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1.
Artigo em Inglês | MEDLINE | ID: mdl-35738156

RESUMO

Albendazole (ABZ) is an anthelmintic frequently used to treat haemonchosis, a common parasitosis of ruminants caused by the gastrointestinal nematode Haemonchus contortus. This parasite is able to protect itself against ABZ via the formation of inactive ABZ-glycosides. The present study was designed to deepen the knowledge about the role of UDP-glycosyltransferases (UGTs) in ABZ glycosylation in H. contortus. The induction effect of phenobarbital, a classical inducer of UGTs, as well as ABZ and ABZ-sulphoxide (ABZSO, the main active metabolite of ABZ) on UGTs expression and UGT activity toward ABZ was studied ex vivo in isolated adult nematodes. The effect of three potential UGT inhibitors (5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine and sulfinpyrazone) on ABZ glycosylation was tested. Pre-incubation of nematodes with ABZ and ABZSO led to increased expression of several UGTs as well as ABZ-glycosides formation in subsequent treatment. Phenobarbital also induced UGTs expression, but did not affect ABZ biotransformation. In the nematode's subcellular fraction, sulfinpyrazone inhibited UGT activity toward ABZ, although no effect of other inhibitors was observed. The inhibitory potential of sulfinpyrazone on the formation of ABZ-glycosides was also proved ex vivo in living nematodes. The obtained results confirmed the role of UGTs in ABZ biotransformation in H. contortus adults and revealed sulfinpyrazone as a potent inhibitor of ABZ glycosylation in this parasite. The possible use of sulfinpyrazone with ABZ in combination therapy merits further research.


Assuntos
Anti-Helmínticos , Haemonchus , Nematoides , Doenças dos Ovinos , Albendazol , Animais , Anti-Helmínticos/uso terapêutico , Glicosídeos/metabolismo , Glicosídeos/farmacologia , Glicosídeos/uso terapêutico , Glicosiltransferases , Fenobarbital/metabolismo , Fenobarbital/farmacologia , Fenobarbital/uso terapêutico , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Sulfimpirazona/metabolismo , Sulfimpirazona/farmacologia , Sulfimpirazona/uso terapêutico , Difosfato de Uridina
2.
Molecules ; 26(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205704

RESUMO

The discovery of drugs capable of inhibiting SARS-CoV-2 is a priority for human beings due to the severity of the global health pandemic caused by COVID-19. To this end, repurposing of FDA-approved drugs such as NSAIDs against COVID-19 can provide therapeutic alternatives that could be utilized as an effective safe treatment for COVID-19. The anti-inflammatory activity of NSAIDs is also advantageous in the treatment of COVID-19, as it was found that SARS-CoV-2 is responsible for provoking inflammatory cytokine storms resulting in lung damage. In this study, 40 FDA-approved NSAIDs were evaluated through molecular docking against the main protease of SARS-CoV-2. Among the tested compounds, sulfinpyrazone 2, indomethacin 3, and auranofin 4 were proposed as potential antagonists of COVID-19 main protease. Molecular dynamics simulations were also carried out for the most promising members of the screened NSAID candidates (2, 3, and 4) to unravel the dynamic properties of NSAIDs at the target receptor. The conducted quantum mechanical study revealed that the hybrid functional B3PW91 provides a good description of the spatial parameters of auranofin 4. Interestingly, a promising structure-activity relationship (SAR) was concluded from our study that could help in the future design of potential SARS-CoV-2 main protease inhibitors with expected anti-inflammatory effects as well. NSAIDs may be used by medicinal chemists as lead compounds for the development of potent SARS-CoV-2 (Mpro) inhibitors. In addition, some NSAIDs can be selectively designated for treatment of inflammation resulting from COVID-19.


Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/uso terapêutico , Tratamento Farmacológico da COVID-19 , Reposicionamento de Medicamentos/métodos , Anti-Inflamatórios não Esteroides/metabolismo , Antivirais/química , Antivirais/farmacologia , Auranofina/química , Auranofina/farmacologia , Sítios de Ligação , COVID-19/complicações , Biologia Computacional , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/etiologia , Bases de Dados de Compostos Químicos , Humanos , Indometacina/química , Indometacina/farmacologia , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ligação Proteica , SARS-CoV-2/química , SARS-CoV-2/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfimpirazona/química , Sulfimpirazona/farmacologia , Estados Unidos , United States Food and Drug Administration
3.
PLoS Negl Trop Dis ; 13(9): e0007687, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31513587

RESUMO

Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans.


Assuntos
Brugia Malayi/efeitos dos fármacos , Brugia Malayi/enzimologia , Glucuronosiltransferase/metabolismo , Albendazol/farmacologia , Animais , Antígenos de Helmintos/sangue , Brugia Malayi/imunologia , Brugia Malayi/metabolismo , Quimioterapia Combinada , Filariose Linfática/tratamento farmacológico , Filariose Linfática/prevenção & controle , Feminino , Filaricidas/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/genética , Humanos , Imunoglobulina E/sangue , Intestinos/enzimologia , Microfilárias/efeitos dos fármacos , Movimento , Probenecid/farmacologia , RNA Interferente Pequeno , Sulfimpirazona/farmacologia
4.
Parasit Vectors ; 11(1): 593, 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30428915

RESUMO

BACKGROUND: Ixodes scapularis organic anion transporting polypeptides (OATPs) play important roles in tick-rickettsial pathogen interactions. In this report, we characterized the role of these conserved molecules in ticks infected with either Lyme disease agent Borrelia burgdorferi or tick-borne Langat virus (LGTV), a pathogen closely related to tick-borne encephalitis virus (TBEV). RESULTS: Quantitative real-time polymerase chain reaction analysis revealed no significant changes in oatps gene expression upon infection with B. burgdorferi in unfed ticks. Synchronous infection of unfed nymphal ticks with LGTV in vitro revealed no significant changes in oatps gene expression. However, expression of specific oatps was significantly downregulated upon LGTV infection of tick cells in vitro. Treatment of tick cells with OATP inhibitor significantly reduced LGTV loads, kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), levels and expression of several oatps in tick cells. Furthermore, bioinformatics characterization of OATPs from some of the medically important vectors including ticks, mosquitoes and lice revealed the presence of several glycosylation, phosphorylation and myristoylation sites. CONCLUSIONS: This study provides additional evidence on the role of arthropod OATPs in vector-intracellular pathogen interactions.


Assuntos
Vetores Aracnídeos/genética , Borrelia burgdorferi/isolamento & purificação , Vírus da Encefalite Transmitidos por Carrapatos/isolamento & purificação , Ixodes/genética , Transportadores de Ânions Orgânicos/genética , Animais , Vetores Aracnídeos/microbiologia , Vetores Aracnídeos/virologia , Borrelia burgdorferi/patogenicidade , Linhagem Celular , Biologia Computacional , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Expressão Gênica , Ixodes/química , Ixodes/microbiologia , Ixodes/virologia , Ninfa/microbiologia , Ninfa/virologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Sulfimpirazona/farmacologia , Transaminases/genética , Viroses , Xanturenatos/metabolismo
5.
Pest Manag Sci ; 73(7): 1402-1409, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27786405

RESUMO

BACKGROUND: UDP-glycosyltransferases (UGTs) are phase II detoxification enzymes widely distributed within living organisms. Their involvement in the biotransformation of various lipophilic endogenous compounds and phytoalexins in insects has been documented. However, the roles of this enzyme family in insecticide resistance have rarely been reported. Here, the functions of UGTs in chlorantraniliprole resistance in Plutella xylostella were investigated. RESULTS: Treatment with sulfinpyrazone and 5-nitrouracil (both inhibitors of UGT enzymes) significantly increased the toxicity of chlorantraniliprole against the third instar larvae of P. xylostella. Among the 23 UGT transcripts examined, only UGT2B17 was found to be over-expressed (with a range from 30.7- to 77.3-fold) in all four chlorantraniliprole-resistant populations compared to the susceptible one (CHS). The knock-down of UGT2B17 by RNA interference (RNAi) dramatically increased the toxicity of chlorantraniliprole by 27.4% and 29.8% in the CHS and CHR (resistant) populations, respectively. In contrast, exposure to phenobarbital significantly increased the relative expression of UGT2B17 while decreasing the toxicity of chlorantraniliprole to the larvae by 14.0%. CONCLUSION: UGT2B17 is involved in the detoxification of chlorantraniliprole, and its over-expression may play an important role in chlorantraniliprole resistance in P. xylostella. These results shed some light upon and further our understanding of the mechanisms of diamide insecticide resistance in insects. © 2016 Society of Chemical Industry.


Assuntos
Glicosiltransferases/genética , Resistência a Inseticidas , Mariposas/efeitos dos fármacos , ortoaminobenzoatos/toxicidade , Animais , Inibidores Enzimáticos/farmacologia , Glicosiltransferases/metabolismo , Inseticidas/toxicidade , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Mariposas/enzimologia , Mariposas/genética , Interferência de RNA , Sulfimpirazona/farmacologia , Uracila/análogos & derivados , Uracila/farmacologia
6.
Antimicrob Agents Chemother ; 58(12): 7475-83, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25288079

RESUMO

We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC50) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field.


Assuntos
Anti-Helmínticos/farmacologia , Glucuronosiltransferase/metabolismo , Haemonchus/efeitos dos fármacos , Proteínas de Helminto/metabolismo , Larva/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Fenobarbital/farmacologia , Animais , Anti-Helmínticos/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/genética , Haemonchus/enzimologia , Haemonchus/genética , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Inativação Metabólica/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Compostos Organofosforados/metabolismo , Probenecid/farmacologia , Sulfimpirazona/farmacologia , Uracila/análogos & derivados , Uracila/farmacologia
7.
Ideggyogy Sz ; 66(11-12): 407-14, 2013 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-24555241

RESUMO

GOALS: The available scientific data indicate that the pathomechanism of Parkinson's disease (PD) involves the accumulation of endogenous and exogenous toxic substances. The disruption of the proper functioning of certain transporters in the blood-brain barrier and in the blood-cerebrospinal fluid barrier in PD would accompany to that accumulation. Although there is an emerging role of the dysfunction of multidrug resistance-associated proteins (MRPs), members of ATP-b nding cassette (ABC) transporter superfamily, in neurodegenerative disorders, there is only a few available data as regards PD. So the aim of our study was the assessment of the role of certain MRPs (1 ,2, 4 and 5) in neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine METHODS: Following the intraperitoneal administration of silymarin (with MRP1, 2, 4 and 5 inhibitory effects), naringenin (with MRP1, 2 and 4 stimulatory effects), sulfinpyrazone (with MRP1, 4 and 5 inhibitory and MRP2 stimulatory effects) and allopurinol (with MRP4 stimulatory effect in doses of 100 mg/kg, 100 mg/kg, 100 mg/kg and 60 mg/kg, respectively, for one week before and after the administration of MPTP in C57B/6 mice in acute dosing regimen the striatal concentrations of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid has been measured using high-performance liquid chromatography. RESULTS: Although the results of these experiments showed that neither of these substances exerted significant influence on MPTP-induced striatal depletion of dopamine and its metabolites, naringenin exerted a slight prevention of dopamine decrease, while allopurinol considerably enhanced the MPTP-induced lethality in mice. The explanation of these findings would be that the stimulation of MRP1- and MRP2-mediated transport of glutathione conjugates of toxic substances may have slight beneficial effects, while stimulation of MRP4-mediated efflux of brain urate, which has an important antioxidant potency, may worsen the effects of oxidative stress.


Assuntos
Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Corpo Estriado/metabolismo , Dopaminérgicos/farmacologia , Dopamina/metabolismo , Ácido Homovanílico/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Alopurinol/farmacologia , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Corpo Estriado/efeitos dos fármacos , Dopaminérgicos/administração & dosagem , Esquema de Medicação , Flavanonas/farmacologia , Infusões Parenterais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Associada à Farmacorresistência Múltipla , Neurotoxinas , Estresse Oxidativo , Doença de Parkinson/etiologia , Silimarina/farmacologia , Sulfimpirazona/farmacologia , Ácido Úrico/metabolismo
8.
Assay Drug Dev Technol ; 10(6): 533-41, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22681402

RESUMO

The transient receptor potential channel subtype A member 1 (TRPA1) is a nonselective cation channel widely viewed as having therapeutic potential, particularly for pain-related indications. Realization of this potential will require potent, selective modulators; however, currently the pharmacology of TRPA1 is poorly defined. As TRPA1 is calcium permeable, calcium indicators offer a simple assay format for high-throughput screening. In this report, we show that probenecid, a uricosuric agent used experimentally in screening to increase loading of calcium-sensitive dyes, activates TRPA1. Prolonged probenecid incubation during the dye-loading process reduces agonist potency upon subsequent challenge. When Chinese Hamster Ovary (CHO)-hTRPA1 or STC-1 cells, which endogenously express TRPA1, were dye loaded in the presence of 2 mM probenecid TRPA1, agonists appeared less potent; EC(50) for allyl isothiocyante agonists in CHO-hTRPA1 was increased from 1.5±0.19 to 7.32±1.20 µM (P<0.01). No significant effect on antagonist potency was observed when using the agonist EC(80) concentration determined under the appropriate dye-loading conditions. We suggest an alternative protocol for calcium imaging using another blocker of anion transport, sulfinpyrazone. This blocker significantly augments indicator dye loading and the screening window, but is not a TRPA1 agonist and has no effect on agonist potency.


Assuntos
Canais Iônicos/efeitos dos fármacos , Proteínas do Tecido Nervoso/agonistas , Probenecid/farmacologia , Fármacos Renais/farmacologia , Canais de Potencial de Receptor Transitório/agonistas , Animais , Células CHO , Canais de Cálcio , Corantes , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Técnicas de Diluição do Indicador , Técnicas de Patch-Clamp , Sulfimpirazona/farmacologia , Canal de Cátion TRPA1
9.
Nephrology (Carlton) ; 16(2): 156-62, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21272127

RESUMO

AIM: Hyperuricaemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. The kidney plays a dominant role in maintaining plasma urate levels through the excretion process. Human renal urate transporter URAT1 is thought to be an essential molecule that mediates the reabsorption of urate on the apical side of the proximal tubule. In this study the pharmacological characteristics and clinical implications of URAT1 were elucidated. METHODS: Madin-Darby canine kidney (MDCK) cells stably expressing URAT1 (MDCK-URAT1) were established and examined the interactions of URAT1 with various drugs such as benzbromarone and its metabolites including 6-hydroxybenzbromarone, angiotensin-converting enzyme inhibitors, non-steroidal anti-inflammatory drugs and urate transport inhibitors including E3040 and probenecid. RESULTS: MDCK-URAT1 cells exhibited a time- and dose-dependent increase in urate uptake, with a Km value of 570.7 µmol/L. When an URAT1-green fluorescent protein fusion protein construct was expressed in MDCK cells, the protein was sorted mainly to the apical side of the membrane. The drugs except for captoril dose-dependently inhibited urate uptake mediated by URAT1, with half maximal inhibitory concentration (IC(50) ) values ranging 0.05-716 µmol/L. CONCLUSION: Comparing these IC(50) values with intratubular concentrations of unbound drugs in humans, it is thought that URAT1 is a target molecule of uricosuric drugs, including 6-hydroxybenzbromarone, probenecid, indomethacin and salicylate, to inhibit urate reabsorption in vivo. In addition, a cell line that stably expressing URAT1 could be a useful tool for the development of uricosuric drugs.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ácido Úrico/metabolismo , Uricosúricos/farmacologia , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Benzobromarona/análogos & derivados , Benzobromarona/farmacologia , Benzotiazóis/farmacologia , Transporte Biológico/efeitos dos fármacos , Captopril/farmacologia , Células Cultivadas , Cães , Enalapril/farmacologia , Indometacina/farmacologia , Concentração Inibidora 50 , Fenilbutazona/farmacologia , Probenecid/farmacologia , Piridinas/farmacologia , Salicilatos/farmacologia , Sulfimpirazona/farmacologia , Uricosúricos/administração & dosagem
10.
Mol Cancer Ther ; 7(5): 1150-5, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18445659

RESUMO

We used the paclitaxel-resistant human small cell lung cancer subline PC-6/TAX1-1, selected from PC-6 cells by paclitaxel, to test whether MRP7/ABCC10 (ABCC10) confers paclitaxel resistance. We found that gene expression of both ABCB1/MDR1 (ABCB1) and ABCC10 was higher in PC-6/TAX1-1 cells than in PC-6 cells. The expression levels of ABCC10 showed a significant inverse correlation with paclitaxel sensitivity (r = 0.574; P < 0.05) in 17 non-small cell lung cancer (NSCLC) cells unlike the expression levels of ABCB1. Pretreatment with the ABCC10 inhibitor sulfinpyrazone altered the sensitivity to paclitaxel in ABCC10-expressing NSCLC cells, concomitant with increased intracellular paclitaxel accumulation. These findings suggest that expression of the ABCC10 gene is induced by paclitaxel and that ABCC10 confers paclitaxel resistance by enhancing the efflux for paclitaxel. To confirm this hypothesis, we tested the effect on paclitaxel cytotoxicity of decreasing the expression of ABCC10 by small interfering RNA and found that this enhanced paclitaxel cytotoxicity in NCI-H23 cells concomitant with increased intracellular paclitaxel accumulation. These data indicate that ABCC10 may be one of the biomarkers for paclitaxel resistance in NSCLC.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Paclitaxel/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Paclitaxel/uso terapêutico , RNA Interferente Pequeno/metabolismo , Sulfimpirazona/farmacologia , Células Tumorais Cultivadas
11.
Am J Trop Med Hyg ; 77(2): 221-7, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17690390

RESUMO

In vitro pyrimethamine response of Plasmodium falciparum isolates and dihydrofolate reductase (dhfr) gene sequences were analyzed in 2004-2005 and compared with our previous data. Most isolates (n = 103, all dhfr mutants) had 50% inhibitory concentrations (IC(50)s) > or = 119 nM, and six isolates had low IC(50)s (five wild-type or mixed dhfr, 0.04-1.37 nM; one triple mutant, 6.4 nM). Of 194 isolates, only 7 had the wild-type dhfr and 187 were mutants. The results of the two methods were highly concordant and indicated a significant increase (P < 0.05) in the prevalence of mutant, pyrimethamine-resistant P. falciparum between 1994 and 2005. The addition of probenecid or sulfinpyrazone to pyrimethamine resulted in a slight-to-moderate decrease in the level of in vitro pyrimethamine resistance without rendering the parasites susceptible to pyrimethamine. Analysis of molecular markers may be useful for the long-term surveillance of antifolate-resistant malaria.


Assuntos
Antimaláricos/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Pirimetamina/farmacologia , Adolescente , Animais , Camarões/epidemiologia , DNA de Protozoário/química , DNA de Protozoário/genética , Resistência a Medicamentos , Quimioterapia Combinada , Humanos , Malária Falciparum/sangue , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Epidemiologia Molecular , Mutação , Testes de Sensibilidade Parasitária , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Mutação Puntual , Reação em Cadeia da Polimerase , Probenecid/farmacologia , Análise de Sequência de DNA , Sulfimpirazona/farmacologia , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
12.
Drug Metab Dispos ; 35(6): 981-6, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17325024

RESUMO

In allopurinol-allergic patients, uricosuric agents are often used in the treatment of hyperuricemia. The existing uricosuric agents are not without problems and the availability of better and safer alternatives is highly desirable. Our previous study (J Pharmacol Exp Ther (2006) 316:169-175) has demonstrated that morin (3,5,7,2',4'-pentahydroxyflavone), which occurs in the twigs of Morus alba L. documented in traditional Chinese medicinal literature for treatment of conditions akin to gout, is a potent inhibitor of urate uptake in rat renal brush-border membrane vesicles. It is also effective in lowering uric acid level in a hyperuricemic rat model in vivo. Whether morin is an equally effective uricosuric agent in human requires verification. The human urate anion transporter (hURAT1) has recently been cloned and identified to be the organic anion transporter that mediates renal urate reabsorption in the human kidney. In the present investigation, human embryonic kidney cells were transfected with hURAT1 and the expression was validated by reverse transcription-polymerase chain reaction and subcellular distribution of the exogenously introduced transporter by confocal microscopy. The inhibitory actions of morin on human renal urate reabsorption were demonstrated using this system. The IC50 value of the inhibition by morin was determined to be 2.0 microM, compared with 50 microM for probenecid, 100 microM for sulfinpyrazone, and 0.3 microM for benzbromarone. Kinetic analysis of the uptake inhibition by morin indicates that this compound is a competitive inhibitor of urate uptake on the human urate transporter with a K(i) value of 5.74 microM.


Assuntos
Flavonoides/farmacologia , Rim/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Ácido Úrico/metabolismo , Benzobromarona/farmacologia , Linhagem Celular , Humanos , Transportadores de Ânions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Probenecid/farmacologia , Sulfimpirazona/farmacologia , Transfecção , Uricosúricos/farmacologia
13.
Eur J Pharmacol ; 560(2-3): 127-31, 2007 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-17320853

RESUMO

The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that belongs to the same family as multidrug resistance-associated proteins whose main function is to expel xenobiotics and physiological organic anions from the cell interior. Despite the overall structural similarity with these membrane proteins, CFTR is not an active transporter but is instead a Cl- channel. We have tested the ability of known inhibitors of multidrug resistance-associated proteins to affect CFTR Cl- currents. We have found that sulfinpyrazone, probenecid, and benzbromarone are also inhibitors of CFTR activity, with a mechanism involving blockage of the channel pore.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Canais de Cloreto/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Animais , Benzobromarona/farmacologia , Células Cultivadas , Probenecid/farmacologia , Ratos , Ratos Endogâmicos F344 , Sulfimpirazona/farmacologia
14.
J Biomol Screen ; 12(2): 248-54, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17259590

RESUMO

Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.


Assuntos
Bioensaio/métodos , Criopreservação/métodos , Preparações Farmacêuticas/metabolismo , Receptores de Esteroides/metabolismo , Ativação Transcricional , Células CACO-2 , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Neoplasias Hepáticas/patologia , Mifepristona/metabolismo , Mifepristona/farmacologia , Receptor de Pregnano X , Receptores de Esteroides/agonistas , Receptores de Esteroides/antagonistas & inibidores , Reprodutibilidade dos Testes , Rifampina/metabolismo , Rifampina/farmacologia , Sulfimpirazona/metabolismo , Sulfimpirazona/farmacologia , Transfecção
15.
Drug Metab Dispos ; 34(10): 1742-8, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16837568

RESUMO

Cytochrome P4503A4 (CYP3A4) is the principal drug-metabolizing enzyme in human liver. Drug-drug interactions (DDIs) caused by induction of CYP3A4 can result in decreased exposure to coadministered drugs, with potential loss of efficacy. Immortalized hepatocytes (Fa2N-4 cells) have been proposed as a tool to identify CYP3A4 inducers. The purpose of the current studies was to characterize the effect of known inducers on CYP3A4 in Fa2N-4 cells, and to determine whether these in vitro data could reliably project the magnitude of DDIs caused by induction. Twenty-four compounds were chosen for these studies, based on previously published data using primary human hepatocytes. Eighteen compounds had been shown to be positive for induction, and six compounds had been shown to be negative for induction. In Fa2N-4 cells, all 18 positive controls produced greater than 2-fold maximal CYP3A4 induction, and all 6 negative controls produced less than 1.5-fold maximal CYP3A4 induction. Subsequent studies were conducted to determine the relationship between in vitro induction data and in vivo induction response. The approach was to relate in vitro induction data (E(max) and EC(50) values) with efficacious free plasma concentrations to calculate a relative induction score. This score was then correlated with decreases in area under the plasma concentration versus time curve values for coadministered CYP3A4 object drugs (midazolam or ethinylestradiol) from previously published clinical DDI studies. Excellent correlations (r(2) values >0.92) were obtained, suggesting that Fa2N-4 cells can be used for identification of inducers as well as prediction of the magnitude of clinical DDIs.


Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Interações Medicamentosas , Hepatócitos/efeitos dos fármacos , Algoritmos , Carbamazepina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP3A , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Nifedipino/farmacologia , Fenobarbital/farmacologia , Fenitoína/farmacologia , Pioglitazona , Reprodutibilidade dos Testes , Rifampina/farmacologia , Rosiglitazona , Sulfimpirazona/farmacologia , Tiazolidinedionas/farmacologia
16.
Free Radic Biol Med ; 39(11): 1449-59, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16274880

RESUMO

To test whether ascorbic acid might be involved in the antioxidant defenses of inflammatory cells, we studied ascorbate uptake and recycling by quiescent and lipopolysaccharide-activated RAW264.7 murine macrophages. These cells concentrated ascorbate 100-fold in overnight culture, achieving steady-state concentrations of more than 10 mM at extracellular concentrations of 20-100 muM. This steep gradient was generated by high-affinity sodium-dependent ascorbate transport. The latter likely reflects function of the SVCT2 (SLC23A2), since this protein was detected on immunoblots. Dehydroascorbate, the two-electron oxidized form of ascorbate, was also taken up and reduced to ascorbate by the cells. Dehydroascorbate reduction required rapid recycling of GSH from GSSG by glutathione reductase. Activation of ascorbate-containing macrophages with lipopolysaccharide transiently depleted intracellular ascorbate without affecting GSH. Recovery of intracellular ascorbate required function of the SVCT2 transporter, the activity of which was modestly enhanced by lipopolysaccharide. Lipopolysaccharide treatment nearly doubled intracellular GSH concentrations over 2 h. Despite lipopolysaccharide-induced oxidant stress, this GSH increase was associated with a comparable increase in reduction of dehydroascorbate to ascorbate. These results show that macrophages maintain millimolar concentrations of ascorbate through function of the SVCT2 and that activated cells have an enhanced ability to transport and recycle ascorbate, possibly reflecting its role as an intracellular antioxidant.


Assuntos
Ácido Ascórbico/farmacocinética , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Sulfimpirazona/farmacologia , Animais , Ácido Ascórbico/metabolismo , Ácido Desidroascórbico/metabolismo , Glutationa/metabolismo , Glutationa Redutase/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Camundongos
17.
Biochim Biophys Acta ; 1712(2): 212-21, 2005 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-15921655

RESUMO

Collagen II is the major protein component of articular cartilage and forms the collagen fibril network, which provides the tensile strength of cartilage. Collagen II synthesis is enhanced by ascorbic acid (vitamin C) at both a transcriptional and post-transcriptional level. While the importance of ascorbic acid in the synthesis of collagen has been established, the mechanism by which this essential nutrient is transported into chondrocytes has not been investigated previously. We have characterized the transport of the reduced form of ascorbic acid in passaged primary human chondrocytes to discern the physiologically relevant pathways of ascorbic acid transport in cartilage. We have found that chondrocytes are robust concentrators of ascorbic acid, capable of transporting the reduced form, and concentrating total ascorbic acid, in the reduced form and its metabolites, 960-fold over the concentration in the extracellular milieu. Chondrocyte transport of ascorbic acid was sodium and temperature dependent, stereoselective for the L-forms, and inhibited by the anion transport inhibitor, sulfinpyrazone. Chondrocytes preferentially expressed the full-length and functional isoform of sodium-dependent vitamin C transporter 2 (SVCT2). When this transcript was suppressed with sequence-specific siRNAs, the active transport component of ascorbic acid was abolished. Thus, we provide the first evidence that SVCT2 mediates the secondary active and concentrative transport of ascorbic acid in human chondrocytes.


Assuntos
Ácido Ascórbico/química , Condrócitos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/fisiologia , Simportadores/fisiologia , Aminoácidos/química , Ácido Ascórbico/metabolismo , Cartilagem/metabolismo , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Ácido Desidroascórbico/metabolismo , Glucose/metabolismo , Humanos , Cinética , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , RNA/química , RNA/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/química , Transportadores de Sódio Acoplados à Vitamina C , Sulfimpirazona/farmacologia , Simportadores/química , Temperatura , Fatores de Tempo
18.
Mol Cell Biochem ; 271(1-2): 43-9, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15881654

RESUMO

Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When 14C-ascorbate uptake in endothelial cells was examined as 14C or by HPLC, the two values did not differ from each other. 14C-ascorbate uptake was Na(+)-dependent, stereoselective for L-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: Km = 27 +/- 3 microM (Hill coefficient = 1) for ascorbate and Km = 73 +/- 14 mM (Hill coefficient = 2) for Na+. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect.


Assuntos
Ácido Ascórbico/farmacocinética , Vasos Coronários/citologia , Vasos Coronários/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Vasos Coronários/efeitos dos fármacos , Ácido Desidroascórbico/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Dados de Sequência Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Sódio/metabolismo , Transportadores de Sódio Acoplados à Vitamina C , Sulfimpirazona/farmacologia , Suínos , Simportadores/genética , Simportadores/metabolismo , Fatores de Tempo
19.
J Am Soc Nephrol ; 15(11): 2828-35, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15504935

RESUMO

p-Aminohippurate (PAH) is the classical substrate used in the characterization of organic anion transport in renal proximal tubular cells. Although basolateral transporters for PAH uptake from blood into the cell have been well characterized, there is still little knowledge on the apical urinary efflux transporters. The multidrug resistance protein 2 (MRP2/ABCC2) is localized to the apical membrane and mediates ATP-dependent PAH transport, but its contribution to urinary PAH excretion is not known. In this report, we show that renal excretion of PAH in isolated perfused kidneys from wild-type and Mrp2-deficient (TR(-)) rats is not significantly different. Uptake of [(14)C]PAH in membrane vesicles expressing two different MRP2 clones isolated from Sf9 and MDCKII cells exhibited a low affinity for PAH (Sf9, 5 +/- 2 mM; MDCKII, 2.1 +/- 0.6 mM). Human MRP4 (ABCC4), which has recently been localized to the apical membrane, expressed in Sf9 cells had a much higher affinity for PAH (K(m) = 160 +/- 50 microM). Various inhibitors of MRP2-mediated PAH transport also inhibited MRP4. Probenecid stimulated MRP2 at low concentrations but had no effect on MRP4; but at high probenecid concentrations, both MRP2 and MRP4 were inhibited. Sulfinpyrazone only stimulated MRP2, but inhibited MRP4. Real-time PCR and Western blot analysis showed that renal cortical expression of MRP4 is approximately fivefold higher as compared with MRP2. MRP4 is a novel PAH transporter that has higher affinity for PAH and is expressed more highly in kidney than MRP2, and may therefore be more important in renal PAH excretion.


Assuntos
Proteínas de Transporte/metabolismo , Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Ácido p-Aminoipúrico/urina , Animais , Ligação Competitiva , Western Blotting , Linhagem Celular , Sistemas Computacionais , Cães , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Insetos , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/deficiência , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Concentração Osmolar , Reação em Cadeia da Polimerase , Probenecid/administração & dosagem , Probenecid/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Mutantes , Ratos Wistar , Sulfimpirazona/farmacologia
20.
Eur J Clin Pharmacol ; 60(6): 407-12, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15197517

RESUMO

PURPOSE: The objective of the study was to determine the effect of a potent and selective CYP2C9 inhibitor, sulfinpyrazone (Anturane), on the pharmacokinetics of nateglinide (Starlix), a novel antidiabetic drug which is primarily (approximately 70%) metabolized via CYP2C9. METHODS: This was a randomized, open-label, two-period, crossover study in 18 healthy volunteers. Nateglinide was administered as a single 120-mg oral dose alone (reference) on day 1 or in combination with sulfinpyrazone (test) on day 7, following twice-daily 200-mg oral doses (i.e., 400 mg/day) of sulfinpyrazone for 7 days. Pharmacokinetic parameters of nateglinide were determined following the administration of nateglinide alone, and when administered in combination with sulfinpyrazone. Plasma nateglinide concentrations were determined using a validated high-performance liquid chromatography method. RESULTS: The administration of nateglinide in combination with sulfinpyrazone resulted in approximately 28% higher mean AUC of nateglinide (90% CI for test-reference ratio: 1.20-1.39) with no differences in mean peak plasma concentration (Cmax; 90% CI test-reference ratio: 0.86-1.12) compared with nateglinide-alone treatment. The time to reach Cmax (tmax) and the elimination half-life of nateglinide were similar between the two treatments. Both treatments were safe and well tolerated. CONCLUSIONS: Sulfinpyrazone increased the mean exposure of nateglinide by 28% when both drugs were administered in combination. Nateglinide, given as a single dose or co-administered with multiple doses of sulfinpyrazone, was safe and well tolerated in healthy subjects.


Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Cicloexanos/farmacocinética , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacocinética , Fenilalanina/análogos & derivados , Fenilalanina/farmacocinética , Sulfimpirazona/farmacologia , Adolescente , Adulto , Área Sob a Curva , Estudos Cross-Over , Citocromo P-450 CYP2C9 , Inibidores Enzimáticos/efeitos adversos , Feminino , Meia-Vida , Humanos , Masculino , Pessoa de Meia-Idade , Nateglinida , Sulfimpirazona/efeitos adversos
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