RESUMO
A new class of highly fluorescent (phi(F) 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1'-carbonyldiimidazole to form an N,N'-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced at C(8) by reaction with concd H(2)SO(4). The resulting sulfonated N,N'-carbonyl-bridged dipyrrinones ("sulfoglows") are isolated as their sodium salts. When the alkyl substituents of the lactam ring are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely, blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2 (ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination is impaired by cholestatic liver disease.
Assuntos
Colchicina/análise , Corantes Fluorescentes/síntese química , Hepatopatias/diagnóstico , Fígado/metabolismo , Pirimidinonas/síntese química , Pirimidinonas/farmacocinética , Pirróis/síntese química , Pirróis/farmacocinética , Ácidos Sulfônicos/síntese química , Ácidos Sulfônicos/farmacocinética , Animais , Bile/química , Bile/metabolismo , Bilirrubina/análogos & derivados , Bilirrubina/química , Colchicina/química , Avaliação Pré-Clínica de Medicamentos , Estudos de Viabilidade , Corantes Fluorescentes/química , Fígado/efeitos dos fármacos , Hepatopatias/genética , Hepatopatias/metabolismo , Masculino , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Pirimidinonas/análise , Pirróis/análise , Pirróis/química , Ratos , Ratos Gunn , Ratos Sprague-Dawley , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Espectrometria de Fluorescência , Ácidos Sulfônicos/análise , Urina/químicaRESUMO
Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <<1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3 x 10(12) viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIalpha and XIIIalpha in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6 x 10(11) viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity.
Assuntos
Adenoviridae , Terapia Genética , Vetores Genéticos , Glucuronosiltransferase/genética , Hiperbilirrubinemia/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Bilirrubina/sangue , DNA/farmacologia , Vetores Genéticos/toxicidade , Glucuronosiltransferase/metabolismo , Humanos , RNA Mensageiro/metabolismo , Ratos , Ratos GunnRESUMO
The biliary excretion of the sodium salts of 8-(2-ethanesulfonic acid)-3-ethyl-2,7,9-trimethyl-1,10-dihydro-11H-dipyrrin-1-one (xanthosulfonic acid) and a fluorescent analogue (8-desethyl-N,N'-carbonyl-kryptopyrromethenone-8-sulfonic acid) was compared in Mrp2-deficient (TR(-)) and normal rats. Both organic anions were excreted rapidly in bile in Mrp2-deficient rats, but the biliary excretion of the fluorescent sulfonate was impaired relative to normal controls. The rat clearly has efficient Mrp2-independent mechanisms for biliary efflux of these anions that are not used by bilirubin or its mono- and diglucuronides.
Assuntos
Alcanossulfonatos/farmacocinética , Sistema Biliar/metabolismo , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Alcanossulfonatos/administração & dosagem , Alcanossulfonatos/metabolismo , Animais , Ácidos e Sais Biliares , Cromatografia Líquida de Alta Pressão , Corantes Fluorescentes/administração & dosagem , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Ratos , Ratos Mutantes , Ratos Sprague-DawleyRESUMO
Multidrug resistance protein 2 (Mrp2) is considered the major mammalian membrane transporter of non-bile salt organic anions from liver to bile. Using Mrp2-deficient rats, we show that the protein is not essential for biliary excretion of biliverdin, its IIIalpha and XIIIalpha isomers, mesobiliverdin XIIIalpha or biliverdins bearing bulky lipophilic groups that are not reduced by biliverdin reductase in vivo. Yet, Mrp2 deficiency does retard the biliary excretion of these verdins to different degrees. The data indicate that there are Mrp2-independent mechanisms in the rat for biliary excretion of dicarboxylate organic anions related to biliverdin.