RESUMEN
BACKGROUND: New therapies for the treatment of mucopolysaccharidoses that target the brain, including intrathecal enzyme replacement, are being explored. Quantitative analysis of the glycosaminoglycans (GAGs) that accumulate in these disorders is required to assess the disease burden and monitor the effect of therapy in affected patients. Because current methods lack the required limit of quantification and specificity to analyze GAGs in small volumes of cerebrospinal fluid (CSF), we developed a method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: Samples of CSF (25 µL) were evaporated to dryness and subjected to methanolysis. The GAGs were degraded to uronic acid-N-acetylhexosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from heparan, dermatan and chondroitin sulfates (HS, DS and CS) were separated by UPLC and analyzed by electrospray ionization MS/MS using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. RESULTS: CSF from control pediatric subjects (n = 22) contained <0.38 mg/L HS, 0.26 mg/L DS, and 2.8 mg/L CS, whereas CSF from patients with Hurler syndrome (n = 7) contained concentrations of DS and HS that were at least 6-fold greater than the upper control limits. These concentrations were reduced by 17.5% to 82.5% after allogeneic transplantation and treatment with intrathecal and intravenous enzyme replacement therapy. CONCLUSIONS: The method described here has potential value in monitoring patients with mucopolysaccharidoses receiving treatment targeted to the brain.
Asunto(s)
Sulfatos de Condroitina/líquido cefalorraquídeo , Dermatán Sulfato/líquido cefalorraquídeo , Heparitina Sulfato/líquido cefalorraquídeo , Mucopolisacaridosis I/líquido cefalorraquídeo , Biomarcadores/líquido cefalorraquídeo , Calibración , Niño , Sulfatos de Condroitina/normas , Cromatografía Líquida de Alta Presión , Dermatán Sulfato/normas , Deuterio , Dimerización , Terapia de Reemplazo Enzimático , Trasplante de Células Madre Hematopoyéticas , Heparitina Sulfato/normas , Hexosaminas/líquido cefalorraquídeo , Humanos , Técnicas de Dilución del Indicador , Inyecciones Intravenosas , Inyecciones Espinales , Mucopolisacaridosis I/terapia , Estándares de Referencia , Valores de Referencia , Espectrometría de Masas en Tándem , Ácidos Urónicos/líquido cefalorraquídeoRESUMEN
A cellulose acetate plate electrophoresis method for analysis of pharmaceutical heparin and its potential glycosaminoglycan impurities, e.g. dermatan sulfate, chondroitin sulfate and oversulfated chondroitin sulfate, is presented. Heparin is chemically degraded by application of nitrous acid and residual glycosaminoglycans are electrophoretically separated thereafter. After staining using Alcian blue 8GS, these glycosaminoglycan impurities can be quantified by means of comparison to a dermatan sulfate standard. Results of a validation study of this analytical method are shown, demonstrating its feasibility for routine use in analytical quality control labs under GMP conditions.
Asunto(s)
Anticoagulantes/análisis , Dermatán Sulfato/análisis , Electroforesis en Acetato de Celulosa/métodos , Glicosaminoglicanos/análisis , Heparina/análisis , Azul Alcián/análisis , Colorantes/análisis , Dermatán Sulfato/metabolismo , Dermatán Sulfato/normas , Contaminación de Medicamentos/prevención & control , Estudios de Factibilidad , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/normas , Control de Calidad , Estándares de Referencia , Sensibilidad y Especificidad , Coloración y Etiquetado/métodosRESUMEN
Heparin-induced thrombocytopenia (HIT) is a hypercoagulable syndrome strongly associated with thrombosis that is usually treated with drugs that inhibit factor Xa (danaparoid) or thrombin (lepirudin). In the present study the outcome of HIT-patients treated with danaparoid or lepirudin was compared using the single or combined endpoints of new thromboembolic complications (new TECs), amputations and/or death, and major bleeding. HIT-patients treated with lepirudin were enrolled in two prospective trials and patients, who were identified in the same two laboratories during the same time period, who were not enrolled into these studies but treated with danaparoid, were assessed retrospectively according to a standardized questionnaire. 126 danaparoid (60.3% female) and 175 lepirudin treated patients (58.3% female) fulfilled the same inclusion and exclusion criteria. In a time-to-event-analysis the cumulative risk of combined endpoint was higher in HIT-patients without thromboembolic complication at baseline treated with danaparoid (usually in prophylactic dose 750 anti-factor Xa units b.i.d. or t.i.d.s.c.) as compared to lepirudin (aPTT adjusted) (P = 0.020). Whereas HIT-patients with TEC at baseline who were usually treated with therapeutic dose had a similar outcome in both treatment groups (P = 0.913). Major bleeding occurred in 2.5% (95% CI 0.5-7.0%) of danaparoid treated patients as compared to 10.4% (95% CI 6.3-15.9%) of lepirudin treated patients until day 42 (P = 0.009). This indicates that the efficacies of therapeutic doses of danaparoid or lepirudin in preventing death, amputation or new TEC in HIT-patients do not differ largely, but the risk of bleeding seems to be higher in lepirudin treated patients. The prophylactic dose of danaparoid approved in the European Union for HIT without TEC at baseline seems suboptimal. A prospective comparative trial is required to verify these preliminary conclusions.
Asunto(s)
Anticoagulantes/normas , Sulfatos de Condroitina/normas , Dermatán Sulfato/normas , Heparina/efectos adversos , Heparitina Sulfato/normas , Hirudinas/análogos & derivados , Hirudinas/normas , Proteínas Recombinantes/normas , Trombocitopenia/tratamiento farmacológico , Factores de Edad , Anciano , Amputación Quirúrgica/estadística & datos numéricos , Anticoagulantes/administración & dosificación , Sulfatos de Condroitina/administración & dosificación , Dermatán Sulfato/administración & dosificación , Combinación de Medicamentos , Evaluación de Medicamentos , Femenino , Hemorragia/etiología , Heparitina Sulfato/administración & dosificación , Hirudinas/administración & dosificación , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Estudios Retrospectivos , Encuestas y Cuestionarios , Trombocitopenia/inducido químicamente , Trombocitopenia/complicaciones , Trombocitopenia/mortalidad , Tromboembolia/etiología , Resultado del TratamientoRESUMEN
Heparin-induced thrombocytopenia (HIT) has become more prevalent in today's cardiac setting and has resulted in the need for alternative anticoagulant therapies. Danaparoid sodium, one alternative to heparin, has been used in six cardiopulmonary bypass procedures in this hospital. This clinical experience has resulted in the progressive refinement of a protocol for the 'safe' clinical use of danaparoid sodium. Although there were six positive outcomes with the use of danaparoid sodium, alternatives must be explored in order to find the optimal anticoagulant for the treatment of HIT.