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1.
Nature ; 593(7859): 418-423, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33727703

RESUMO

The COVID-19 pandemic is the third outbreak this century of a zoonotic disease caused by a coronavirus, following the emergence of severe acute respiratory syndrome (SARS) in 20031 and Middle East respiratory syndrome (MERS) in 20122. Treatment options for coronaviruses are limited. Here we show that clofazimine-an anti-leprosy drug with a favourable safety profile3-possesses inhibitory activity against several coronaviruses, and can antagonize the replication of SARS-CoV-2 and MERS-CoV in a range of in vitro systems. We found that this molecule, which has been approved by the US Food and Drug Administration, inhibits cell fusion mediated by the viral spike glycoprotein, as well as activity of the viral helicase. Prophylactic or therapeutic administration of clofazimine in a hamster model of SARS-CoV-2 pathogenesis led to reduced viral loads in the lung and viral shedding in faeces, and also alleviated the inflammation associated with viral infection. Combinations of clofazimine and remdesivir exhibited antiviral synergy in vitro and in vivo, and restricted viral shedding from the upper respiratory tract. Clofazimine, which is orally bioavailable and comparatively cheap to manufacture, is an attractive clinical candidate for the treatment of outpatients and-when combined with remdesivir-in therapy for hospitalized patients with COVID-19, particularly in contexts in which costs are an important factor or specialized medical facilities are limited. Our data provide evidence that clofazimine may have a role in the control of the current pandemic of COVID-19 and-possibly more importantly-in dealing with coronavirus diseases that may emerge in the future.


Assuntos
Antivirais/farmacologia , Clofazimina/farmacologia , Coronavirus/classificação , Coronavirus/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antivirais/farmacocinética , Antivirais/uso terapêutico , Disponibilidade Biológica , Fusão Celular , Linhagem Celular , Clofazimina/farmacocinética , Clofazimina/uso terapêutico , Coronavirus/crescimento & desenvolvimento , Coronavirus/patogenicidade , Cricetinae , DNA Helicases/antagonistas & inibidores , Sinergismo Farmacológico , Feminino , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Masculino , Mesocricetus , Profilaxia Pré-Exposição , SARS-CoV-2/crescimento & desenvolvimento , Especificidade da Espécie , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/genética
2.
Int J Mol Sci ; 21(17)2020 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-32824985

RESUMO

The aim of this study is to examine the use of an inflammasome competitor as a preventative agent. Coronaviruses have zoonotic potential due to the adaptability of their S protein to bind receptors of other species, most notably demonstrated by SARS-CoV. The binding of SARS-CoV-2 to TLR (Toll-like receptor) causes the release of pro-IL-1ß, which is cleaved by caspase-1, followed by the formation and activation of the inflammasome, which is a mediator of lung inflammation, fever, and fibrosis. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is implicated in a variety of human diseases including Alzheimer's disease (AD), prion diseases, type 2 diabetes, and numerous infectious diseases. By examining the use of 4,4'-diaminodiphenyl sulfone (DDS) in the treatment of patients with Hansen's disease, also diagnosed as Alzheimer's disease, this study demonstrates the diverse mechanisms involved in the activation of inflammasomes. TLRs, due to genetic polymorphisms, can alter the immune response to a wide variety of microbial ligands, including viruses. In particular, TLR2Arg677Trp was reported to be exclusively present in Korean patients with lepromatous leprosy (LL). Previously, mutation of the intracellular domain of TLR2 has demonstrated its role in determining the susceptibility to LL, though LL was successfully treated using a combination of DDS with rifampicin and clofazimine. Of the three tested antibiotics, DDS was effective in the molecular regulation of NLRP3 inflammasome activators that are important in mild cognitive impairment (MCI), Parkinson's disease (PD), and AD. The specific targeting of NLRP3 itself or up-/downstream factors of the NLRP3 inflammasome by DDS may be responsible for its observed preventive effects, functioning as a competitor.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Dapsona/farmacologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia Viral/tratamento farmacológico , Doença de Alzheimer/patologia , COVID-19 , Clofazimina/farmacologia , Disfunção Cognitiva/patologia , Humanos , Interleucina-1beta/metabolismo , Hanseníase/tratamento farmacológico , Hanseníase/genética , Pandemias , Transtornos Parkinsonianos/patologia , Rifampina/farmacologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 2 Toll-Like/genética
3.
Biochem Pharmacol ; 177: 113993, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32339493

RESUMO

Leprosy is a chronic infectious disease caused my Mycobacterium leprae that primarily affects peripheral nervous system and extremities and is prevalent in tropical countries. Treatment for leprosy with multidrug regimens is very effective compared to monotherapy especially in multibacillary cases. The three major antileprosy drugs currently in use are 4, 4'-diaminodiphenyl sulfone (DDS, dapsone), rifampicin, and clofazimine. During multidrug therapy, the potent antibiotic rifampicin induces the metabolism of dapsone, which results in decreased plasma half-life of dapsone and its metabolites. Furthermore, rifampicin induces its own metabolism and decreases its half-life during monotherapy. Rifampicin upregulates several hepatic microsomal drug-metabolizing enzymes, especially cytochrome P450 (CYP) family that in turn induce the metabolism of dapsone. Clofazimine lacks significant induction of any drug-metabolizing enzyme including CYP family and does not interact with dapsone metabolism. Rifampicin does not induce clofazimine metabolism during combination treatment. Administration of dapsone in the acetylated form (acedapsone) can release the drug slowly into circulation up to 75 days and could be useful for the effective treatment of paucibacillary cases along with rifampicin. This review summarizes the major aspects of antileprosy drug metabolism and drug interactions and the role of cytochrome P450 family of drug metabolizing enzymes, especially CYP3A4 during multidrug regimens for the treatment of leprosy.


Assuntos
Acedapsona/sangue , Clofazimina/sangue , Citocromo P-450 CYP3A/metabolismo , Dapsona/sangue , Hansenostáticos/sangue , Hanseníase/tratamento farmacológico , Rifampina/sangue , Acedapsona/farmacocinética , Acedapsona/farmacologia , Disponibilidade Biológica , Biotransformação , Clofazimina/farmacocinética , Clofazimina/farmacologia , Dapsona/farmacocinética , Dapsona/farmacologia , Interações Medicamentosas , Quimioterapia Combinada , Meia-Vida , Humanos , Hansenostáticos/farmacocinética , Hansenostáticos/farmacologia , Hanseníase/sangue , Hanseníase/microbiologia , Hanseníase/patologia , Taxa de Depuração Metabólica , Redes e Vias Metabólicas/fisiologia , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/patogenicidade , Rifampina/farmacocinética , Rifampina/farmacologia
4.
Haematologica ; 105(4): 971-986, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31371410

RESUMO

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38- progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.


Assuntos
Hanseníase , Leucemia Mielogênica Crônica BCR-ABL Positiva , Preparações Farmacêuticas , Apoptose , Clofazimina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , PPAR gama
5.
Tuberculosis (Edinb) ; 115: 63-66, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30948178

RESUMO

The Mycobacterium tuberculosis mec+-cysO-cysM gene cluster was shown to be part of a novel cysteine biosynthesis pathway in vitro, but little is known about its essentiality or role in M. tuberculosis physiology. In this study, we generate a knock out of the mec+-cysO-cysM gene cluster in M. tuberculosis and show that the gene cluster is not essential under a variety of conditions, suggesting redundancy in pathways for cysteine biosynthesis in M. tuberculosis. The cysteine biosynthesis gene cluster is essential for resistance for clofazimine, a peroxide-producing anti-leprosy drug. Therefore, although under most conditions the pathway is not essential, it likely has an important role in defense against oxidative stress in M. tuberculosis.


Assuntos
Antituberculosos/farmacologia , Clofazimina/farmacologia , Cisteína/biossíntese , Genes Bacterianos/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Vias Biossintéticas/genética , Cisteína/genética , Farmacorresistência Bacteriana/genética , Deleção de Genes , Hansenostáticos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estresse Oxidativo/efeitos dos fármacos
6.
J Clin Invest ; 128(10): 4682-4696, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30198908

RESUMO

Polyamine inhibition for cancer therapy is, conceptually, an attractive approach but has yet to meet success in the clinical setting. The aryl hydrocarbon receptor (AHR) is the central transcriptional regulator of the xenobiotic response. Our study revealed that AHR also positively regulates intracellular polyamine production via direct transcriptional activation of 2 genes, ODC1 and AZIN1, which are involved in polyamine biosynthesis and control, respectively. In patients with multiple myeloma (MM), AHR levels were inversely correlated with survival, suggesting that AHR inhibition may be beneficial for the treatment of this disease. We identified clofazimine (CLF), an FDA-approved anti-leprosy drug, as a potent AHR antagonist and a suppressor of polyamine biosynthesis. Experiments in a transgenic model of MM (Vk*Myc mice) and in immunocompromised mice bearing MM cell xenografts revealed high efficacy of CLF comparable to that of bortezomib, a first-in-class proteasome inhibitor used for the treatment of MM. This study identifies a previously unrecognized regulatory axis between AHR and polyamine metabolism and reveals CLF as an inhibitor of AHR and a potentially clinically relevant anti-MM agent.


Assuntos
Poliaminas Biogênicas/biossíntese , Clofazimina/farmacologia , Mieloma Múltiplo , Proteínas de Neoplasias , Neoplasias Experimentais , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo
7.
Acta Biochim Biophys Sin (Shanghai) ; 50(10): 1062-1067, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30137201

RESUMO

Human Raf1 kinase inhibitory protein (hRKIP) is an important modulator of the Ras/Raf1/MEK/ERK signaling pathway. Here, we demonstrated that anti-leprosy drug Clofazimine can bind to hRKIP with a significantly stronger affinity than the endogenous substrate phosphatidylethanolamine (PE) by using Biolayer interference technology. Moreover, we identified that residues P74, S75, K80, P111, P112, V177, and P178 play crucial roles in the binding of hRKIP to Clofazimine by using a combination of Nuclear Magnetic Resonance spectroscopy and molecular docking approach. These residues are located at the conserved ligand-binding pocket of hRKIP. Furthermore, we found that 3.2 µM Clofazimine could significantly increase the ERK phosphorylation level by about 37%. Our results indicate that Clofazimine can enhance Ras/Raf1/MEK/ERK signaling transduction pathway via binding to hRKIP. This work provides valuable hints for exploiting Clofazimine as a potential lead compound to efficiently treat the diseases related to RKIP or the Ras/Raf/MEK/ERK pathway.


Assuntos
Clofazimina/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Ligação Competitiva , Clofazimina/química , Clofazimina/farmacologia , Células HEK293 , Humanos , Hansenostáticos/química , Hansenostáticos/metabolismo , Hansenostáticos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteína de Ligação a Fosfatidiletanolamina/química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Domínios Proteicos
8.
Biomed Pharmacother ; 103: 1392-1396, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29864923

RESUMO

The aim of this work was to assess the feasibility of drug nanosystems combination for oral therapy of multibacillary leprosy. The anti-leprotic drugs dapsone (DAP) and clofazimine (CLZ) were incorporated within polymeric nanosystems and studied per se and in combination. DAP was loaded in Eudragit L100 nanoparticles (NPs-DAP) while CLZ was loaded in (poly(lactic-co-glycolic acid) (NPs-CLZ). The nanosystems exhibited around 200 nm in size and a drug loading of 12% for each drug. In vitro cytotoxicity on intestinal Caco-2 cells revealed that after 8 h incubation, DAP alone and within NPs were not toxic up to 100 µg mL-1, while CLZ per se was toxic, reducing cell viability to 30% at 50 µg mL-1. Caco-2 exposed to the combination of NPs-DAP (100 µg mL-1) and NPs-CLZ (50 µg mL-1) exhibited 80% of viability. Caco-2 monolayer permeability assays revealed that DAP and CLZ in the nanosystems per se or in NPs-DAP/ NPs-CLZ combination crossed the intestinal barrier. No significant differences were observed between the single nanosystems or in combination with the apparent permeability values and the amount of permeated drug. Thus, the NPs-DAP/NPs-CLZ combination seems to be a promising platform to deliver both drugs in association, representing an important step towards the improvement of multibacillary leprosy therapy.


Assuntos
Clofazimina/farmacologia , Dapsona/farmacologia , Sistemas de Liberação de Medicamentos , Intestinos/fisiologia , Nanopartículas/química , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Impedância Elétrica , Humanos , Intestinos/efeitos dos fármacos
9.
J R Soc Interface ; 15(139)2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29436513

RESUMO

The aim of this work was to develop solid lipid nanoparticles (SLNs) loaded with clofazimine (CLZ) (SLNs-CLZ) to overcome its intrinsic toxicity and low water solubility, for oral drug delivery. A Box-Behnken design was constructed to unravel the relations between the independent variables in the selected responses. The optimized SLNs-CLZ exhibited the following properties: particle size ca 230 nm, zeta potential of -34.28 mV, association efficiency of 72% and drug loading of 2.4%, which are suitable for oral delivery. Further characterization included Fourier transformed infrared spectroscopy that confirmed the presence of the drug and the absence of chemical interactions. By differential scanning calorimetry was verified the amorphous state of CLZ. The storage stability studies ensured the stability of the systems over a period of 12 weeks at 4°C. In vitro cytotoxicity studies evidenced no effect of both drug-loaded and unloaded SLNs on MKN-28 gastric cells and on intestinal cells, namely Caco-2 and HT29-MTX cells up to 25 µg ml-1 in CLZ. Free CLZ solutions exhibited IC50 values of 16 and 20 µg ml-1 for Caco-2 and HT29-MTX cells, respectively. It can be concluded that the optimized system, designed considering important variables for the formulation of poorly soluble drugs, represents a promising platform for oral CLZ delivery.


Assuntos
Clofazimina , Portadores de Fármacos , Lipídeos , Teste de Materiais , Modelos Biológicos , Nanopartículas , Células CACO-2 , Clofazimina/química , Clofazimina/farmacocinética , Clofazimina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Humanos , Lipídeos/química , Lipídeos/farmacocinética , Lipídeos/farmacologia , Nanopartículas/química , Nanopartículas/uso terapêutico
10.
Int J Antimicrob Agents ; 51(2): 235-238, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28711677

RESUMO

In the Horn of Africa, there is a high prevalence of tuberculosis that is reported to be partly driven by multidrug-resistant (MDR) Mycobacterium tuberculosis strictu sensu strains. We conducted a prospective study to investigate M. tuberculosis complex species causing tuberculosis in Djibouti, and their in vitro susceptibility to standard anti-tuberculous antibiotics in addition to clofazimine, minocycline, chloramphenicol and sulfadiazine. Among the 118 mycobacteria isolates from 118 successive patients with suspected pulmonary tuberculosis, 111 strains of M. tuberculosis, five Mycobacterium canettii, one 'Mycobacterium simulans' and one Mycobacterium kansasii were identified. Drug-susceptibility tests performed on the first 78 isolates yielded nine MDR M. tuberculosis isolates. All isolates were fully susceptible to clofazimine, minocycline and chloramphenicol, and 75 of 78 isolates were susceptible to sulfadiazine. In the Horn of Africa, patients with confirmed pulmonary tuberculosis caused by an in vitro susceptible strain may benefit from anti-leprosy drugs, sulfamides and phenicol antibiotics.


Assuntos
Antituberculosos/uso terapêutico , Mycobacterium kansasii/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Adulto , Cloranfenicol/farmacologia , Clofazimina/farmacologia , Djibuti , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Minociclina/farmacologia , Mycobacterium kansasii/isolamento & purificação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Estudos Prospectivos , Sulfadiazina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/microbiologia
11.
J Antimicrob Chemother ; 72(6): 1678-1687, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28333192

RESUMO

Background: The second-line drugs recommended to treat drug-resistant TB are toxic, expensive and difficult to procure. Given increasing resistance, the need for additional anti-TB drugs has become more urgent. But new drugs take time to develop and are expensive. Some commercially available drugs have reported anti-mycobacterial activity but are not routinely used because supporting laboratory and clinical evidence is sparse. Methods: We analysed 217 MDR M. tuberculosis isolates including 153 initial isolates from unique patients and 64 isolates from follow-up specimens during the course of treatment. The resazurin microdilution assay was performed to determine MICs of trimethoprim/sulfamethoxazole, mefloquine, thioridazine, clofazimine, amoxicillin/clavulanate, meropenem/clavulanate, nitazoxanide, linezolid and oxyphenbutazone. Isoniazid was used for validation. We calculated the MIC 50 and MIC 90 as the MICs at which growth of 50% and 90% of isolates was inhibited, respectively. Results: The MIC 50 s, in mg/L, for initial isolates were as follows: trimethoprim/sulfamethoxazole, 0.2/4; mefloquine, 8; thioridazine, 4; clofazimine, 0.25; amoxicillin/clavulanate, 16/8; meropenem/clavulanate, 1/2.5; nitazoxanide, 16; linezolid, 0.25; and oxyphenbutazone, 40. The MIC 90 s, in mg/L, for initial isolates were as follows: trimethoprim/sulfamethoxazole, 0.4/8; mefloquine, 8; thioridazine, 8; clofazimine, 0.5; amoxicillin/clavulanate, 32/16; meropenem/clavulanate, 8/2.5; nitazoxanide, 16; linezolid, 0.25; and oxyphenbutazone, 60. By comparison, the MIC 90 of isoniazid was >4 mg/L, as expected. There was no evidence that previous treatment affected susceptibility to any drug. Conclusions: Most drugs demonstrated efficacy against M. tuberculosis . When these MICs are compared with the published pharmacokinetic/pharmacodynamic profiles of the respective drugs in humans, trimethoprim/sulfamethoxazole, meropenem/clavulanate, linezolid, clofazimine and nitazoxanide appear promising and warrant further clinical investigation.


Assuntos
Antituberculosos/farmacologia , Descoberta de Drogas/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Combinação Amoxicilina e Clavulanato de Potássio/farmacologia , Antituberculosos/farmacocinética , Ácido Clavulânico/farmacologia , Clofazimina/farmacologia , Farmacorresistência Bacteriana Múltipla , Humanos , Hansenostáticos/farmacologia , Meropeném , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/isolamento & purificação , Tienamicinas/farmacocinética , Tienamicinas/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Inibidores de beta-Lactamases/farmacologia
12.
PLoS Negl Trop Dis ; 11(2): e0005373, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28158186

RESUMO

Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine has a long history of use and has demonstrated a good safety profile for a disease that requires chronic dosing for a period of time ranging 3-36 months. These results, taken with clofazimine's status as an FDA-approved drug with over four decades of use for the treatment of leprosy, support the continued investigation of clofazimine both as a new chemical tool for understanding cryptosporidium biology and a potential new treatment of cryptosporidiosis.


Assuntos
Antiprotozoários/farmacologia , Clofazimina/farmacologia , Criptosporidiose/tratamento farmacológico , Cryptosporidium parvum/efeitos dos fármacos , Reposicionamento de Medicamentos , Animais , Automação Laboratorial , Linhagem Celular , Criptosporidiose/parasitologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/parasitologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Resultado do Tratamento
13.
Oncotarget ; 8(24): 38276-38293, 2017 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-27542263

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer with rising incidence in developing countries. Unfortunately, the overall 5-year survival rate is still less than 5%. The most frequent oncogenic mutations in PDAC are loss-of function mutations in p53 and gain-of-function mutations in KRAS. Here we show that clofazimine (Lamprene), a drug already used in the clinic for autoimmune diseases and leprosy, is able to efficiently kill in vitro five different PDAC cell lines harboring p53 mutations. We provide evidence that clofazimine induces apoptosis in PDAC cells with an EC50 in the µM range via its specific inhibitory action on the potassium channel Kv1.3. Intraperitoneal injection of clofazimine resulted in its accumulation in the pancreas of mice 8 hours after administration. Using an orthotopic PDAC xenotransplantation model in SCID beige mouse, we show that clofazimine significantly and strongly reduced the primary tumor weight. Thus, our work identifies clofazimine as a promising therapeutic agent against PDAC and further highlights ion channels as possible oncological targets.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/patologia , Clofazimina/farmacologia , Canal de Potássio Kv1.3/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Canal de Potássio Kv1.3/antagonistas & inibidores , Camundongos , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Antimicrob Agents Chemother ; 60(6): 3470-9, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27021320

RESUMO

Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1ß (IL-1ß) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1ß cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan- and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.


Assuntos
Anti-Inflamatórios/farmacologia , Clofazimina/farmacologia , Inflamassomos/imunologia , Proteína Acessória do Receptor de Interleucina-1/biossíntese , Macrófagos/efeitos dos fármacos , Animais , Carragenina , Caspase 1/metabolismo , Inflamação/tratamento farmacológico , Proteína Acessória do Receptor de Interleucina-1/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Fígado/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/imunologia , Baço/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
15.
Br J Pharmacol ; 172(21): 5161-73, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26276903

RESUMO

BACKGROUND AND PURPOSE: Kv 1.3 potassium channels are promising pharmaceutical targets for treating immune diseases as they modulate Ca(2+) signalling in T cells by regulating the membrane potential and with it the driving force for Ca(2+) influx. The antimycobacterial drug clofazimine has been demonstrated to attenuate antigen-induced Ca(2+) oscillations, suppress cytokine release and prevent skin graft rejection by inhibiting Kv 1.3 channels with high potency and selectivity. EXPERIMENTAL APPROACH: We used patch-clamp methodology to investigate clofazimine's mechanism of action in Kv 1.3 channels expressed in HEK293 cells. KEY RESULTS: Clofazimine blocked Kv 1.3 channels by involving two discrete mechanisms, both of which contribute to effective suppression of channels: (i) a use-dependent open-channel block during long depolarizations, resulting in accelerated K(+) current inactivation and (ii) a block of closed deactivated channels after channels were opened by brief depolarizations. Both modes of block were use-dependent and state-dependent in that they clearly required prior channel opening. The clofazimine-sensitive closed-deactivated state of the channel was distinct from the resting closed state because channels at hyperpolarized voltages were not inhibited by clofazimine. Neither were channels in the C-type inactivated state significantly affected. Kv 1.3 channels carrying the H399T mutation and lacking C-type inactivation were insensitive to clofazimine block of the closed-deactivated state, but retained their susceptibility to open-channel block. CONCLUSIONS AND IMPLICATIONS: Given the prominent role of Kv 1.3 in shaping Ca(2+) oscillations, the use-dependent and state-dependent block of Kv 1.3 channels by clofazimine offers therapeutic potential for selective immunosuppression in the context of autoimmune diseases in which Kv 1.3-expressing T cells play a significant role.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Clofazimina/farmacologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Hansenostáticos/farmacologia , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Canal de Potássio Kv1.3/genética , Mutação , Técnicas de Patch-Clamp
16.
J Antimicrob Chemother ; 70(9): 2507-10, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26045528

RESUMO

OBJECTIVES: Although clofazimine has been traditionally used to treat leprosy, there is recent interest in using clofazimine for the treatment of MDR-TB and drug-susceptible TB. However, the mechanisms of resistance to clofazimine are poorly understood. Here, we investigated the molecular basis of clofazimine resistance using resistant mutants isolated in vitro. METHODS: We isolated 96 mutants of Mycobacterium tuberculosis resistant to clofazimine and performed WGS and Sanger sequencing to identify possible mutations associated with clofazimine resistance. RESULTS: We found that 97% (93/96) of clofazimine-resistant mutants had a mutation in rv0678 encoding a transcription repressor for efflux pump MmpL5. Two mutational hot spots at nucleotide positions 193 and 466 in rv0678 accounted for 43.8% (42/96) and 11.5% (11/96) of the mutations, respectively. The previously reported A202G mutation (S68G) in rv0678 occurred less frequently, in 5 of 96 mutants. The remaining 34 mutations were scattered along the entire rv0678 gene. We discovered two new genes (rv1979c and rv2535c) associated with clofazimine resistance in mutants without rv0678 mutations. CONCLUSIONS: Mutations in rv0678 are a major mechanism of clofazimine resistance. Our findings provide useful information for the design of new molecular tests for rapid detection of clofazimine resistance. Further studies are needed to address the role of rv1979c and rv2535c in clofazimine resistance and mechanisms of action.


Assuntos
Antituberculosos/farmacologia , Clofazimina/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mutação Puntual , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Genes Bacterianos , Genoma Bacteriano , Humanos , Análise de Sequência de DNA
17.
Cytometry A ; 87(9): 855-67, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26109497

RESUMO

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/análise , Macrófagos/química , Macrófagos/fisiologia , Xantenos/análise , Xenobióticos/análise , Animais , Linhagem Celular , Clofazimina/análise , Clofazimina/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Xenobióticos/farmacologia
18.
Mol Pharm ; 12(7): 2517-27, 2015 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-25909959

RESUMO

Clofazimine (CFZ) is an FDA-approved leprostatic and anti-inflammatory drug that massively accumulates in macrophages, forming insoluble, intracellular crystal-like drug inclusions (CLDIs) during long-term oral dosing. Interestingly, when added to cells in vitro, soluble CFZ is cytotoxic because it depolarizes mitochondria and induces apoptosis. Accordingly, we hypothesized that, in vivo, macrophages detoxify CFZ by sequestering it in CLDIs. To test this hypothesis, CLDIs of CFZ-treated mice were biochemically isolated and then incubated with macrophages in vitro. The cell biological effects of phagocytosed CLDIs were compared to those of soluble CFZ. Unlike soluble CFZ, phagocytosis of CLDIs did not lead to mitochondrial destabilization or apoptosis. Rather, CLDIs altered immune signaling response pathways downstream of Toll-like receptor (TLR) ligation, leading to enhanced interleukin-1 receptor antagonist (IL-1RA) production, dampened NF-κB activation and tissue necrosis factor alpha (TNFα) production, and ultimately decreased TLR expression levels. In aggregate, our results constitute evidence that macrophages detoxify soluble CFZ by sequestering it in a biocompatible, insoluble form. The altered cellular response to TLR ligation suggests that CLDI formation may also underlie CFZ's anti-inflammatory activity.


Assuntos
Clofazimina/farmacologia , Imunidade Inata/efeitos dos fármacos , Proteína Antagonista do Receptor de Interleucina 1/antagonistas & inibidores , Fagocitose/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/imunologia
19.
Biochem Pharmacol ; 87(4): 571-8, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24355563

RESUMO

Research on existing drugs often discovers novel mechanisms of their action and leads to the expansion of their therapeutic scope and subsequent remarketing. The Wnt signaling pathway is of the immediate therapeutic relevance, as it plays critical roles in cancer development and progression. However, drugs which disrupt this pathway are unavailable despite the high demand. Here we report an attempt to identify antagonists of the Wnt-FZD interaction among the library of the FDA-approved drugs. We performed an in silico screening which brought up several potential antagonists of the ligand-receptor interaction. 14 of these substances were tested using the TopFlash luciferase reporter assay and four of them identified as active and specific inhibitors of the Wnt3a-induced signaling. However, further analysis through GTP-binding and ß-catenin stabilization assays showed that the compounds do not target the Wnt-FZD pair, but inhibit the signaling at downstream levels. We further describe the previously unknown inhibitory activity of an anti-leprosy drug clofazimine in the Wnt pathway and provide data demonstrating its efficiency in suppressing growth of Wnt-dependent triple-negative breast cancer cells. These data provide a basis for further investigations of the efficiency of clofazimine in treatment of Wnt-dependent cancers.


Assuntos
Clofazimina/farmacologia , Inibidores do Crescimento/farmacologia , Hansenostáticos/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Proteína Wnt3A/antagonistas & inibidores , Proteína Wnt3A/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Linhagem Celular Tumoral , Clofazimina/uso terapêutico , Cristalografia por Raios X , Inibidores do Crescimento/uso terapêutico , Células HEK293 , Humanos , Hansenostáticos/uso terapêutico , Camundongos , Neoplasias de Mama Triplo Negativas/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteína Wnt3A/química
20.
Int J Tuberc Lung Dis ; 17(8): 1001-7, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23541151

RESUMO

The increased incidence of drug-resistant tuberculosis has created an urgent necessity for the development of new and effective anti-tuberculosis drugs and for alternative therapeutic regimens. Clofazimine (CFZ) is a fat-soluble riminophenazine dye used in the treatment of leprosy worldwide. CFZ has also been used as a Group 5 drug in the treatment of tuberculosis (TB). A large cohort study from Bangladesh published in 2010 described a treatment regimen for multidrug-resistant tuberculosis (MDR-TB) including CFZ as being highly effective against MDR-TB. We searched multiple databases for studies published through February 2012 that reported use of CFZ in MDR- and extensively drug-resistant TB (XDR-TB) treatment regimens. We identified nine observational studies (6 MDR-TB and 3 XDR-TB) including patients with drug-resistant TB treated with CFZ. Overall, 65% (95% confidence interval [95%CI] 54-76) of the patients experienced favorable outcomes, defined as either cure or treatment completion. Using random effects meta-analysis, 65% (95%CI 52-79) of those with MDR-TB and 66% (95%CI 42-89) of those with XDR-TB experienced favorable treatment outcomes. High-quality prospective cohort studies and clinical trials examining the effect of CFZ as part of drug-resistant TB treatment regimens are needed.


Assuntos
Clofazimina/uso terapêutico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Animais , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Clofazimina/farmacologia , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Resultado do Tratamento , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia
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