RESUMEN
The metabolism of diclofenac has been reported to produce reactive benzoquinone imine intermediates. We describe the identification of mercapturic acid derivatives of diclofenac in rats and humans. Three male Sprague-Dawley rats were administered diclofenac in aqueous solution (pH 7) at 50 mg/kg by intraperitoneal injection, and urine was collected for 24 h. Human urine specimens were obtained, and samples were pooled from 50 individuals. Urine samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Two metabolites with MH(+) ions at m/z 473 were detected in rat urine and identified tentatively as N-acetylcysteine conjugates of monohydroxydiclofenac. Based upon collision-induced fragmentation of the MH(+) ions, accurate mass measurements of product ions, and comparison of LC/MS/MS properties of the metabolites with those of synthetic reference compounds, one metabolite was assigned as 5-hydroxy-4-(N-acetylcystein-S-yl)diclofenac and the other as 4'-hydroxy-3'-(N-acetylcystein-S-yl)diclofenac. The former conjugate also was detected in the pooled human urine sample by multiple reaction-monitoring LC/MS/MS analysis. It is likely that these mercapturic acid derivatives represent degradation products of the corresponding glutathione adducts derived from diclofenac-2,5-quinone imine and 1',4'-quinone imine, respectively. Our data are consistent with previous findings, which suggest that oxidative bioactivation of diclofenac in humans proceeds via benzoquinone imine intermediates.
Asunto(s)
Acetilcisteína/orina , Benzoquinonas/metabolismo , Diclofenaco/farmacocinética , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Humanos , Iminas/metabolismo , Masculino , Espectrometría de Masas , Ratas , Ratas Sprague-DawleyRESUMEN
Cyanobacteria (blue-green algae) produce a wide range of low molecular weight metabolites that include potent neurotoxins, hepatotoxins, and cytotoxins. The accumulation of such toxins in freshwaters, and in brackish and marine waters presents hazards to human and animal health by a range of exposure routes. A review is presented of developments in the detection and analysis of cyanobacterial toxins, other than bioassays, including application of physicochemical, immunoassays, and enzyme-based methods. Analytical requirements are considered with reference to recently derived guideline levels for the protection of health and to the availability, or otherwise, of purified, quantitative cyanobacterial toxin standards.
Asunto(s)
Cianobacterias/química , Toxinas Biológicas/análisis , Toxinas Bacterianas/análisis , Fenómenos Químicos , Química Física , Inmunoensayo , Microcistinas , Péptidos Cíclicos/análisis , Toxinas Biológicas/química , Abastecimiento de Agua/análisis , Abastecimiento de Agua/normasRESUMEN
Microcystins are a group of structurally similar cyclic heptapeptide hepatotoxins and tumor promoters, produced by cyanobacteria. A microbore liquid chromatography electrospray ionization ion-trap mass spectrometry (LC-ESI-ITMS) method has been developed which is capable of separating and detecting trace amounts of microcystin variants in environmental samples. Extracted water sample was loaded onto a LC trapping column and, using a column switching technique, the compounds of interest were back-flushed onto a 1-mm LC column. Structural elucidation was achieved using ion-trap with tandem mass spectrometry in the data dependent scan mode. Collision-induced dissociation to MS3 allowed tentative identification of these cyclic peptides. Full-scan LC-ESI-MS mass spectrum was obtained when 250 pg of the authentic compound was injected onto the HPLC column, which represents the detection limit for microcystin-LR. This study demonstrated that LC-ESI-ITMS is a reliable and sensitive technique for analysing trace levels of microcystins.
Asunto(s)
Toxinas Bacterianas/análisis , Péptidos Cíclicos/análisis , Cromatografía Liquida , Cianobacterias/química , Cromatografía de Gases y Espectrometría de Masas , Microcistinas , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
During the early phase of a drug discovery process, in order to facilitate the selection of drug candidates from a discovery research program, a liquid chromatography tandem mass spectrometry (LC/MS/MS) strategy has been developed to assess the preliminary pharmacokinetics and metabolism of new drug entities. For pharmacokinetic studies using multiple reaction monitoring (MRM), the parent drug concentration and its 'suspected' (predictable) metabolites were monitored in the biological samples simultaneously. For metabolic identification, the general methodologies most frequently used to search for metabolites include full scan, precursor-ion scan, product-ion scan and neutral-loss scan. However, when the metabolites do not produce intense signals for tandem mass spectrometry, a more sensitive and selective assay has to be developed, and MRM would be the method of choice. Likewise, an intelligent guess as to which MRM transitions ought to be used for the metabolites will be considered, based on the product ion mass spectrum of the parent drug.
Asunto(s)
Cromatografía Liquida/métodos , Drogas en Investigación/farmacocinética , Espectrometría de Masas/métodos , Acetonitrilos , Animales , Biotransformación , Drogas en Investigación/metabolismo , Glucurónidos/metabolismo , Haplorrinos , Humanos , Hígado/metabolismo , Metilación , RatasRESUMEN
BACKGROUND: After a drought in February, 1996, all 126 patients in a haemodialysis unit in Caruaru, north-east Brazil, developed signs and symptoms of acute neurotoxicity and subacute hepatotoxicity following the use of water from a lake with massive growth of cyanobacteria (blue-green algae). 60 patients died. METHODS: Besides recording clinical details and outcome at follow-up, we arranged laboratory, radiological, and histological investigations on the patients and toxicological studies of serum and haemodialysis water filters. FINDINGS: The acute presentation was with malaise, myalgia and weakness, nausea and vomiting, and tender hepatomegaly, with a range of neurological symptoms from tinnitus, vertigo, headaches, and deafness to blindness and convulsions. Liver injury ranged from abnormal liver-function test results to rapidly progressive and fatal hepatic failure. Biochemical investigations revealed gross hyperbilirubinaemia, abnormal liver enzyme activities, and hypertriglyceridaemia, but there was no evidence of haemolysis or microangiopathy. Histology revealed a novel acute toxic hepatitis with diffuse panlobular hepatocyte necrosis, neutrophil infiltration, canalicular cholestasis, and regenerative multinucleate hepatocytes. Samples of serum, dialysis filters, and water-treatment columns contained microcystins, the highly toxic low-molecular-weight hepatotoxins produced by cyanobacteria. INTERPRETATION: Cyanobacteria present water-borne hazards to health via drinking water and recreational water. Haemodialysis presents an additional high-risk exposure route: when they enter directly into the circulation, microcystins can lead to fatal clinical syndromes ranging from acute neurotoxic illness to subacute liver failure.
Asunto(s)
Toxinas Bacterianas/envenenamiento , Cianobacterias , Unidades de Hemodiálisis en Hospital , Péptidos Cíclicos/envenenamiento , Microbiología del Agua , Brasil/epidemiología , Enfermedad Hepática Inducida por Sustancias y Drogas , Femenino , Humanos , Masculino , Microcistinas , Persona de Mediana Edad , Enfermedades del Sistema Nervioso/inducido químicamente , Intoxicación/mortalidadRESUMEN
PURPOSE: D-Limonene is a natural monoterpene with pronounced chemotherapeutic activity and minimal toxicity in preclinical studies. A phase I clinical trial to assess toxicity, the maximum tolerated dose (MTD) and pharmacokinetics in patients with advanced cancer was followed by a limited phase II evaluation in breast cancer. METHODS: A group of 32 patients with refractory solid tumors completed 99 courses of D-limonene 0.5 to 12 g/m2 per day administered orally in 21-day cycles. Pharmacokinetics were analyzed by liquid chromatography-mass spectrometry. Ten additional breast cancer patients received 15 cycles of D-limonene at 8 g/m2 per day. Intratumoral monoterpene levels were measured in two patients. RESULTS: The MTD was 8 g/m2 per day; nausea, vomiting and diarrhea were dose limiting. One partial response in a breast cancer patient on 8 g/m2 per day was maintained for 11 months; three patients with colorectal carcinoma had prolonged stable disease. There were no responses in the phase II study. Peak plasma concentration (Cmax) for D-limonene ranged from 10.8+/-6.7 to 20.5+/-11.2 microM. Predominant circulating metabolites were perillic acid (Cmax 20.7+/-13.2 to 71+/-29.3 microM), dihydroperillic acid (Cmax 16.6+/-7.9 to 28.1+/-3.1 microM), limonene-1,2-diol (Cmax 10.1+/-8 to 20.7+/-8.6 microM), uroterpenol (Cmax 14.3+/-1.5 to 45.1+/-1.8 microM), and an isomer of perillic acid. Both isomers of perillic acid, and cis and trans isomers of dihydroperillic acid were in urine hydrolysates. Intratumoral levels of D-limonene and uroterpenol exceeded the corresponding plasma levels. Other metabolites were trace constituents in tissue. CONCLUSIONS: D-Limonene is well tolerated in cancer patients at doses which may have clinical activity. The favorable toxicity profile supports further clinical evaluation.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Neoplasias/metabolismo , Terpenos/farmacocinética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/metabolismo , Ciclohexenos , Femenino , Humanos , Limoneno , Masculino , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Terpenos/administración & dosificación , Terpenos/efectos adversos , Terpenos/metabolismo , Resultado del TratamientoRESUMEN
Antibody-directed enzyme prodrug therapy (ADEPT) was administered to ten patients in a phase I clinical trial. The aim was to measure plasma levels of the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA) and the bifunctional alkylating drug (CJS11) released from it by the action of tumour-localised carboxypeptidase G2 (CPG2) enzyme. New techniques were developed to extract the prodrug and drug from plasma by solid-phase absorption and elution and to measure CPG2 activity in plasma and tissue. All extracts were analysed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). CPG2 activity was found in metastatic tumour biopsies but not in normal tissue, indicating that localisation had been successful. The clearing agent SB43-gal, given at 46.5 mg/m2, achieved the aim of clearing non-tumour-localised enzyme in the circulation, indicating that conversion of prodrug to drug could take place only at the site of localised conjugate. Plasma prodrug did not always remain above its required threshold of 3 microM for the "therapeutic window" of 120 min after dosing, but the presence of residual prodrug after the first administration of each day indicated that this could be achieved during the remaining four doses over the following 8 h. Despite considerable inter-patient prodrug plasma concentration variability, the elimination half-life of the prodrug was remarkably reproducible at 18 +/- 8 min. Rapid appearance of the drug in plasma indicated that successful conversion from the prodrug had taken place, but also undesirable leakback from the site of localisation into the bloodstream. However, drug plasma levels fell rapidly by at least 50% at between 10 and 60 min with a half-life of 36 +/- 14 min. Analysis of the plasma extracts by LC/MS indicated that this technique might be used to confirm qualitatively the presence of prodrug, drug and their metabolites.
Asunto(s)
Antineoplásicos/farmacocinética , Neoplasias Colorrectales/metabolismo , Glutamatos/farmacocinética , Compuestos de Mostaza Nitrogenada/farmacocinética , Profármacos/farmacocinética , gamma-Glutamil Hidrolasa/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Cromatografía Líquida de Alta Presión , Neoplasias Colorrectales/tratamiento farmacológico , Femenino , Glutamatos/química , Glutamatos/uso terapéutico , Humanos , Inmunoconjugados/farmacocinética , Inmunoterapia , Masculino , Espectrometría de Masas , Compuestos de Mostaza Nitrogenada/química , Compuestos de Mostaza Nitrogenada/uso terapéutico , Profármacos/química , Profármacos/uso terapéuticoRESUMEN
Limonene is a farnesyl transferase inhibitor that has shown antitumor properties. The drug had been given orally to cancer patients. Plasma and urine samples collected from the patients were examined by reversed-phase HPLC-atmospheric pressure chemical ionization and electrospray ionization MS. The drug underwent rapid conversion to hydroxylated and carboxylated derivatives. Characterization and structural elucidation of the metabolites were achieved by LC/MS and NMR. Five major metabolites were detected in the plasma extracts, namely limonene-1,2-diol, limonene-8,9-diol, perillic acid, an isomer of perillic acid, and dihydroperillic acid. Urinary metabolites comprised the glucuronides of the two isomers of perillic acid, dihydroperillic acid, limonene-8,9-diol, and a monohydroxylated limonene.
Asunto(s)
Transferasas Alquil y Aril , Antineoplásicos Fitogénicos/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias Colorrectales/metabolismo , Terpenos/metabolismo , Adulto , Anciano , Antineoplásicos Fitogénicos/sangre , Antineoplásicos Fitogénicos/orina , Cromatografía Liquida , Ciclohexenos , Farnesiltransferasa , Femenino , Humanos , Limoneno , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Estructura Molecular , Terpenos/sangre , Terpenos/orina , Transferasas/antagonistas & inhibidoresRESUMEN
DNA from white blood cells of seven women receiving tamoxifen as adjuvant therapy for breast cancer and of three women who served as healthy controls was analysed for the presence of tamoxifen-DNA adducts using 32P-postlabelling with a limit of detection of 8 adducts/10(10) nucleotides. No postlabelled adducts with the chromatographic properties of known tamoxifen-DNA adducts were detected in any of the samples. It is concluded that at therapeutic levels of exposure there is no significant formation of DNA adducts by tamoxifen or its metabolites in circulating white blood cells.
Asunto(s)
Antineoplásicos Hormonales/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Aductos de ADN/análisis , Antagonistas de Estrógenos/metabolismo , Leucocitos/metabolismo , Tamoxifeno/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Tamoxifeno/uso terapéuticoRESUMEN
The potential for the anti-breast cancer drug tamoxifen [(Z)-1-[4-[2-( dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-butene] to induce genotoxic damage (DNA adducts) in the human endometrium was investigated in vivo and in vitro. Endometria from hysterectomy patients who were not on tamoxifen were sectioned and maintained in short-term organ culture. The cultures were treated with either solvent vehicle (DMSO), tamoxifen, alpha-hydroxytamoxifen [(E)-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1,2-diphenyl-1-buten-3- ol; the major DNA-reactive metabolite in the rat], or benzo(a)pyrene. DNA was isolated and analyzed by 32P postlabeling. Chromatography on polyethyleneimine-cellulose TLC plates revealed DNA adducts in endometria treated with alpha-hydroxytamoxifen identical to those seen previously in the rat liver. However, no adducts were seen from treatment with tamoxifen itself. The viability of the enzyme-metabolizing systems of the endometrial samples was demonstrated by the detection of expected DNA adducts induced by benzo(a)pyrene. Examination by liquid chromatography-mass spectrometry of the explant culture media from endometria treated with tamoxifen revealed the presence of the alpha-hydroxy metabolite in a dose-dependent manner, although apparently at levels insufficient to produce detectable DNA adducts. Endometrial DNA obtained from 18 patients undergoing daily treatment with 10-40 mg tamoxifen for 3 months-9 years was also analyzed. No evidence for any DNA adducts induced by tamoxifen was found in any of the patients examined. These data suggest that the genotoxic events observed with tamoxifen in the rat may not apply to the human endometrium.
Asunto(s)
Daño del ADN , Endometrio/efectos de los fármacos , Antagonistas de Estrógenos/toxicidad , Tamoxifeno/toxicidad , Adulto , Anciano , Anciano de 80 o más Años , Animales , Técnicas de Cultivo , Aductos de ADN/análisis , Femenino , Humanos , Persona de Mediana Edad , RatasRESUMEN
On-line capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESMS) has been employed for the analysis of metabolites of the anticancer drug tamoxifen. Nonaqueous (methanol) CE electrolyte provided better resolution and detection sensitivity compared to aqueous systems or highly aqueous water-methanol electrolyte mixtures. Nonaqueous methanol also permitted the use of lower ES voltages presumably owing to its lower surface tension, which facilitated droplet breakup. This decreased the tendency to produce electric discharges, thus improving the stability of electrospray conditions. The relative ease of methanol solvent evaporation may contribute to an improved yield of protonated analytes as compared to highly aqueous solutions. Enhanced CE resolution can be at least partially attributed to the improved solubility of analytes in methanol relative to water. Higher solubility implies less aggregation of hydrophobic analytes, thus improving homogeneity in solution. Moreover, electroosmotic flow toward the detector decreased in methanol relative to water. The reduction of this force pushing all analytes through the capillary, but not aiding in separation, implies that other factors such as slight differences in electrophoretic mobilities are more apt to lead to successful separations. Surfactants were employed as nonaqueous CE-ESMS buffer additives. An SDS concentration of 7 mM lowered the ESMS signal response for N-desmethyltamoxifen by a factor of approximately 3. However, separation of tamoxifen metabolites using 7 mM SDS was augmented relative to the unadulterated methanol electrolyte. This enabled the separation of alpha-hydroxytamoxifen and 4-hydroxytamoxifen, which were not resolvable in methanol electrolyte devoid of SDS. The methanol-surfactant electrolyte system has been successfully used to determine metabolites formed after incubation of tamoxifen with mouse hepatocytes.
Asunto(s)
Antineoplásicos Hormonales/análisis , Tamoxifeno/análisis , Animales , Antineoplásicos Hormonales/farmacocinética , Biotransformación , Células Cultivadas , Electroforesis Capilar , Espectrometría de Masas , Ratones , Microsomas Hepáticos/química , Ratas , Tensoactivos , Tamoxifeno/farmacocinéticaRESUMEN
Liquid chromatography with an electrospray ionization (ESI) interface has been applied to study the anticancer drug taxol and its metabolites after incubation with human hepatic microsomes. The parent drug and its metabolites were monitored in the positive-ionization mode. Since ESI gave only quasi-molecular ions for taxol and its analogues, collision-induced dissociation experiments were carried out in order to generate fragment ions, by increasing the cone voltage at the ESI source. The product-ion mass spectra of taxol and its metabolites contained diagnostic fragment ions, which enabled the presence of hydroxylated and deacetylated metabolites of taxol to be established.
Asunto(s)
Antineoplásicos Fitogénicos/análisis , Microsomas Hepáticos/química , Paclitaxel/análisis , Antineoplásicos Fitogénicos/farmacocinética , Biotransformación , Cromatografía Líquida de Alta Presión , Remoción de Radical Alquila , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hidroxilación , Paclitaxel/farmacocinéticaRESUMEN
This study evaluates the metabolic profile of JM216 [bis(acetato)ammine-dichloro(cyclohexylamine) platinum(IV)], the first orally administrable platinum complex, in plasma ultrafiltrates of 12 patients (n = 2-4 time points per patient) following different doses of drug (120, 200, 340, 420, 560 mg/m2). The biotransformation profile was evaluated by high-performance liquid chromatography (HPLC) followed by atomic absorption spectrophotometry (AA). The AA profiles were compared with those previously identified by HPLC on line with mass spectrometry (HPLC-MS) in plasma incubated with JM216. A total of six platinum peaks (Rt = 5.5, 7.2, 10.6, 12.4, 15.6, and 21.6 min, respectively) were observed in patients' plasma ultrafiltrate samples, of which only four appeared during the first 6 h post-treatment. Four of these coeluted with those observed and identified previously in plasma incubation medium. No parent JM216 was detected. The major metabolite seen in patients was the Pt II complex JM118 [cis-amminedichloro-(cyclohexylamine)platinum(II)] and was observed in all the patients. Interestingly, the second metabolite was shown to coelute with the Pt IV species JM383 [bis-acetatoammine(cyclohexylamine)dihydroxoplatinum (IV)]. Both JM118 and JM383 were identified by HPLC-MS in a clinical sample. Peak C, which was a minor product (less than 5% of the free platinum), coeluted with JM559 [bis-acetatoammine-chloro(cyclohexylalamine)hydroxoplatin um(IV)]. The cytotoxicity profile of all three metabolites in a panel of cisplatin-sensitive and -resistant human ovarian carcinoma cell lines was very close to that of the parent drug. In addition, the concentrations of JM118 reached in patients' plasma ultrafiltrate were comparable with the cytotoxic levels of the compound determined in the ovarian carcinoma panel of cell lines. Two metabolites were seen in patients but not in the in vitro incubation medium, suggesting the involvement of a possible enzymatic reaction. Thus, the biotransformation profile following oral administration of JM216 shows a variety of Pt(IV) and Pt(Il) metabolites in plasma that differ significantly from other systemically applied platinum drugs.
Asunto(s)
Antineoplásicos/metabolismo , Compuestos Organoplatinos/metabolismo , Administración Oral , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Muerte Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Estructura Molecular , Compuestos Organoplatinos/administración & dosificación , Compuestos Organoplatinos/química , Compuestos Organoplatinos/uso terapéutico , Neoplasias Ováricas , Platino (Metal)/sangre , Células Tumorales CultivadasRESUMEN
The metabolic activation of tamoxifen and its metabolite alpha-hydroxytamoxifen in primary cultures of rat, mouse and human hepatocytes has been compared. The extent of formation of DNA adducts in these cells was measured by 32P-postlabelling, using either nuclease P1 digestion or sorbent extraction of DNA digests to enhance the sensitivity of the assay. DNA adducts were readily detected in rat hepatocytes treated with 1 or 10 microM tamoxifen (mean levels 18.2 and 89.8 adducts/10(8) nucleotides respectively). Similar levels of adducts were formed by mouse hepatocytes (15.0 +/- 1.8 adducts/10(8) nucleotides, 10 microM tamoxifen). However DNA adducts were not detected in tamoxifen-treated human hepatocytes with a detection limit for the assay of 4 adducts/10(10) nucleotides. Treatment of rat cells with alpha-hydroxytamoxifen resulted in 15- to 63-fold higher levels of adducts than with comparable concentrations of tamoxifen. A similar level of adducts was also seen in mouse hepatocytes treated with alpha-hydroxytamoxifen at the 1 microM concentration (173.9 +/- 4.1 adducts/10(8) nucleotides). Treatment of human cells with alpha-hydroxytamoxifen resulted in DNA adduct formation at levels (1.94 +/- 0.89 and 18.9 +/- 17.9 adducts/10(8) nucleotides at 1 and 10 microM respectively) approximately 300-fold lower than those in rat hepatocytes. The presence of alpha-hydroxytamoxifen in the culture medium from experiments where cells were incubated with tamoxifen was monitored by mass spectrometry. Concentrations were found to be approximately 50-fold lower in the medium from human hepatocytes than from rat and mouse hepatocytes.
Asunto(s)
Aductos de ADN/metabolismo , Antagonistas de Estrógenos/metabolismo , Hígado/metabolismo , Tamoxifeno/metabolismo , Adolescente , Adulto , Animales , Biotransformación , Niño , Femenino , Humanos , Hígado/citología , Masculino , Ratones , Persona de Mediana Edad , Ratas , Ratas Endogámicas F344 , Especificidad de la EspecieRESUMEN
The present study describes the application of on-line liquid chromatography-electrospray ionisation in conjunction with a high resolution magnetic sector mass spectrometer to identify metabolites of a platinum(IV) anticancer drug JM216 [bis(acetato)amminedichloro(cyclohexylamine)platinum(IV)] in human plasma. Four metabolites were identified following incubation of JM216 in human plasma: JM118 [amminedichlorocyclohexylamineplatinum(II)], a platinum(II) complex; JM383 [bis(acetato)amminedihydroxo(cyclohexylamine)platinum(IV)]; JM518 [bis(acetato)amminechloro(cyclohexylamine)hydroxoplatinum (IV)] and its isomer JM559. The platinum complexes mass spectra were dominated by the natriated [M + Na]+ ion. Elemental compositions of these natriated ions were confirmed by accurate mass measurement on a magnetic sector mass spectrometer in the course of LC/MS analysis. This study demonstrates the capability of direct LC-ESI/MS with accurate mass measurement for analysis of platinum complexes in biological samples. Our results suggest that LC-ESI/MS is a powerful technique for structure elucidation of novel metabolites, and could make valuable contributions to drug metabolism research.
Asunto(s)
Antineoplásicos/sangre , Antineoplásicos/farmacocinética , Compuestos Organoplatinos/sangre , Compuestos Organoplatinos/farmacocinética , Biotransformación , Cromatografía Liquida , Ionización de Llama , Humanos , Isomerismo , Espectrometría de Masas , Platino (Metal)/sangre , Polietilenglicoles/análisis , UltrafiltraciónRESUMEN
Bis(acetato)amminedichloro(cyclohexylamine) platinum(IV) (JM216) is a new orally administered platinum complex with antitumor properties, and is currently undergoing phase II clinical trials. When JM216 was incubated with human plasma ultrafiltrate, 93% of the platinum species were protein-bound and 7% were unbound. The unbound platinum complexes in the ultrafiltrates of human plasma were analysed using a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method. Apart from the parent drug, four metabolites were identified and characterised. These include JM118 [amminedichloro(cyclohexylamine) platinum(II)], JM383 [bis(acetato)ammine(cyclohexylamine)dihydroxo platinum(IV)] and the two isomers JM559 and JM518 [bis(acetato)amminechloro(cyclohexylamine) hydroxo platinum(IV)]. Their elemental compositions were determined by accurate mass measurement during the LC analysis, to confirm their identities. Quantitation of these metabolites by off-line LC atomic absorption spectroscopy demonstrated that JM118 is the major metabolite in plasma from patients receiving JM216 treatment.
Asunto(s)
Antineoplásicos/sangre , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Compuestos Organoplatinos/sangre , Antineoplásicos/uso terapéutico , Cromatografía Líquida de Alta Presión/métodos , Femenino , Humanos , Iones , Espectrometría de Masas/métodos , Compuestos Organoplatinos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Células Tumorales Cultivadas , UltrafiltraciónRESUMEN
This study describes the application of on line HPLC-electrospray ionization MS in the structural determination of the metabolites formed following incubation with rat liver microsomes of an equimolar mixture of the anticancer drug tamoxifen and its [D5-ethyl]-analogue. The ratio of ca 3:1 between unlabelled and D4-labelled alpha-hydroxytamoxifen, indicating a large isotope effect for this metabolic process, accounted for the previously observed lower yield of DNA adducts formed in the livers of rats treated with D5-tamoxifen compared with unlabelled drug. The loss of one deuterium atom on metabolism discriminated hydroxyethylated metabolites from others and enabled two further such metabolites to be detected, namely alpha-hydroxytamoxifen N-oxide and alpha-hydroxy-N-desmethyltamoxifen of which the latter is novel. Furthermore, the use of [alpha-D2-ethyl]- and [beta-D3-ethyl] tamoxifens discriminated alpha- from beta-hydroxylated metabolites and proved that the metabolites described here were alpha-hydroxylated. In contrast to the alpha-hydroxylated metabolites, the other metabolites identified, namely tamoxifen N-oxide, N-desmethyltamoxifen, 4-hydroxytamoxifen and their deuterated counterparts were not depleted in the deuterated components. The use of on line HPLC-electrospray ionization MS combined with isotopic labelling is a powerful technique for probing the structures of metabolites, and, applied to tamoxifen, has provided further evidence that alpha-hydroxylation is an important pathway for the conversion of the drug into a DNA-reactive metabolite.
Asunto(s)
Deuterio/farmacología , Microsomas Hepáticos/metabolismo , Tamoxifeno/metabolismo , Tamoxifeno/farmacocinética , Animales , Cromatografía Líquida de Alta Presión/métodos , Hidroxilación , Inactivación Metabólica , Espectrometría de Masas/métodos , Oxidación-Reducción , Ratas , Sensibilidad y Especificidad , Tamoxifeno/toxicidadRESUMEN
The metabolism of tamoxifen was examined in human liver homogenate and human Hep G2 cell line preparations by LC/electro spray ionization/MS. Several metabolites were detected in the human liver homogenate extracts, namely N-didesmethyltamoxifen (metabolite I), alpha-hydroxytamoxifen (metabolite II), 4-hydroxytamoxifen (metabolite III), N-desmethyltamoxifen (metabolite IV), and tamoxifen N-oxide (metabolite V). Metabolites II, III, IV, and V were observed in the samples after incubating tamoxifen with the human Hep G2 cell line. When these results were compared with the metabolic profiles in patients, apart from metabolites I-V, alpha-hydroxy-N-desmethyltamoxifen (VI) and 4-hydroxy N-desmethyltamoxifen (VII) were present in all the plasma samples. In addition, in patients who had received tamoxifen treatment daily for > 6 months, several minor metabolites were detected, namely 4-hydroxytamoxifen N-oxide (metabolite VIII) and two dihydroxylated analogs (metabolites IX and X). The precise positions of the hydroxylation have yet to be determined.
Asunto(s)
Neoplasias de la Mama/metabolismo , Hígado/metabolismo , Tamoxifeno/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Hígado/citología , Masculino , Persona de Mediana Edad , Estructura Molecular , Tamoxifeno/uso terapéutico , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
It has been proposed that the antiestrogen tamoxifen induces liver tumors in rats and genotoxic effects in vitro through metabolic activation involving, initially, alpha-hydroxylation of the ethyl group. To test this hypothesis, the extent of DNA adduct formation in primary rat hepatocytes treated with tamoxifen and alpha-hydroxytamoxifen was investigated. Hepatocytes from female Fischer F-344 rats were treated with 1 or 10 microM concentrations of either alpha-hydroxytamoxifen or tamoxifen. DNA was isolated and analyzed for the presence of DNA adducts by 32P postlabeling. Chromatography on polyethyleneimine cellulose thin layer chromatography and reverse-phase high performance liquid chromatography revealed that the same pattern of adducts was formed by both compounds. However, the level of adduct formation was 25 and 49 times greater with alpha-hydroxytamoxifen than with tamoxifen at 1 and 10 microM, respectively. The formation of alpha-hydroxytamoxifen as a metabolite of tamoxifen was demonstrated by mass spectrometric analysis of the extracted culture medium. alpha-Hydroxytamoxifen was found to react with DNA in the absence of metabolizing enzymes. These results demonstrate the involvement of alpha-hydroxylation in the metabolic activation of tamoxifen.
Asunto(s)
Carcinógenos/metabolismo , ADN/metabolismo , Antagonistas de Estrógenos/metabolismo , Hígado/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Animales , Femenino , Cromatografía de Gases y Espectrometría de Masas , Ratas , Ratas Endogámicas F344RESUMEN
Antibody-directed enzyme prodrug therapy (ADEPT) separates the cytotoxic function from the targeting function (5). An antibody-carboxypeptidase G2 (CPG2) enzyme is delivered prior to the nontoxic prodrug, CMDA, which is converted to a cytotoxic drug by the action of the localized conjugate at the tumor site. An indirect in vitro assay was developed to detect the presence of functional CPG2 in the plasma of patients in an ADEPT clinical trial. Compounds in the plasma of patients were characterized using liquid chromatography-mass spectrometry. Plasma at three different time points (prior to treatment, post-antibody-enzyme conjugate, and post-galactosylated anti-enzyme antibody clearing agent) was added to the CMDA prodrug and analyzed. Conversion of the CMDA prodrug to its active drug indicates that CPG2-conjugate remains in the plasma. This technique will provide essential data for the timing of prodrug administration in ADEPT.