RESUMO
An analytical method based on capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICP-MS) detection was developed for studies on the interaction of gold-containing drugs and plasma proteins using auranofin as example. A detection limit of 18 ng/mL of auranofin corresponding to 5.2 ng/mL Au and a precision of 1.5 % were obtained. Kinetic studies of the interaction between auranofin and protein were performed by incubation in aqueous solutions as well as 20 % human plasma at 37 °C. The reaction of auranofin with human serum albumin (HSA) and plasma proceeded fast; 50 % of un-bound auranofin disappeared within 2 and 3 min, respectively. By blocking the free cysteine (Cys-34) by iodoacetamide on HSA, it was shown that Cys-34 was the main reaction site for auranofin. By selective labeling of HSA present in 20 % human plasma with iophenoxate, it was demonstrated that HSA was the major auranofin-interacting protein in plasma. The CE-ICP-MS method is proposed as a novel approach for kinetic studies of the interactions between gold-based drugs and plasma proteins. Graphical Abstract Development of a CE-ICP-MS based method allows for studies on interaction of the gold containing drug auranofin with plasma proteins.
Assuntos
Antirreumáticos/sangue , Auranofina/sangue , Eletroforese Capilar/métodos , Ouro/química , Albumina Sérica/química , Espectrofotometria Atômica/métodos , Antirreumáticos/química , Auranofina/química , Cisteína/química , Humanos , Iodoacetamida/química , Ácido Iopanoico/química , Cinética , Limite de Detecção , Albumina Sérica/antagonistas & inibidores , Albumina Sérica/metabolismo , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: Deiodinases (DIO1, 2, and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation, and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation (e.g., the low triiodothyronine syndrome in critical illness). Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil is in use for treatment of severe hyperthyroidism. Other well-known inhibitors (e.g., iopanoic acid or aurothioglucose) are nonselective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening is needed. We recently described a nonradioactive screening assay, utilizing the classic Sandell-Kolthoff reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine DIO1 activity testing. Here, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2, and DIO3. METHODS: As proof-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various nonradioactive substrates and confirmed by liquid chromatography-tandem mass spectrometry. RESULTS: The contrast agent and known DIO inhibitor iopanoic acid was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor, while xanthohumol turned out to potently block the activity of all three isoenzymes. CONCLUSIONS: A rapid nonradioactive screening method based on the Sandell-Kolthoff reaction is suitable for identification of environmental, nutritive and pharmacological compounds modulating activities of human deiodinase enzymes.
Assuntos
Flavonoides/uso terapêutico , Genisteína/uso terapêutico , Iodeto Peroxidase/antagonistas & inibidores , Propiofenonas/uso terapêutico , Animais , Catálise , Diferenciação Celular , Cromatografia Líquida , Proteínas de Ligação a DNA/química , Avaliação Pré-Clínica de Medicamentos , Enzimas/química , Células HEK293 , Humanos , Concentração Inibidora 50 , Iodeto Peroxidase/química , Ácido Iopanoico/química , Isoenzimas/química , Espectrometria de Massas , Camundongos , Fases de Leitura Aberta , Proteínas Recombinantes/química , Hormônios Tireóideos/química , Iodotironina Desiodinase Tipo IIRESUMO
Human serum albumin (HSA) is the most abundant protein in the human plasma. HSA has several physiological roles in the human body, including storage and transport. Owing to the predominance of albumin in plasma, HSA is often involved in the protein binding of drugs. The aim of this work was to develop a selective, quantitative method for determining albumin in plasma with the purpose of clarifying the fate of metal-based drugs in biological systems. The method can also be applied for determination of urine albumin, which is of relevance in diagnostics of kidney disease. A selective method for quantification of HSA based on labelling the protein with iophenoxic acid (IPA) was developed. Samples were subjected to size exclusion chromatography (SEC) and detection by inductively coupled plasma mass spectrometry (ICP-MS) monitoring iodine and platinum. The iodine signal for the HSA-IPA complex showed linearity in the range 1 to 250 mg L(-1). The precision was 3.7% and the accuracy 100.7% determined by analysis of a certified HSA reference material. The limit of detection (LOD) and limit of quantification (LOQ) were 0.23 and 9.79 mg L(-1), respectively. The method was applied for analysis of HSA in human plasma and urine samples and for studying the binding of cisplatin to proteins in the human plasma.
Assuntos
Cromatografia em Gel/métodos , Ácido Iopanoico/química , Espectrometria de Massas/métodos , Albumina Sérica/análise , Albuminúria/urina , Cisplatino/metabolismo , Humanos , Limite de Detecção , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Albumina Sérica/química , Albumina Sérica/metabolismoRESUMO
Simultaneous specific and nonspecific binding of bendroflumethiazide (BFZ) to human serum albumin (HSA) and concentration profile of BFZ in HSA buffer (pH 7.40) solution were investigated by ¹9F nuclear magnetic resonance (NMR) method. The ¹9F NMR spectrum of BFZ (200 µM) in a buffer solution showed a sharp signal of its CF3 group at 17.8 ppm from the reference trifluoroethanol. Addition of 0.60mM HSA to the sample solution caused the CF(3) signal splitting into three broadened peaks at 18.4 (A), 17.9 (B) and 17.4 ppm (C). By its chemical shift and spectral behavior, B was assigned to unbound BFZ. Competition experiments with Site I and II ligands lead to C being assigned to Site II bound BFZ. However, the peak intensity (areas) of A was not reduced by these ligands, suggesting that A arises from nonspecific binding. Using the peak intensities at several total concentrations of BFZ, Scatchard plot was performed. The plot for A provided a straight line parallel to the x-axis confirming nonspecific binding and that for C was consistent with specific binding. The binding constants for nonspecific and specific Site II binding were 1.02 and 1.00 × 104 (M⻹) (n=1.1), respectively. The presence of 0.10 M Clâ» in the sample solution affected the binding constant of Site II binding, but not that of nonspecific binding. The concentration profile of BFZ calculated using the binding constants revealed that nonspecific binding is more effective than Site II binding for the binding of BFZ to HSA. It was also confirmed that considerable amounts of BFZ liberated from Site II by the Site II ligands or Clâ» ions bind again nonspecifically.
Assuntos
Anti-Hipertensivos/metabolismo , Bendroflumetiazida/metabolismo , Diuréticos/metabolismo , Albumina Sérica/metabolismo , Anti-Hipertensivos/química , Bendroflumetiazida/química , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Diazepam/química , Diazepam/metabolismo , Diuréticos/química , Humanos , Ácido Iopanoico/análogos & derivados , Ácido Iopanoico/química , Ácido Iopanoico/metabolismo , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Concentração Osmolar , Fenilbutazona/química , Fenilbutazona/metabolismo , Ligação Proteica , Albumina Sérica/química , Albumina Sérica Humana , Triptofano/química , Triptofano/metabolismoRESUMO
A method is described for screening compounds that inhibit crystallization in solution to enable more accurate measurement of amorphous drug solubility. Three polymers [polyvinylpyrrolidone, hydroxypropyl methylcellulose, and hydroxypropyl methylcellulose acetate succinate (HPMCAS)] were screened for their ability to inhibit the crystallization of neat amorphous drugs during measurement of solubility of the amorphous form in water. Among the polymers evaluated, HPMCAS was found to be most promising. The use of HPMCAS provided an "apparent solubility" of amorphous drugs that was closer to the theoretically calculated values. With danazol, agreement was essentially quantitative, and for griseofulvin and iopanoic acid, agreement was within a factor of two; these maximum concentrations were sustained for a period of 40-90 min. Dynamic light scattering of filtered samples (0.22 µ) revealed the presence of colloidal drug-polymer assemblies in solution (100-150 nm). The supernatant resulting from this centrifugation gradually decreased in concentration, but remained supersaturated with respect to crystalline drug for several hours. Thus, HPMCAS has been shown to be a useful additive in dissolution media to allow a more accurate determination of the solubility of fast crystallizing neat amorphous drugs, at least for the drugs studied, and it should also serve to retard crystallization in vivo and therefore, facilitate improved bioavailability.
Assuntos
Metilcelulose/análogos & derivados , Preparações Farmacêuticas/química , Química Farmacêutica , Coloides , Cristalização , Danazol/química , Griseofulvina/química , Derivados da Hipromelose/química , Ácido Iopanoico/química , Cinética , Luz , Metilcelulose/química , Povidona/química , Espalhamento de Radiação , Solubilidade , Tecnologia Farmacêutica/métodos , UltracentrifugaçãoRESUMO
BACKGROUND: Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. RESULTS: We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which--in drugs sites 1 and 2 on the protein--are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. CONCLUSIONS: The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin.
Assuntos
Ácido Iopanoico/análogos & derivados , Albumina Sérica/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Ácido Iopanoico/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Albumina Sérica/genética , Albumina Sérica/metabolismoRESUMO
The binding of two cholecystographic agents to human serum albumin (HSA) was evaluated by means of two different complementary methodologies. In particular, the inhibition of drug HSA binding caused by iopanoic- and iophenoxic-acid was investigated by circular dichroism (CD) and resonant mirror (RM) optical biosensor techniques. The CD study allowed to obtain information both on the cholecystographic agent binding site and on the effect of the binding on the protein conformation. Iopanoic acid (IOP), a drug potentially useful for thyrotoxic disorders, resulted a direct competitor for ligands that selectively bind to site II, in agreement to literature data. No definite evidence was obtained for the highest affinity binding site of iophenoxic acid (IOPH), however, this diagnostic tool markedly affected the binding of ligands to the most characterized high affinity sites on HSA, namely sites I, II and III. Binding parameters were obtained by optical biosensor analysis: K(D) values were 3.6 x 10(-7) and 2.8 x 10(-8) M for IOP and IOPH, respectively.
Assuntos
Meios de Contraste/química , Ácido Iopanoico/análogos & derivados , Ácido Iopanoico/química , Albumina Sérica/química , Sítios de Ligação , Técnicas Biossensoriais , Colecistografia , Dicroísmo Circular , Humanos , Ligação ProteicaRESUMO
RATIONALE AND OBJECTIVES: To assess a surface-modified emulsion as a percutaneous CT lymphographic agent in normal dogs. METHODS: An iodinated chylomicron remnant-like microemulsion was formulated with a mean particle size of 91.3 nm and an iodine concentration of 91 mg I/mL. Contrast material (2 mL) was injected into the subcutaneous tissues of the metatarsus and metacarpus of six normal dogs to enhance popliteal and cervical lymph nodes, respectively. CT images were acquired at 0, 15, 30, 45, 60, 240, 480, and 1440 minutes. RESULTS: Significant lymph node enhancement occurred in as little as 15 minutes after injection and persisted at least 8 hours. Node opacification was most pronounced at 1 to 4 hours postinjection and exceeded 200 HU in some nodes (precontrast attenuation = 45 HU). Marked enhancement of popliteal efferent lymphatics and of iliac and sacral node groups also occurred indicating distribution to second order nodes. Attenuation of enhanced nodes reverted to precontrast levels by 24 hours. CONCLUSION: The new surface-modified, chylomicron remnant-like emulsion provided marked, selective enhancement of targeted lymph nodes after subcutaneous administration. Moreover, the formulation produced significant opacification of more distant node groups from a single injection.
Assuntos
Meios de Contraste/química , Iodo , Ácido Iopanoico/química , Linfonodos/diagnóstico por imagem , Linfografia/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Meios de Contraste/farmacocinética , Cães , Emulsões , Iodo/farmacocinética , Ácido Iopanoico/análogos & derivados , Ácido Iopanoico/farmacocinética , Lipídeos/farmacocinética , Linfografia/instrumentação , Microscopia Eletrônica , Tamanho da Partícula , Ratos , Tomografia Computadorizada por Raios X/instrumentaçãoRESUMO
Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed.
Assuntos
Ácido Iopanoico/análogos & derivados , Oxigenases de Função Mista/química , Alanina/química , Varredura Diferencial de Calorimetria , Cisteína/química , Ácido Ditionitrobenzoico/química , Estabilidade Enzimática , Flavina-Adenina Dinucleotídeo/química , Temperatura Alta , Ácido Iopanoico/química , Cinética , Oxigenases de Função Mista/genética , Modelos Químicos , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
Automatic titrimetric methods with catalytic, spectrophotometric and potentiometric monitoring of the course of titration were applied for determination of iodide content in iopanoic acid, 2-(3-amino-2,4,6-tri-iodobenzyl)butyric acid, as well as in the mixture of sodium amidotrizoate (sodium 3,5-diacetamido-2,4,6-tri-iodobenzoate), and meglumine amidotrizoate (N-methylglucamine salt of 3,5-diacetamido-2,4,6-tri-iodobenzoic acid), in some pharmaceutical contrasting preparations. After combustion of the sample in an oxygen atmosphere and absorption of products in hydrazine sulphate solution, the iodide present was titrated with a standard solution of silver nitrate. In catalytic titrations, the system persulphate-sulphanilic acid (acetate buffer pH 4.35) in the presence of 2,2'-bipyridine as an activator served as the indicator reaction. The results obtained were compared with those of the official methods. Amounts of iodide of about 1.2 mg per aliquot of analyte were determined with a standard deviation less than 1.9%.
Assuntos
Meios de Contraste/química , Iodetos/análise , Ácido Iopanoico/química , Mesilatos/química , Diatrizoato , Potenciometria , Espectrofotometria UltravioletaRESUMO
The effect of various surfactants [the cationics cetyl trimethyl ammonium bromide (CTAB) and cetyl pyridinium chloride (CPC), the anionic sodium dodecyl sulphate (SDS), and the nonionic polysorbate 80 (Tween 80)] on the solubility and ionization constant of some sparingly soluble weak acids of pharmaceutical interest was studied. Benzoic acid (and its 3-methyl-, 3-nitro-, and 4-tert-butyl-derivatives), acetylsalicylic acid, naproxen and iopanoic acid were chosen as model examples. Precise and accurate acid-base titrations in micellar systems were made feasible using a microcomputer-controlled titrator. The response curve, response time and potential drift of the glass electrode in the micellar systems were examined. The cationics CTAB and CPC were found to increase considerably the ionization constant of the weak acids (delta pKa ranged from -0.21 to -3.57), while the anionic SDS showed negligible effect and the nonionic Tween 80 generally decreased the ionization constants. The solubility of the acids in aqueous micellar and acidified micellar solutions was studied spectrophotometrically and it was found increased in all cases. Acetylsalicylic acid, naproxen, benzoic acid and iopanoic acid could be easily determined in raw material and some of them in pharmaceutical preparations by direct titration in CTAB-micellar system instead of using the traditional non-aqueous or back titrimetry. Precisions of 0.3-4.3% RSD and good correlation with the official tedious methods were obtained. The interference study of some excipients showed that a preliminary test should be carried out before the assay of formulations.