (SE) by recording the brain metabolic activity with an [18F]FDG-PET analysis. METHODS: Wistar rats received KA (10 mg/kg, i.p., single dose) to produce sustained seizures. [18F]FDG-PET and electroencephalographic (EEG) studies were then performed. DDS or vehicle were administered 30 min before KA. [18F]FDG uptake and EEG were evaluated at baseline, 2 and 25 h after KA injection. Likewise, caspase-8, 3 hippocampal activities and Fluoro-Jade B neuronal degeneration and Hematoxylin-eosin staining were measured 25 h after KA. RESULTS: PET data evaluated at 2 h showed hyper-uptake of [18F]FDG in the control group, which was decreased by DDS. At 25 h, hypo-uptake was observed in the control group and higher values due to DDS effect. EEG spectral power was increased 2 h after KA administration in the control group during the generalized tonic-clonic seizures, which was reversed by DDS, correlated with [18F]FDG-PET uptake changes. The values of caspases-8 activity decreased 48 and 43 % vs control group in the groups treated with DDS (12.5 y 25 mg/kg respectively), likewise; caspase-3 activity diminished by 57 and 53 %. Fewer degenerated neurons were observed due to DDS treatments. CONCLUSIONS: This study pinpoints the anticonvulsant therapeutic potential of DDS. Given its safety and effectiveness, DDS may be a viable alternative for patients with drug-resistant epilepsy.

Drug Resistance (Dapsone, Rifampicin, Ofloxacin) and Resistance-Related Gene Mutation Features in Leprosy Patients: A Systematic Review and Meta-Analysis.

Dapsone (DDS), Rifampicin (RIF) and Ofloxacin (OFL) are drugs recommended by the World Health Organization (WHO) for the treatment of leprosy. In the context of leprosy, resistance to these drugs occurs mainly due to mutations in the target genes (Folp1, RpoB and GyrA). It is important to monitor antimicrobial resistance in patients with leprosy. Therefore, we performed a meta-analysis of drug resistance in Mycobacterium leprae and the mutational profile of the target genes. In this paper, we limited the study period to May 2022 and searched PubMed, Web of Science (WOS), Scopus, and Embase databases for identified studies. Two independent reviewers extracted the study data. Mutation and drug-resistance rates were estimated in Stata 16.0. The results demonstrated that the drug-resistance rate was 10.18% (95% CI: 7.85-12.51). Subgroup analysis showed the highest resistance rate was in the Western Pacific region (17.05%, 95% CI:1.80 to 13.78), and it was higher after 2009 than before [(11.39%, 7.46-15.33) vs. 6.59% (3.66-9.53)]. We can conclude that the rate among new cases (7.25%, 95% CI: 4.65-9.84) was lower than the relapsed (14.26%, 95 CI%: 9.82-18.71). Mutation rates of Folp1, RpoB and GyrA were 4.40% (95% CI: 3.02-5.77), 3.66% (95% CI: 2.41-4.90) and 1.28% (95% CI: 0.87-1.71) respectively, while the rate for polygenes mutation was 1.73% (0.83-2.63). For further analysis, we used 368 drug-resistant strains as research subjects and found that codons (Ser, Pro, Ala) on RpoB, Folp1 and GyrA are the most common mutation sites in the determining region (DRDR). In addition, the most common substitution patterns of Folp1, RpoB, and GyrA are Pro→Leu, Ser→Leu, and Ala→Val. This study found that a higher proportion of patients has developed resistance to these drugs, and the rate has increased since 2009, which continue to pose a challenge to clinicians. In addition, the amino acid alterations in the sequence of the DRDR regions and the substitution patterns mentioned in the study also provide new ideas for clinical treatment options.

A comprehensive insight into the anti-inflammatory properties of dapsone.

The 4,4'-diaminodiphenyl sulfone (DDS), also known as dapsone, is traditionally used as a potent anti-bacterial agent in clinical management of leprosy. For decades, dapsone has been among the first-line medications used in multidrug treatment of leprosy recommended by the World Health Organization (WHO). Shortly after dapsone's discovery as an antibiotic in 1937, the dual function of dapsone (anti-microbial and anti-inflammatory) was elucidated. Dapsone exerts its anti-bacterial effects by inhibiting dihydrofolic acid synthesis, leading to inhibition of bacterial growth, while its anti-inflammatory properties are triggered by inhibiting reactive oxygen species (ROS) production, reducing the effect of eosinophil peroxidase on mast cells and downregulating neutrophil-mediated inflammatory responses. Among the leading mechanisms associated with its anti-microbial/anti-protozoal effects, dapsone clearly has multiple antioxidant, anti-inflammatory, and anti-apoptotic functions. In this regard, it has been described in treating a wide variety of inflammatory and infectious skin conditions. Previous reports have explored different molecular targets for dapsone and provided insight into the anti-inflammatory mechanism of dapsone. This article reviews several basic, experimental, and clinical approaches on anti-inflammatory effect of dapsone.

Investigating drug resistance of Mycobacterium leprae in the Comoros: an observational deep-sequencing study.

BACKGROUND: Despite strong leprosy control measures, including effective treatment, leprosy persists in the Comoros. As of May, 2022, no resistance to anti-leprosy drugs had been reported, but there are no nationally representative data. Post-exposure prophylaxis (PEP) with rifampicin is offered to contacts of patients with leprosy. We aimed to conduct a countrywide drug resistance survey and investigate whether PEP led to the emergence of drug resistance in patients with leprosy. METHODS: In this observational, deep-sequencing analysis we assessed Mycobacterium leprae genomes from skin biopsies of patients in Anjouan and Mohéli, Comoros, collected as part of the ComLep (NCT03526718) and PEOPLE (NCT03662022) studies. Skin biopsies that had sufficient M leprae DNA (>2000 bacilli in 2 µl of DNA extract) were assessed for the presence of seven drug resistance-associated genes (ie, rpoB, ctpC, ctpI, folP1, gyrA, gyrB, and nth) using Deeplex Myc-Lep (targeted next generation deep sequencing), with a limit of detection of 10% for minority M leprae bacterial populations bearing a polymorphism in these genes. All newly registered patients with leprosy for whom written informed consent was obtained were eligible for inclusion in the survey. Patients younger than 2 years or with a single lesion on the face did not have biopsies taken. The primary outcome of our study was the proportion of patients with leprosy (ie, new cases, patients with relapses or reinfections, patients who received single (double) dose rifampicin-PEP, or patients who lived in villages where PEP was distributed) who were infected with M leprae with a drug-resistant mutation for rifampicin, fluoroquinolone, or dapsone in the Comoros. FINDINGS: Between July 1, 2017, and Dec 31, 2020, 1199 patients with leprosy were identified on the basis of clinical criteria, of whom 1030 provided a skin biopsy. Of these 1030 patients, 755 (73·3%) tested positive for the M leprae-specific repetitive element-quantitative PCR (qPCR) assay. Of these 755 patients, 260 (34·4%) were eligible to be analysed using Deeplex Myc-Lep. 251 (96·5%) were newly diagnosed with leprosy, whereas nine (3·4%) patients had previously received multidrug therapy. 45 (17·3%) patients resided in villages where PEP had been administered in 2015 or 2019, two (4·4%) of whom received PEP. All seven drug resistance-associated targets were successfully sequenced in 216 samples, 39 samples had incomplete results, and five had no results. No mutations were detected in any of the seven drug resistance-related genes for any patient with successfully sequenced results. INTERPRETATION: This drug resistance survey provides evidence to show that M leprae is fully susceptible to rifampicin, fluoroquinolones, and dapsone in the Comoros. Our results also show, for the first time, the applicability of targeted sequencing directly on skin biopsies from patients with either paucibacillary or multibacillary leprosy. These data suggest that PEP had not selected rifampicin-resistant strains, although further support for this finding should be confirmed with a larger sample size. FUNDING: Effect:Hope, The Mission To End Leprosy, the Fonds Wetenschappelijk Onderzoek, the EU.

Risk factors for dapsone resistance in leprosy patients: a systematic meta-analysis.

OBJECTIVES: Dapsone is one of the important drugs in the treatment of leprosy. The present study aims to evaluate the resistance of Mycobacterium leprae isolates to dapsone, in turn assisting in implementing better control strategies for leprosy elimination. METHODS: A systematic literature search was conducted in PubMed, Embase, Medline, and Web of Science. Two independent reviewers selected the literature according to the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA), extracted data, and evaluated the risk of bias. Drug resistance data were pooled using the random-effects model. Subgroup analysis was performed based on across sampling time, region, study population (treatment status, relapses status), and sample size. RESULTS: A total of 30 studies were included. The results of meta-analysis showed that the dapsone resistance rate of leprosy patients after treatment was 8% (95% confidence interval [CI], 6%-10%). Compared to the rates of primary resistance of new cases without treatment therapy (pooled incidence, 4% [95% CI, 2%-5%]), treatment cases (13% [95% CI 9%-16%]) had secondary resistance, and relapse cases (26% [95% CI, 18%-33%]) had drug resistance. In addition, the drug resistance rate of monotherapy was significantly increased than that of relapsed patients treated with diamino-diphenylsulfone monotherapy. Subgroup analysis showed that the patients in the Western Pacific have the highest dapsone resistance, and the resistance to dapsone was slightly lower after 2005. For sample size, the rate in the group under 100 samples was significantly higher than in the other. CONCLUSION: Dapsone resistance is closely related to leprosy relapse and long-term drug use. Dapsone monotherapy is one of important reasons for drug resistance in relapsed cases. Drug resistance varies among different populations and regions of the world.

Ofloxacin resistance in multibacillary new leprosy cases from Purulia, West Bengal: a threat to effective secondary line treatment for rifampicin-resistant leprosy cases.

OBJECTIVES: Purulia is one of the high-endemic districts for leprosy in West Bengal (the eastern part of India). The annual new case detection rate (ANCDR) of leprosy in West Bengal is 6.04/100000 (DGHS 2019-20). Our earlier report provided evidence of secondary drug resistance in relapse cases of leprosy. The aim of the current study was to observe primary drug resistance patterns for dapsone, rifampicin, and ofloxacin amongst new leprosy patients from Purulia, West Bengal in order to better understand the emergence of primary resistance to these drugs. METHODS: In the present study, slit-skin smear samples were collected from 145 newly diagnosed leprosy cases from The Leprosy Mission (TLM) Purulia hospital between 2017 and 2018. DNA was extracted from these samples and the Mycobacterium leprae genome was analyzed for genes associated with drug resistance by polymerase chain reaction (PCR), followed by Sanger sequencing. Wild-type strain (Thai-53) and mouse footpad-derived drug-resistant strain (Z-4) were used as reference strains. RESULTS: Of 145 cases, 25 cases showed mutations in genes associated with resistance to rifampicin, dapsone, and ofloxacin (as described by the World Health Organization, rpoB, folP, and gyrA, respectively) through Sanger sequencing. Of these 25 cases, 16 cases showed mutations in ofloxacin, two cases showed mutations in combinations of ofloxacin and rifampicin, four cases showed a mutation only in rifampicin, one case showed mutations in combinations of rifampicin and dapsone, and two cases showed mutations only in dapsone. CONCLUSION: Results from this study indicated the emergence of resistance to antileprosy drugs in new cases of leprosy. As ofloxacin is the alternate drug for the treatment of rifampicin-resistant cases, the emergence of new cases with resistance to ofloxacin indicates that ofloxacin-resistant M. leprae strains are actively circulating in this endemic region (i.e., Purulia, West Bengal), posing challenges for the effective treatment of rifampicin-resistant cases.

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.

Protective effect of dapsone against bleomycin-induced lung fibrosis in rat.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial disease of the lung tissue that causes symptoms such as coughing and asthma. It is caused by inflammatory factors and oxidative stress. In vivo model of IPF is induced by bleomycin (BLM,) a chemotherapeutic agent. We have investigated the effect of dapsone on bleomycin-induced IPF in adult male Wistar rats due to its anti-inflammatory and anti-oxidative stress effects. The animals were randomly divided into 5 groups (Control, BLM, BLM + dapsone 1, BLM + Dapsone 3, BLM + Dapsone 10). The control group received normal water and food. In the fibrosis group, bleomycin (BLM) (5 mg/kg) was used to induce pulmonary fibrosis by intratracheal administration. Three groups of animals were treated daily with single doses of 1, 3, and 10 mg dapsone by intraperitoneal injection 1 h after receiving BLM for 2 weeks. The activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), and oxidative stress markers such as myeloperoxidase (MPO), malondialdehyde (MDA), protein carbonyl (PC) and nitrite were measured to evaluate bleomycin and therapeutic effect of dapsone. The histological assays of lung tissues were done by Hematoxylin-eosin (H & E) and Masson's trichrome staining. BLM reduced the activity of oxidative enzymes and increased the oxidative stress markers, while treatment with dapsone has reversed the results. In addition, the total number of cells as inflammatory cells such as neutrophils and eosinophils were examined. It has been indicated BLM increased these cells, and dapsone decreased them. The results of H & E and Masson's trichrome staining showed that dapsone reduced inflammation and alveolar wall thickness and BLM-induced pulmonary fibrosis. According to the findings of this study, dapsone seems to have therapeutic effects on pulmonary fibrosis through its anti-inflammatory and anti-oxidative stress properties and reduction of the toxic effects of bleomycin.

Disease Burden and Current Therapeutical Status of Leprosy with Special Emphasis on Phytochemicals.

BACKGROUND: Leprosy (Hansen's disease) is a neglected tropical disease affecting millions of people globally. The combined formulations of dapsone, rifampicin and clofazimine (multidrug therapy, MDT) is only supportive in the early stage of detection, while "reemergence" is a significant problem. Thus, there is still a need to develop newer antileprosy molecules either of natural or semi-synthetic origin. OBJECTIVES: The review intends to present the latest developments in the disease prevalence, available therapeutic interventions and the possibility of identifying new molecules from phytoextracts. METHODS: Literature on the use of plant extracts and their active components to treat leprosy was searched. Selected phytoconstituents were subjected to molecular docking study on both wild and mutant types of the Mycobacterium leprae. Since the M. leprae dihydropteroate synthase (DHPS) is not available in the protein data bank (PDB), it was modelled by the homology model method and validated with the Ramachandran plot along with other bioinformatics approaches. Two mutations were introduced at codons 53 (Thr to Ile) and 55 (Pro to Leu) for docking against twenty-five selected phytoconstituents reported from eight plants that recorded effective anti-leprosy activity. The chemical structure of phytochemicals and the standard dapsone structure were retrieved from the PubChem database and prepared accordingly for docking study with the virtual-screening platform of PyRx-AutoDock 4.1. RESULTS: Based on the docking score (kcal/mol), most of the phytochemicals exhibited a higher docking score than dapsone. Asiaticoside, an active saponin (-11.3, -11.2 and -11.2 kcal/mol), was proved to be the lead phytochemical against both wild and mutant types DHPS. Some other useful phytoconstituents include echinocystic acid (-9.6, -9.5 and -9.5 kcal/mol), neobavaisoflavone (-9.2, -9.0 and -9.0 kcal/mol), boswellic acid (-8.90, -8.90 and -8.90 kcal/mol), asiatic acid (-8.9, -8.8 and -8.9 kcal/mol), corylifol A (-8.8, 8.0, and -8.0), etc. Overall, the computational predictions support the previously reported active phytoextracts of Centella asiatica (L.) Urban, Albizia amara (Roxb.) Boivin, Boswellia serrata Roxb. and Psoralea corylifolia L. to be effective against leprosy. CONCLUSION: A very small percentage of well-known plants have been evaluated scientifically for antileprosy activity. Further in vivo experiments are essential to confirm anti-leprosy properties of such useful phytochemicals.

Interaction of constituents of MDT regimen for leprosy with Mycobacterium leprae HSP18: impact on its structure and function.

Mycobacterium leprae, the causative organism of leprosy, harbors many antigenic proteins, and one such protein is the 18-kDa antigen. This protein belongs to the small heat shock protein family and is commonly known as HSP18. Its chaperone function plays an important role in the growth and survival of M. leprae inside infected hosts. HSP18/18-kDa antigen is often used as a diagnostic marker for determining the efficacy of multidrug therapy (MDT) in leprosy. However, whether MDT drugs (dapsone, clofazimine, and rifampicin) do interact with HSP18 and how these interactions affect its structure and chaperone function is still unclear. Here, we report evidence of HSP18-dapsone/clofazimine/rifampicin interaction and its impact on the structure and chaperone function of HSP18. These three drugs interact efficiently with HSP18 (having submicromolar binding affinity) with 1 : 1 stoichiometry. Binding of these MDT drugs to the 'α-crystallin domain' of HSP18 alters its secondary structure and tryptophan micro-environment. Furthermore, surface hydrophobicity, oligomeric size, and thermostability of the protein are reduced upon interaction with these three drugs. Eventually, all these structural alterations synergistically decrease the chaperone function of HSP18. Interestingly, the effect of rifampicin on the structure, stability, and chaperone function of this mycobacterial small heat shock protein is more pronounced than the other two MDT drugs. This reduction in the chaperone function of HSP18 may additionally abate M. leprae survivability during multidrug treatment. Altogether, this study provides a possible foundation for rational designing and development of suitable HSP18 inhibitors in the context of effective treatment of leprosy.

Dapsone Prevents Allodynia and Hyperalgesia and Decreased Oxidative Stress After Spinal Cord Injury in Rats.

STUDY DESIGN: Prospective longitudinal experimental study. OBJECTIVE: We evaluate the effect of dapsone on tactile allodynia and mechanical hyperalgesia and to determine its anti-oxidant effect in a spinal cord injury (SC) model in rats. SUMMARY OF BACKGROUND DATA: Neuropathic pain (NP) as result of traumatic spinal cord injury is a deleterious medical condition with temporal or permanent time-course. Painful stimuli trigger a cascade of events that activate the N-methyl-D-aspartate (NMDA) receptor, inducing an increase in oxidative stress. Since there is no effective treatment for this condition, dapsone (4,4'diaminodiphenylsulfone) is proposed as potential treatment for NP. Its anti-oxidant, neuroprotective, and anti-inflammatory properties have been documented, however, there is no evidence regarding its use for treatment of NP induced by SCI. METHODS: In this study, we evaluated the anti-allodynic and anti-hyperalgesic effect of dapsone as preventive or acute treatment after NP was already established. Furthermore, participation of oxidative stress was evaluated by measuring lipid peroxidation (LP) and glutathione concentration (GSH) in rats with SCI. RESULTS: Acute treatment with dapsone (3.1-25 mg/kg, i.p.) decreased nociceptive behaviors in a dose-dependent manner, decreased LP, and increased GSH in the injured tissue 15 days after the injury was produced. On the other hand, preventive treatment (3 h post-injury, once daily for 3 days) with dapsone (3.1-25 mg/kg, i.p.) yielded similar results. CONCLUSION: The findings suggest that the anti-nociceptive effect of dapsone is regulated through the decrease of oxidative stress and the excitotoxicity is associated with the activation of NMDA receptors.Level of Evidence: N/A.

The effect of dapsone in testosterone enanthate-induced polycystic ovary syndrome in rat.

BACKGROUND: One of the most common reasons for infertility is polycystic ovary syndrome (PCOS). PCOS is related to metabolic syndrome, weight gain, type 2 diabetes mellitus, and cardiovascular diseases. Some of the causes of PCOS are dysfunction in the hypothalamus-pituitary-ovarian axis, insulin activity as well as over-activity of sympathetic nerves and elevation in serum levels of pro-inflammatory cytokines. Dapsone, a sulfonamide antibacterial agent, has anti-inflammatory effects such as decreasing inflammatory cytokine levels like TNF-α and IL-1ß. METHODS: PCOS was induced by subcutaneous injection of testosterone enanthate (1 mg/100 g) in 21 days old female rats for 35 days. Then, the MET control received metformin (300 mg/kg/day, orally) for 28 days, and to evaluate the efficacy of dapsone (DAP), the DAP group received (12.5 mg/kg, orally) for 28 days. Then, on the last day of the study, the rats were euthanized and the blood was collected to measure the serum levels of hormones, glucose, LDL, LDL/HDL and the left ovaries were dissected for histopathological assay. RESULTS: In the PCOS group, the serum levels of glucose, LDL and LDL/HDL were significantly higher than in the control group (P < 0.001). In addition, the levels of LH, FSH and testosterone changed in the PCOS group compared to the control (P < 0.001). The histopathological morphology changes of the ovary of the PCOS group were significant. Treatment with dapsone and metformin reversed the effects of testosterone in the DAP and MET groups. CONCLUSIONS: Based on the data, dapsone displayed a good antiandrogenic role via decreasing the testosterone levels in PCOS-induced rats.

Synthesis, characterization and drug delivery application of Dapsone based double tailed biocompatible nonionic surfactant.

The increase in antimicrobial resistance has created a crisis that has become top priority for global policy and public health. Antibiotics are constantly being rendered in-effective due to the emergence of bacterial resistance; therefore, novel strategies for improving therapeutic efficacies of existing drugs must be focused. Advancements in nanotechnology have opened up new avenues for enhancing therapeutic efficacy of existing drugs via construction of intelligent and efficient delivery systems. This study reports the synthesis of Dapsone based nonionic surfactant and its utilization as delivery system for Ceftriaxone sodium. The synthesized nonionic surfactant was characterized via mass spectrometry and 1H NMR and IR spectroscopic techniques. The drug loaded vesicles of newly synthesized sulfur based nonionic were formed through thin film hydration method and characterized for drug entrapment efficiency, vesicles size, zeta potential, morphology using UV-vis spectrometry, dynamic light scattering (DLS) and atomic force microscopic (AFM) techniques. The biocompatibility of newly synthesized surfactant was assessed using blood hemolysis and in-vitro cells cytotoxicity. Antibacterial potential of drug loaded vesicles was assessed in gram positive and gram negative bacterial cultures. The spectroscopic results confirm successful synthesis of novel sulfur based nonionic surfactant that formed spherical shaped drug loaded vesicles with an average size of 97.95 ± 3.45 nm and 56.3 ± 3.15 % entrapment of the model drug (Ceftriaxone sodium). The vesicles displayed negative surface charge of -16.8 ± 3.72 mV and released the entrapped drug in a controlled way in-vitro drug release. The drug loaded vesicular formulation showed enhanced cellular uptake and greater antibacterial potentials when compared with control. Results of this study show that the Dapsone based surfactant is safe, biocompatible, non-toxic and can be used as promising vesicular carrier for enhancing therapeutic efficacy of antibacterial drug, Ceftriaxone sodium.

Dapsone Suppresses Disease in Preclinical Murine Models of Pemphigoid Diseases.

Epidermolysis bullosa acquisita and mucous membrane pemphigoid are autoimmune blistering diseases characterized by mucocutaneous blisters elicited by an autoantibody-mediated immune response against specific proteins of the epidermal basement membrane. The antibiotic dapsone is frequently used to treat both diseases, but its therapeutic effectiveness is uncertain, and its mode of action in these diseases is largely unknown. We evaluated the effect of dapsone in antibody transfer mouse models of epidermolysis bullosa acquisita and mucous membrane pemphigoid, which do not allow the drawing of conclusions on clinical treatment regimens but can be instrumental to partially uncover the mode(s) of action of dapsone in these diseases. Dapsone significantly mitigated inflammation in both models, reducing the recruitment of neutrophils into the skin and disrupting their release of leukotriene B4 (LTB4) and ROS in response to immune complexes. LTB4 has been implicated in numerous diseases, but effective LTB4 inhibitors for clinical use are not available. Our findings indicate that the mode of action of dapsone in these models may be based on the inhibition of LTB4 and ROS release from neutrophils. Moreover, they encourage testing the use of dapsone as an effective, albeit nonspecific, inhibitor of LTB4 biosynthesis in other LTB4-driven diseases.

HLA Class-II‒Restricted CD8+ T Cells Contribute to the Promiscuous Immune Response in Dapsone-Hypersensitive Patients.

HLA-B∗13:01 is associated with dapsone (DDS)-induced hypersensitivity, and it has been shown that CD4+ and CD8+ T cells are activated by DDS and its nitroso metabolite (nitroso dapsone [DDS-NO]). However, there is a need to define the importance of the HLA association in the disease pathogenesis. Thus, DDS- and DDS-NO‒specific CD8+ T-cell clones (TCCs) were generated from hypersensitive patients expressing HLA-B∗13:01 and were assessed for phenotype and function, HLA allele restriction, and killing of target cells. CD8+ TCCs were stimulated to proliferate and secrete effector molecules when exposed to DDS and/or DDS-NO. DDS-responsive and several DDS-NO‒responsive TCCs expressing a variety of TCR sequences displayed HLA class-I restriction, with the drug (metabolite) interacting with multiple HLA-B alleles. However, activation of certain DDS-NO‒responsive CD8+ TCCs was inhibited with HLA class-II block, with DDS-NO binding to HLA-DQB1∗05:01. These TCCs were of different origin but expressed TCRs displaying the same amino acid sequences. They were activated through a hapten pathway; displayed CD45RO, CD28, PD-1, and CTLA-4 surface molecules; secreted the same panel of effector molecules as HLA class-I‒restricted TCCs; but displayed a lower capacity to lyse target cells. To conclude, DDS and DDS-NO interact with a number of HLA molecules to activate CD8+ TCCs, with HLA class-II‒restricted CD8+ TCCs that display hybrid CD4‒CD8 features also contributing to the promiscuous immune response that develops in patients.