RESUMO
In human trials certain heat shock protein 90 (Hsp90) inhibitors, including 17-DMAG and NVP-AUY922, have caused visual disorders indicative of retinal dysfunction; others such as 17-AAG and ganetespib have not. To understand these safety profile differences we evaluated histopathological changes and exposure profiles of four Hsp90 inhibitors, with or without clinical reports of adverse ocular effects, using a rat retinal model. Retinal morphology, Hsp70 expression (a surrogate marker of Hsp90 inhibition), apoptotic induction and pharmacokinetic drug exposure analysis were examined in rats treated with the ansamycins 17-DMAG and 17-AAG, or with the second-generation compounds NVP-AUY922 and ganetespib. Both 17-DMAG and NVP-AUY922 induced strong yet restricted retinal Hsp70 up-regulation and promoted marked photoreceptor cell death 24h after the final dose. In contrast, neither 17-AAG nor ganetespib elicited photoreceptor injury. When the relationship between drug distribution and photoreceptor degeneration was examined, 17-DMAG and NVP-AUY922 showed substantial retinal accumulation, with high retina/plasma (R/P) ratios and slow elimination rates, such that 51% of 17-DMAG and 65% of NVP-AUY922 present at 30 min post-injection were retained in the retina 6h post-dose. For 17-AAG and ganetespib, retinal elimination was rapid (90% and 70% of drugs eliminated from the retina at 6h, respectively) which correlated with lower R/P ratios. These findings indicate that prolonged inhibition of Hsp90 activity in the eye results in photoreceptor cell death. Moreover, the results suggest that the retina/plasma exposure ratio and retinal elimination rate profiles of Hsp90 inhibitors, irrespective of their chemical class, may predict for ocular toxicity potential.
Assuntos
Modelos Animais de Doenças , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Animais , Benzoquinonas/toxicidade , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP90/metabolismo , Lactamas Macrocíclicas/toxicidade , Masculino , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Valor Preditivo dos Testes , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Degeneração Retiniana/patologiaRESUMO
BACKGROUND: This phase I study investigated the maximum tolerated dose (MTD), safety, pharmacokinetics and antitumor activity of ganetespib in patients with solid malignancies. METHODS: Patients were enrolled in cohorts of escalating ganetespib doses, given as 1 hour IV infusion, once weekly for 3 weeks, followed by a 1-week rest until disease progression or unacceptable toxicity. Endpoints included safety, pharmacokinetic and pharmacodynamic parameters and preliminary clinical activity. RESULTS: Fifty-three patients were treated at doses escalating from 7 to 259 mg/m(2). The most common adverse events were Grade 1 and 2 diarrhea, fatigue, nausea or vomiting. Dose-limiting toxicities (DLT) observed were: one Grade 3 amylase elevation (150 mg/m(2)), one Grade 3 diarrhea and one Grade 3 and one Grade 4 asthenia (259 mg/m(2)). The MTD was 216 mg/m(2) and the recommended phase 2 dose was established at 200 mg/m(2) given IV at Days 1, 8, and 15 every 4 weeks. There was a linear relationship between dose and exposure. Plasma HSP70 protein levels remained elevated for over a week post treatment. Disease control rate (objective response and stable disease at ≥ 16 weeks) was 24.4%. CONCLUSIONS: Ganetespib is well tolerated as a weekly infusion for 3 of every 4 weeks cycle. The recommended phase II dose is 200 mg/m(2), and is associated with an acceptable tolerability profile. TRIAL REGISTRATION: NCT00687934.
Assuntos
Neoplasias/tratamento farmacológico , Triazóis/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Esquema de Medicação , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/sangue , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias/metabolismo , Resultado do Tratamento , Triazóis/administração & dosagem , Triazóis/efeitos adversos , Triazóis/farmacocinéticaRESUMO
Between 1995 and 1997, we performed Isshiki's original method of arytenoid adduction alone or as an adjunct to type I thyroplasty for the treatment of unilateral vocal fold paralysis. From 1997 onward, we performed arytenoid adduction by traction of the lateral cricoarytenoid muscle (Iwamura's method), because it reduces discomfort to the patient and avoids rotation of the thyroid cartilage. Preliminary experiments and surgical procedures involving traction of the lateral cricoarytenoid muscle are described. Of 21 patients with a maximum phonation time of less than 9 seconds, 14 underwent type I thyroplasty as an adjunct to our method of arytenoid adduction and 7 underwent arytenoid adduction alone. Sixteen patients (76%) were able after surgery to extend their maximum phonation time beyond 10 seconds; this result compares favorably with the results of Isshiki's original adduction technique. We describe useful anatomic landmarks for approaching the lateral cricoarytenoid muscle in the hope that more voice surgeons will adopt this approach in the treatment of unilateral vocal fold paralysis.
Assuntos
Cartilagem Cricoide/cirurgia , Procedimentos Cirúrgicos Bucais/métodos , Paralisia das Pregas Vocais/cirurgia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cartilagem Aritenoide/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Mecânico , SuturasRESUMO
Systemic lupus erythematosus (SLE) is a complex, systemic autoimmune disease with a diverse range of immunological and clinical manifestations. The introduction of broad spectrum immunosuppressive therapies and better management of acute disease exacerbations have improved outcomes for lupus patients over recent years. However, these regimens are burdened by substantial toxicities and confer significantly higher risks of infection, thus there remains a significant and unmet medical need for alternative treatment options, particularly those with improved safety profiles. Heat shock protein 90 (HSP90) is a ubiquitously expressed molecular chaperone that acts as an important modulator of multiple innate and adaptive inflammatory processes. Of note, accumulating clinical and experimental evidence has implicated a role for HSP90 in the pathogenesis of SLE. Here we evaluated the potential of HSP90 as a therapeutic target for this disease using the selective small molecule inhibitor ganetespib in the well-characterized MRL/lpr autoimmune mouse model. In both the prophylactic and therapeutic dosing settings, ganetespib treatment promoted dramatic symptomatic improvements in multiple disease parameters, including suppression of autoantibody production and the preservation of renal tissue integrity and function. In addition, ganetespib exerted profound inhibitory effects on disease-related lymphadenopathy and splenomegaly, and reduced pathogenic T and B cell lineage populations in the spleen. Ganetespib monotherapy was found to be equally efficacious and tolerable when compared to an effective weekly dosing regimen of the standard-of-care immunosuppressive agent cyclophosphamide. Importantly, co-treatment of ganetespib with a sub-optimal, intermittent dosing schedule of cyclophosphamide resulted in superior therapeutic indices and maximal disease control. These findings highlight the potential of HSP90 inhibition as an alternative, and potentially complementary, strategy for therapeutic intervention in SLE. Such approaches may have important implications for disease management, particularly for limiting or preventing treatment-related toxicities, a major confounding factor in current SLE therapy.
Assuntos
Ciclofosfamida/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Triazóis/uso terapêutico , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Ciclofosfamida/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Imunossupressores/farmacologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Baço/efeitos dos fármacos , Baço/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Triazóis/farmacologiaRESUMO
The clinical benefits of chemotherapy are commonly offset by insufficient drug exposures, narrow safety margins, and/or systemic toxicities. Over recent decades, a number of conjugate-based targeting approaches designed to overcome these limitations have been explored. Here, we report on an innovative strategy that utilizes HSP90 inhibitor-drug conjugates (HDC) for directed tumor targeting of chemotherapeutic agents. STA-12-8666 is an HDC that comprises an HSP90 inhibitor fused to SN-38, the active metabolite of irinotecan. Mechanistic analyses in vitro established that high-affinity HSP90 binding conferred by the inhibitor backbone could be exploited for conjugate accumulation within tumor cells. In vivo modeling showed that the HSP90 inhibitor moiety was required for selective retention of STA-12-8666, and this enrichment promoted extended release of active SN-38 within the tumor compartment. Indeed, controlled intratumoral payload release by STA-12-8666 contributed to a broad therapeutic window, sustained biomarker activity, and remarkable degree of efficacy and durability of response in multiple cell line and patient-derived xenograft models. Overall, STA-12-8666 has been developed as a unique HDC agent that employs a distinct mechanism of targeted drug delivery to achieve potent and sustained antitumor effects. These findings identify STA-12-8666 as a promising new candidate for evaluation as novel anticancer therapeutic.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/análogos & derivados , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Resorcinóis/farmacologia , Triazóis/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Western Blotting , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/farmacologia , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Irinotecano , Camundongos Endogâmicos ICR , Camundongos SCID , Microscopia de Fluorescência , Terapia de Alvo Molecular/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Resorcinóis/química , Resorcinóis/farmacocinética , Inibidores da Topoisomerase I/administração & dosagem , Inibidores da Topoisomerase I/farmacocinética , Inibidores da Topoisomerase I/farmacologia , Resultado do Tratamento , Triazóis/administração & dosagem , Triazóis/farmacocinética , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The separation of two highly negatively charged enantiomeric organic disulfates containing two chiral centers was investigated by capillary electrophoresis using cyclodextrin based chiral selectors added to the run buffer. The optimum separation for the enantiomers was achieved in less than 3 min at 25 degrees C with a run buffer of 10 mM glycine pH 2.4 and 5 mM QA-beta-CD, which is a positively charged quaternary ammonium beta-cyclodextrin derivative. The method resulted in baseline resolution, excellent linearity, and highly reproducible migration times allowing facile evaluation of the enantiomeric purity of the individual isomers. Detection limits for the enantiomeric pair were determined to be 0.3 ng/microl (S/N = 3). The nature of the selector-enantiomer interaction and a quantitative measurement of the apparent stability constants that governed chiral discrimination of the enantiomers with QA-beta-CD were also investigated by UV-Vis spectroscopy and electrospray ionization mass spectrometry.
Assuntos
Eletroforese Capilar/métodos , Cromatografia Líquida de Alta Pressão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta , EstereoisomerismoRESUMO
UNLABELLED: Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor-resistant phenotype of FGFR3 mutant-expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity. IMPLICATIONS: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Triazóis/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Distribuição Aleatória , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Triazóis/farmacocinética , Neoplasias da Bexiga Urinária/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Elesclomol is an investigational drug that exerts potent anticancer activity through the elevation of reactive oxygen species (ROS) levels and is currently under clinical evaluation as a novel anticancer therapeutic. Here we report the first description of selective mitochondrial ROS induction by elesclomol in cancer cells based on the unique physicochemical properties of the compound. Elesclomol preferentially chelates copper (Cu) outside of cells and enters as elesclomol-Cu(II). The elesclomol-Cu(II) complex then rapidly and selectively transports the copper to mitochondria. In this organelle Cu(II) is reduced to Cu(I), followed by subsequent ROS generation. Upon dissociation from the complex, elesclomol is effluxed from cells and repeats shuttling elesclomol-Cu complexes from the extracellular to the intracellular compartments, leading to continued copper accumulation within mitochondria. An optimal range of redox potentials exhibited by copper chelates of elesclomol and its analogs correlated with the elevation of mitochondrial Cu(I) levels and cytotoxic activity, suggesting that redox reduction of the copper triggers mitochondrial ROS induction. Importantly the mitochondrial selectivity exhibited by elesclomol is a distinct characteristic of the compound that is not shared by other chelators, including disulfiram. Together these findings highlight a unique mechanism of action with important implications for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Cobre/metabolismo , Hidrazinas/farmacologia , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Cobre/química , Humanos , Mitocôndrias/efeitos dos fármacos , Neoplasias/metabolismo , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 µm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Triazóis/farmacologia , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Benzoquinonas/efeitos adversos , Benzoquinonas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Cristalografia por Raios X , Feminino , Células HL-60 , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Humanos , Células K562 , Lactamas Macrocíclicas/efeitos adversos , Lactamas Macrocíclicas/farmacologia , Masculino , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Coelhos , Ratos , Ratos Sprague-Dawley , Triazóis/efeitos adversos , Triazóis/química , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
PURPOSE: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. EXPERIMENTAL DESIGN: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. RESULTS: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. CONCLUSIONS: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Triazóis/farmacologia , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Benzoquinonas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Oxirredutases Intramoleculares/metabolismo , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos SCID , Prostaglandina-E Sintases , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Triazóis/uso terapêutico , Triazóis/toxicidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors. METHODS AND FINDINGS: This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25). CONCLUSIONS: This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients.
Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/veterinária , Pró-Fármacos/uso terapêutico , Triazóis/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Cromatografia Líquida de Alta Pressão , Cães , Feminino , Indóis , Masculino , Neoplasias/tratamento farmacológico , Espectrometria de Massas em Tandem , Triazóis/química , Triazóis/farmacocinética , Triazóis/toxicidadeRESUMO
A chemically modified electrode has been developed as a detector for the sensitive and selective determination of thiol-containing compounds following capillary electrophoresis separation. Electrodes were constructed by entrapment of the coenzyme pyrroloquinoline quinone (PQQ) into a polypyrrole (PPy) matrix on a 245-microm graphite electrode during electropolymerization of pyrrole in the presence of PQQ. PQQ serves as an efficient biocatalyst to mediate the oxidation of thiols at a substantially reduced overpotential relative to an unmodified electrode. Furthermore, this design takes advantage of the pH-dependent reversible electrochemical properties of PQQ, which facilitates optimization of separation and detection conditions. The PQQ/PPy-modified electrode was incorporated as an end-column detector, and a separation of homocysteine, cysteine, N-acetylcysteine, and glutathione was developed. Detection limits for these four thiols were determined to be 11, 23, 104, and 134 nM, respectively, with mass detection limits ranging from 0.29 to 3.48 fmol. The PQQ/PPy electrode was also found to be very reproducible in run-to-run, day-to-day, and electrode-to-electrode comparisons. The utility of this electrode was demonstrated for the detection of cysteine in dietary supplements and human urine, resulting in excellent agreement with reported values.
Assuntos
Eletrodos , Eletroforese Capilar/métodos , Quinolonas/química , Quinonas/química , Compostos de Sulfidrila/análise , Suplementos Nutricionais/análise , Humanos , Cofator PQQ , Compostos de Sulfidrila/urinaRESUMO
Enzyme-modified microelectrodes were developed for the indirect amperometric detection of acetylcholine and choline following separation by capillary electrophoresis. Electrodes were prepared by first sequentially electrodepositing polypyrrole and polytyramine from the monomers on a 200-microm platinum electrode. The polymer bilayer provides enhanced selectivity and pendant amino groups for covalent coupling of acetylcholinesterase (AChE) and choline oxidase (ChO) by their reaction with glutaraldehyde. The AChE/ChO-modified electrode was then employed as an end-column detector to determine acetylcholine and choline with and without the internal standard butyrylcholine. Excellent operational stability during 2 days of continuous use was observed, with detection limits of 2 microM or 50 fmol for both acetylcholine and choline. The response from potential interferences, such as dopamine, catechol, and norepinephrine, were significantly reduced in comparison to a bare platinum electrode. The utility of this approach was demonstrated by monitoring the uptake of choline into synaptosomes.