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1.
Haemophilia ; 24(4): 619-627, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29855112

RESUMEN

INTRODUCTION: For individuals with haemophilia A, prophylaxis with factor VIII (FVIII) is typically directed towards trough activity >1 IU/dL; however, some patients still experience spontaneous bleeding events (sBEs). AIM: Aims were to evaluate relationships of endogenous thrombin potential (ETP) and FVIII:C with occurrence of clinical bleeding. METHODS: GENA-21 was a prospective, open-label, phase IIIb study investigating the safety and efficacy of Nuwiq® (human-cl rhFVIII) in previously treated adults with severe haemophilia A. The study included a 72-hour pharmacokinetic (PK) evaluation phase and a 6-month personalized prophylaxis phase in which treatment was guided by PK parameters. This subanalysis assessed FVIII:C by one-stage assay and ETP by thrombin generation assay in blood samples. RESULTS: Baseline mean ETP was lower in the 7 patients who experienced sBEs during personalized prophylaxis versus 25 who did not (n = 32 with data from PK phase and prophylaxis phase; P = .0002). During personalized prophylaxis (n = 49), only patients with lower median trough ETP experienced sBEs (8/49 patients; ROC AUC = 0.9421; P < .0001); there was no significant relationship for FVIII:C in predicting sBEs (ROC AUC = 0.5838; P = .4750). Directly following infusion of human-cl rhFVIII, ETP was lower in patients who experienced sBEs versus those who did not (P = .0002), whereas FVIII:C did not differ significantly between these groups. CONCLUSIONS: In adults with severe haemophilia A and reduced thrombin generation, increased frequency of spontaneous bleeding was observed irrespective of trough FVIII levels. Thus, personalized prophylaxis should take into account variables other than FVIII:C. Large prospective trials are needed to verify ETP as a marker for spontaneous bleeding.


Asunto(s)
Factor VIII/uso terapéutico , Hemofilia A/complicaciones , Hemofilia A/tratamiento farmacológico , Hemorragia/complicaciones , Medicina de Precisión , Proteínas Recombinantes/uso terapéutico , Trombina/biosíntesis , Adulto , Factor VIII/farmacología , Femenino , Hemofilia A/metabolismo , Hemorragia/prevención & control , Humanos , Masculino , Proteínas Recombinantes/farmacología
2.
Haemophilia ; 24(4): 595-603, 2018 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-29582516

RESUMEN

INTRODUCTION: Nuwiq® (human-cl rhFVIII, simoctocog alfa) is a 4th generation recombinant human FVIII, without chemical modification or fusion with any other protein, produced in a human cell line. AIM/METHODS: This study (GENA-13) was an extension of the GENA-03 study in which previously treated children aged 2-12 years with severe haemophilia A received Nuwiq® prophylaxis for ≥6 months. GENA-13 examined long-term tolerability, immunogenicity and efficacy of Nuwiq® prophylaxis in children. RESULTS: Of 59 patients enrolled in GENA-03, 49 continued Nuwiq® prophylaxis in GENA-13 for a median (range) of 30.0 (9.5-52.0) months. No patient withdrew due to drug-related adverse events or developed inhibitors. Only 2 of 20 518 infusions were associated with possibly related adverse events (dyspnoea, fever). The estimated annualized bleeding rate (ABR) was 0.67 (95% CI: 0.44, 1.02) for spontaneous and 2.88 (95% CI: 1.86, 4.46) for all bleeds. Younger children (2-5 years) had lower ABRs than children aged 6-12 years. Annualized bleeding rates were reduced in GENA-13 vs GENA-03, especially for spontaneous bleeds in younger children (71% reduction; ABR ratio 0.29 [95% CI: 0.11, 0.74]). Nuwiq® efficacy was rated as excellent/good in the treatment of 83.0% of 305 evaluated breakthrough bleeds. Surgical prophylaxis with Nuwiq® was rated as excellent for all 17 assessed procedures. CONCLUSION: Long-term treatment with Nuwiq® for the prevention of bleeds in children with severe haemophilia A was well tolerated, effective and reduced spontaneous bleeding by up to 70% compared with GENA-03.


Asunto(s)
Factor VIII/efectos adversos , Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Hemofilia A/inmunología , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Seguridad , Niño , Preescolar , Factor VIII/inmunología , Femenino , Hemofilia A/complicaciones , Hemorragia/complicaciones , Hemorragia/prevención & control , Humanos , Masculino , Proteínas Recombinantes/inmunología , Índice de Severidad de la Enfermedad , Resultado del Tratamiento
3.
Haemophilia ; 24(1): 70-76, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29048712

RESUMEN

INTRODUCTION: Haemophilia A patients are at a high risk of excess bleeding during surgeries. The aim of haemostatic therapy during the perioperative period is to normalize FVIII level perioperatively and postoperatively to maintain normal haemostasis until wound healing is complete. AIMS/METHODS: To examine the efficacy of Nuwiq® (simoctocog alfa, human-cl rhFVIII), a 4th generation recombinant FVIII produced in a human cell line, for surgical prophylaxis in patients with severe haemophilia A. This analysis assessed the efficacy of Nuwiq® during surgical procedures and in the postoperative period in seven clinical studies of previously treated patients (PTPs) with severe haemophilia A. RESULTS: Thirty-six patients, aged 3-55 years, received surgical prophylaxis with Nuwiq® for 60 surgeries (28 major and 32 minor). Efficacy was evaluated for 52 surgeries (25 major and 27 minor). The success rate of Nuwiq® treatment was 98.1% (51 of 52 evaluated surgeries); haemostatic efficacy was assessed as "excellent" or "good" in all but one major surgery (assessed as "moderate"). The number of infusions ranged from 1 to 19 for minor surgeries and from 3 to 76 for major surgeries. The median (range) daily doses were 42.0 (28.2-100.9) IU kg-1 for minor surgeries and 69.3 (43.3-135.6) IU kg-1 for major surgeries. There were no serious treatment-related adverse events, and none of the patients developed FVIII inhibitors. CONCLUSIONS: The results of this pooled analysis show that Nuwiq® was efficacious in maintaining haemostasis during and after major and minor surgical procedures in PTPs with severe haemophilia A.


Asunto(s)
Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Adulto , Niño , Preescolar , Factor VIII/efectos adversos , Hemofilia A/patología , Hemofilia A/cirugía , Humanos , Masculino , Persona de Mediana Edad , Atención Perioperativa , Cuidados Posoperatorios , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Índice de Severidad de la Enfermedad , Resultado del Tratamiento
4.
Haemophilia ; 23(5): 697-704, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28452151

RESUMEN

INTRODUCTION: Nuwiq® (human-cl rhFVIII) is a 4th generation recombinant human FVIII, without chemical modification or protein fusion, produced in a human cell-line. AIMS/METHODS: This study (NuPreviq) was a prospective, open-label, multicentre, phase IIIb study of the efficacy and safety of personalized prophylaxis with Nuwiq® in 66 previously treated adults with severe haemophilia A. NuPreviq had three phases: (i) a 72-h pharmacokinetic (PK) phase; (ii) a 1-3 month standard prophylaxis phase; and (iii) a 6-month personalized prophylaxis phase. The personalized prophylaxis regimen was based on individual PK modelling for each patient according to whether their PK profile most closely fitted a two- or one-compartment model (NuPreviq approach). In cases of uncertainty, a noncompartment model was applied. RESULTS: The median dosing interval during personalized prophylaxis was 3.5 days, with 57% of patients on ≤2 weekly dosing. Mean annualized bleeding rates during personalized prophylaxis were 1.45 (median [interquartile range, IQR]: 0 [0, 1.9]) for all bleeds, 0.79 (median [IQR]: 0 [0, 0]) for spontaneous bleeds, and 0.91 (median [IQR]: 0 [0, 0]) for joint bleeds. During personalized prophylaxis, 83.1% of patients were spontaneous bleed-free. Compared with standard prophylaxis, median weekly prophylaxis dose was reduced by 7.2% from 100.0 to 92.8 IU kg-1 during the last 2 months of personalized prophylaxis. There were no FVIII inhibitors or treatment-related serious or severe adverse events. CONCLUSION: PK-guided personalized prophylaxis with Nuwiq® provided bleeding protection and enabled the dosing interval to be extended to twice weekly or less in many patients and an overall dose reduction.


Asunto(s)
Factor VIII/uso terapéutico , Hemofilia A/diagnóstico , Hemofilia A/tratamiento farmacológico , Hemorragia/prevención & control , Proteínas Recombinantes/uso terapéutico , Adulto , Monitoreo de Drogas , Factor VIII/administración & dosificación , Factor VIII/farmacocinética , Semivida , Hemofilia A/complicaciones , Humanos , Masculino , Medicina de Precisión/métodos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proyectos de Investigación , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto Joven
5.
Haemophilia ; 22(2): 225-231, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26315974

RESUMEN

INTRODUCTION: Nuwiq® [human cell line-derived recombinant factor VIII (human-cl rhFVIII)] is a new generation rFVIII protein, without chemical modification or fusion to any other protein, produced in a human cell line. AIM/METHODS: This prospective, open-label, multinational phase III study assessed the efficacy and safety of human-cl rhFVIII in 32 adult previously treated patients (PTPs) with severe haemophilia A during standard prophylaxis for ≥6 months and ≥50 exposure days. Efficacy in treating bleeds and during surgical prophylaxis was also assessed. RESULTS: Prophylactic efficacy, based on mean monthly bleeding rate, was rated as 'excellent' or 'good' in 97% of patients for all bleeds and in 100% of patients for spontaneous bleeds. Mean (SD) annualized bleeding rate was 2.28 (3.73) [median = 0.9] for all bleeds, 1.16 (2.57) [median = 0] for spontaneous bleeds and 1.00 (1.79) [median = 0] for traumatic bleeds. There were no bleeds in 50% of patients and there were no major, life-threatening bleeds. Efficacy was 'excellent' or 'good' in treating 28 (100%) of 28 bleeds. Overall efficacy was rated as 'excellent' during four surgical procedures (three major, one minor) and 'moderate' during one major surgery. Incremental in vivo recovery (IVR) data were comparable with the one-stage and chromogenic assays. IVR was >2.0% per IU kg-1 for all measurements and stable over 6 months. No patients developed FVIII inhibitors and there were no treatment-related serious or severe adverse events. CONCLUSION: These results in adult PTPs indicate that human-cl rhFVIII is effective for the prevention and treatment of bleeds in adults with severe haemophilia A.

6.
Haemophilia ; 22(3): 374-80, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26582282

RESUMEN

INTRODUCTION: Haemophilia A is treated with FVIII, either prophylactically or on demand. Prophylaxis is the gold standard in children and evidence is accumulating in adults. AIMS/METHODS: The aim of this analysis was to compare prophylaxis vs. on-demand treatment with Nuwiq(®) (Human-cl rhFVIII), a new-generation rFVIII expressed in a human cell line, in previously treated patients (PTPs) with severe haemophilia A. Data were analysed from two similarly designed, multinational, prospective, open-label studies with similar inclusion and exclusion criteria and comparable patient demographics. Human-cl rhFVIII was administered either prophylactically in a study of 32 adults or on-demand in a study of 22 patients (20 adults and two adolescents). RESULTS: Patients treated prophylactically experienced 36 bleeds compared with 997 bleeds in patients treated on-demand (mean observation periods: 180 and 335 days respectively). Based on a negative binomial regression model, annualized bleeding rate (ABR) during prophylaxis was 2.30 (95% CI: 1.54, 3.44) compared with 57.74 (95% CI: 43.36, 76.91) during on-demand treatment, which equates to a 96% lower ABR during prophylaxis. 'Excellent' or 'good' efficacy in the treatment of bleeds was achieved with Human-cl rhFVIII in 100% of 28 evaluated bleeds during the prophylaxis study and 94.5% of 985 evaluated bleeds during the on-demand study. No inhibitors, treatment-related serious adverse events or severe adverse events were recorded during prophylaxis or or-demand treatment. CONCLUSIONS: Prophylaxis with Human-cl rhFVIII reduces recurrent bleeding in adult PTPs with severe haemophilia A and adds further supportive evidence for the benefits of prophylaxis in adults.


Asunto(s)
Coagulantes/uso terapéutico , Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Adolescente , Adulto , Anciano , Niño , Esquema de Medicación , Factor VIII/genética , Factor VIII/metabolismo , Hemofilia A/patología , Hemorragia/epidemiología , Hemorragia/prevención & control , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto Joven
7.
Scand J Rheumatol ; 44(4): 280-3, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25993023

RESUMEN

OBJECTIVES: Chronic anterior uveitis is a serious complication of juvenile idiopathic arthritis (JIA); disease flares are highly associated with loss of vision. Leflunomide (LEF) is used successfully for JIA joint disease but its effectiveness in uveitis has not been determined. The aim of this study was to determine whether LEF improves flare rates of uveitis in JIA patients compared to preceding methotrexate (MTX) therapy. METHOD: A single-centre retrospective study of consecutive children with JIA and chronic anterior uveitis was performed. All children initially received MTX and were then switched to LEF. Demographic, clinical, and laboratory data, dose and duration of MTX and LEF therapy, concomitant medications and rate of anterior uveitis flares, as determined by an expert ophthalmologist, were obtained. Flare rates were compared using a generalized linear mixed model with a negative binomial distribution. RESULTS: A total of 15 children were included (80% females, all antinuclear antibody positive). The median duration of MTX therapy was 51 (range 26-167) months; LEF was given for a median of 12 (range 4-47) months. Anti-tumour necrosis factor (anti-TNF-α) co-medication was given to four children while on MTX. By contrast, LEF was combined with anti-TNF-α treatment in six children. On MTX, JIA patients showed a uveitis flare rate of 0.0247 flares/month, while LEF treatment was associated with a significantly higher flare rate of 0.0607 flares/month (p = 0.008). CONCLUSIONS: Children with JIA had significantly more uveitis flares on LEF compared to MTX despite receiving anti-TNF-α co-medication more frequently. Therefore, LEF may need to be considered less effective in controlling chronic anterior uveitis.


Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Juvenil/complicaciones , Isoxazoles/uso terapéutico , Metotrexato/uso terapéutico , Uveítis/tratamiento farmacológico , Uveítis/etiología , Niño , Preescolar , Enfermedad Crónica , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Humanos , Lactante , Leflunamida , Masculino , Recurrencia , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores
8.
Food Chem Toxicol ; 45(8): 1428-36, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17376579

RESUMEN

Aim of the study was to investigate the impact of coffee on DNA-stability in humans. DNA-damage was monitored in lymphocytes of eight individuals with single cell gel electrophoresis assays before and after consumption of 600 ml coffee (400 ml paper filtered and 200 ml metal filtered/d) for five days. Under standard conditions, no alteration of DNA-migration was seen, but a strong reduction of DNA-migration attributable to endogenous formation of oxidised purines and pyrimidines was detected with restriction enzymes; furthermore DNA-damage caused by reactive oxygen radicals (H2O2 treatment) and by the heterocyclic aromatic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole-acetate was significantly reduced after coffee consumption by 17% and 35%, respectively. Also in in vitro experiments, inhibition of H2O2 induced DNA-damage was observed with coffee at low concentrations (

Asunto(s)
Carbolinas/envenenamiento , Café , Daño del ADN , ADN/efectos de los fármacos , Linfocitos/efectos de los fármacos , Adulto , Supervivencia Celular/efectos de los fármacos , Ensayo Cometa , ADN/metabolismo , Diterpenos/farmacología , Electroforesis , Glutatión Peroxidasa/metabolismo , Humanos , Linfocitos/metabolismo , Persona de Mediana Edad , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo
9.
J Physiol Pharmacol ; 56 Suppl 2: 49-64, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16077190

RESUMEN

It is well documented that reactive oxygen species (ROS) are involved in the aetiology of age related diseases. Over the last decades, strong efforts have been made to identify antioxidants in human foods and numerous promising compounds have been detected which are used for the production of supplements and functional foods. The present paper describes the advantages and limitations of methods which are currently used for the identification of antioxidants. Numerous in vitro methods are available which are easy to perform and largely used in screening trials. However, the results of such tests are only partly relevant for humans as certain active compounds (e.g. those with large molecular configuration) are only poorly absorbed in the gastrointestinal tract and/or may undergo metabolic degradation. Therefore experimental models are required which provide information if protective effects take place in humans under realistic conditions. Over the last years, several methods have been developed which are increasingly used in human intervention trials. The most widely used techniques are chemical determinations of oxidised guanosine in peripheral blood cells or urine and single cell gel electrophoresis (comet) assays with lymphocytes which are based on the measurement of DNA migration in an electric field. By using of DNA-restriction enzymes (formamidopyrimidine DNA glycosylase and endonuclease III) it is possible to monitor the endogenous formation of oxidised purines and pyrimidines; recently also protocols have been developed which enable to monitor alterations in the repair of oxidised DNA. Alternatively, also the frequency of micronucleated cells can be monitored with the cytokinesis block method in peripheral human blood cells before and after intervention with putative antioxidants. To obtain information on alterations of the sensitivity towards oxidative damage, the cells can be treated ex vivo with ROS (H(2)O(2) exposure, radiation). The evaluation of currently available human studies shows that in approximately half of them protective effects of dietary factors towards oxidative DNA-damage were observed. Earlier studies focused predominantly on the effects of vitamins (A, C, E) and carotenoids, more recently also the effects of fruit juices (from grapes, kiwi) and beverages (soy milk, tea, coffee), vegetables (tomato products, berries, Brussels sprouts) and other components of the human diet (coenzyme Q(10), polyunsaturated fatty acids) were investigated. On the basis of the results of these studies it was possible to identify dietary compounds which are highly active (e.g. gallic acid). At present, strong efforts are made to elucidate whether the different parameters of oxidative DNA-damage correlates with life span, cancer and other age related diseases. The new techniques are highly useful tools which provide valuable information if dietary components cause antioxidant effects in humans and can be used to identify individual protective compounds and also to develop nutritional strategies to reduce the adverse health effects of ROS.


Asunto(s)
Envejecimiento/metabolismo , Antioxidantes/farmacología , Bioensayo/métodos , Técnicas de Química Analítica/métodos , Daño del ADN/efectos de los fármacos , Pruebas de Mutagenicidad , Estrés Oxidativo/efectos de los fármacos , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Células Cultivadas , Aberraciones Cromosómicas/efectos de los fármacos , Ensayo Cometa , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Determinación de Punto Final , Humanos , Longevidad , Micronúcleos con Defecto Cromosómico/efectos de los fármacos , Modelos Animales , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados
10.
Toxicology ; 198(1-3): 315-28, 2004 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-15138058

RESUMEN

This article gives an overview of the results of genotoxicity tests, which have been conducted within the last 5 years with the human liver cell line HepG2. It is an update of an earlier review from 1998 (by Knasmüller et al.). In addition, a number of publications are discussed which are relevant for the use of human derived liver cell lines in genetic toxicology. They concern the establishment of new endpoints, the development of new cell lines and possible pitfalls and problems. HepG2 cells have been used to test a wide variety of compounds over the last years. The most interesting observations are that the cells are highly sensitive toward polycyclic aromatic hydrocarbons and that genotoxic effects are seen with a number of carcinogenic mycotoxins, that give negative results in other in vitro assays. Carcinogenic metals such as As and Cd caused positive results as well, whereas only marginal or negative results were seen with nitrosamines. The low sensitivity toward these latter carcinogens is probably due to a lack of cytochrome P4502E1 which catalyses their activation. Also, a number of structurally different synthetic pesticides as well as bioactive plant constituents ("natural pesticides") have been tested and with some of them genotoxic effects were found. In most experiments, the formation of micronuclei was used as an endpoint; however also the single cell gel electrophoresis assay is increasingly used. Several transfectant lines of HepG2 have been constructed which express increased levels of phase I enzymes (such as CYP1A1, CYP1A2, CYP2E1 etc.); furthermore, cell lines became available which express human glutathione-S-transferases. These new clones might be particularly useful for the investigation of specific classes of genotoxicants and also for mechanistic studies. Apart from HepG2 cells, a number of other human derived liver cell lines have been isolated, but so far no data from genotoxicity experiments are available, except for Hep3B cells, which were compared with HepG2 and found to be less sensitive in general. Studies with HepG2 clones of a different origin indicate that the cells differ in regard to their sensitivity toward genotoxicants; also medium effects and the cultivation time might affect the outcome of genotoxicity studies. Overall, the results support the assumption that HepG2 cells are a suitable tool for genotoxicity testing.


Asunto(s)
Hígado/efectos de los fármacos , Micotoxinas/toxicidad , Plaguicidas/toxicidad , Extractos Vegetales/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Línea Celular , Reparación del ADN , Contaminación de Alimentos , Humanos , Hígado/enzimología , Hígado/metabolismo , Micronúcleos con Defecto Cromosómico , Pruebas de Mutagenicidad
11.
Transfus Med ; 14(2): 165-71, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15113381

RESUMEN

In this first-in-man study, we assessed the pharmacokinetics, safety and tolerability of MonoRho, a human recombinant monoclonal anti-RhD immunoglobulin G1 (IgG1) antibody. Eighteen RhD-negative healthy male volunteers were randomized in two groups to receive a single administration of 300 micro g of MonoRho either intravenously or intramuscularly. There were no symptoms of allergic or anaphylactic type reaction in any subject, and there was no evidence of any MonoRho-related changes in laboratory safety parameters. None of the subjects mounted a detectable immune response to MonoRho. Serum samples were obtained up to 91 days after injection to measure anti-D IgG concentrations by flow cytometry. After intramuscular administration of MonoRho, anti-D IgG concentrations gradually increased reaching peak levels after a mean of 3.4 days. After 3 weeks, the mean anti-D IgG concentrations after intravenous and intramuscular administration became virtually equal to each other and remained so thereafter. In both the treatment groups, the mean elimination half-life was about 18 days and thus similar to that described for plasma-derived anti-D IgG. The bioavailability of MonoRho after intramuscular administration was estimated as 46%. The excellent tolerability and safety of MonoRho as well as its expected elimination half-life supports the continued clinical development of this compound.


Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Fragmentos de Inmunoglobulinas/administración & dosificación , Isoanticuerpos/sangre , Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Adulto , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Disponibilidad Biológica , Semivida , Humanos , Fragmentos de Inmunoglobulinas/efectos adversos , Inmunoglobulina G , Masculino , Farmacocinética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinética
12.
Br J Haematol ; 111(1): 157-66, 2000 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11091196

RESUMEN

Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.


Asunto(s)
Biotecnología/métodos , Fragmentos Fab de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Globulina Inmune rho(D)/metabolismo , Animales , Bacteriófagos , Secuencia de Bases , Bromelaínas/farmacología , Células CHO , Clonación Molecular , Cricetinae , Eritrocitos , Humanos , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Isoinmunización Rh/prevención & control
13.
Circulation ; 97(6): 581-8, 1998 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-9494029

RESUMEN

BACKGROUND: Arterial injury after percutaneous transluminal coronary angioplasty (PTCA) triggers acute thrombus formation and thrombin generation. Hirudin, a potent and direct thrombin inhibitor, prevents thrombus formation after arterial injury. Two large clinical trials showed marked reduction in acute clinical events but no long-term benefits in reducing restenosis during short-term administration of thrombin inhibitors. Our hypothesis is that adequate, maintained thrombin inhibition, by inhibiting all the thrombin-dependent mechanisms, will reduce neointima formation after PTCA. METHODS AND RESULTS: Thirty-six pigs received three different regimens of hirudin: bolus (1 mg/kg), short-term (bolus + 0.7 mg/kg per day for 2 days), and long-term (bolus + 0.7 mg/kg per day for 14 days). The results on neointima formation at 4 weeks after coronary angioplasty were compared with the control group (100 IU heparin/kg bolus). Hirudin was continuously administered for 2 weeks through an infusion pump. In vivo thrombin generation was persistently increased up to 2 weeks after angioplasty. Inhibition of thrombin activity for 14 days reduced luminal narrowing by 40% (58+/-3% versus 35+/-3%; P<.001). No differences were observed among the bolus and short-term hirudin groups and the control group. CONCLUSIONS: Our results indicate that there is a continued, marked thrombin generation that lasts for at least 2 weeks after PTCA. Administration of r-hirudin for 2 weeks significantly reduces neointima formation after PTCA. This observation, if extrapolated to humans, could explain the lack of effect on restenosis observed in the clinical trials with antithrombin agents despite the clear benefits on reducing acute thrombotic complications after PTCA. Therefore an adequate and prolonged administration of thrombin inhibitors is needed to "passivate" the thrombogenic substrate (disrupted arterial wall) and achieve full benefit of this therapeutic approach.


Asunto(s)
Angioplastia Coronaria con Balón/efectos adversos , Enfermedad Coronaria/terapia , Vasos Coronarios/lesiones , Trombina/antagonistas & inhibidores , Animales , Antitrombinas/metabolismo , Antitrombinas/farmacología , Enfermedad Coronaria/patología , Enfermedad Coronaria/prevención & control , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/patología , Hirudinas/sangre , Hirudinas/farmacología , Tiempo de Tromboplastina Parcial , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacología , Recurrencia , Porcinos , Trombina/metabolismo
14.
Thromb Res ; 88(6): 473-80, 1997 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-9610958

RESUMEN

Previous work has shown that pre-treatment with the thrombin inhibitor recombinant desulfato-hirudin prevented fibrin formation and respiratory dysfunction in porcine lipopolysaccharide shock. We examined the effects of delayed administration of recombinant desulfato-hirudin in bacterial lipopolysaccharide shock. Miniature pigs were studied under anaesthesia and ventilation, and received a bacterial lipopolysaccharide infusion (2 microg/kg/h) for 7 h; recombinant desulfato-hirudin was started 1 h after bacterial lipopolysaccharide in 10 animals (bolus 12.9 nmol/kg; continuous infusion 6.5 nmol/ kg/h); 10 randomised control animals received saline instead of recombinant desulfato-hirudin. Fibrin and thrombin-antithrombin complex levels in plasma were significantly lower in bacterial lipopolysaccharide+recombinant desulfato-hirudin animals than in controls. Both groups displayed a similar rise in pulmonary vascular resistance and other parameters of lung dysfunction; only lung tissue wet/dry ratio was lower in recombinant desulfato-hirudin-treated than in control animals. Both groups had similar circulatory alterations. Recombinant desulfato-hirudin interrupted coagulation activation during ongoing bacterial lipopolysaccharide-induced shock in pigs even when administered with a delay of one hour after start of the bacterial lipopolysaccharide infusion. A protective effect of delayed recombinant desulfato-hirudin administration on bacterial lipopolysaccharide-induced acute lung injury and alterations in the systemic circulation could not be demonstrated in this experiment.


Asunto(s)
Fibrina/biosíntesis , Fibrinolíticos/uso terapéutico , Hirudinas/análogos & derivados , Lipopolisacáridos , Choque Séptico/tratamiento farmacológico , Porcinos Enanos , Animales , Ascitis , Sistema Cardiovascular/efectos de los fármacos , Terapia con Hirudina , Riñón/efectos de los fármacos , Pulmón/efectos de los fármacos , Cavidad Peritoneal , Proteínas Recombinantes/uso terapéutico , Porcinos , Factores de Tiempo
16.
Biochemistry ; 34(34): 10872-8, 1995 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-7662668

RESUMEN

Advanced glycation end products (AGEs) and glycoxidation products are formed during Maillard or browning reactions between sugars and proteins and are implicated in the pathophysiology of aging and the complications of diabetes. To determine the structure of AGEs, antibodies were prepared to protein browned by incubation with glucose and used in ELISA assays to measure AGEs formed in model reactions between bovine serum albumin (BSA) or N alpha-acetyllysine and glucose, fructose, or glyoxal. AGEs were formed from glucose and fructose only under oxidative conditions, but from glyoxal under both oxidative and antioxidative conditions. Gel permeation chromatographic analysis indicated that a similar AGE was formed in reactions of N alpha-acetyllysine with glucose, fructose, and glyoxal and that this AGE co-eluted with authentic N alpha-acetyl-N epsilon-(carboxymethyl)lysine. Amino acid analysis of AGE proteins revealed a significant content of N epsilon-(carboxymethyl)lysine (CML). In ELISA assays using polyclonal antibodies against AGE proteins, CML-BSA (approximately 25 mol of CML/mol of BSA), prepared by chemical modification of BSA, was a potent inhibitor of the recognition of AGE proteins and of AGEs in human lens proteins. We conclude that AGEs are largely glycoxidation products and that CML is a major AGE recognized in tissue proteins by polyclonal antibodies to AGE proteins.


Asunto(s)
Productos Finales de Glicación Avanzada/química , Lisina/análogos & derivados , Reacción de Maillard , Envejecimiento/fisiología , Antígenos/análisis , Antígenos/inmunología , Cromatografía en Gel , Cristalinas/química , Ensayo de Inmunoadsorción Enzimática , Fructosa/química , Glucosa/química , Productos Finales de Glicación Avanzada/inmunología , Glioxal/química , Humanos , Cinética , Lisina/análisis , Lisina/química , Lisina/inmunología , Albúmina Sérica Bovina/química
17.
Biochemistry ; 34(29): 9277-81, 1995 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-7626597

RESUMEN

Platelet accretion at sites of vascular injury yields a neo-tissue comprising packed platelets and having an interstitial space not supplied with blood. Within growing thrombi platelet masses become anoxic and depolarize to yield interstitial cation concentrations characteristic of the more voluminous platelet cytosol, with extracellular [Ca2+] falling below that adequate to support the plasma clotting system. The platelet-associated clotting system reactivates during disaggregation of the thrombi in vitro, which proceeds with high yield of apparently basal, functional platelets when specific anticoagulants are included in the disaggregating media. The capacity of regulatory demand to lower extracellular [Ca2+] in the microenvironment of platelet aggregates provides a physiological basis for evolution of the highly cooperative calcium interactions of the hemostasis system.


Asunto(s)
Plaquetas/fisiología , Calcio/farmacología , Agregación Plaquetaria/fisiología , Trombina/metabolismo , Adenosina Trifosfato/sangre , Animales , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Arterias Carótidas/patología , Arterias Carótidas/fisiología , Hirudinas/farmacología , Hipoxia/sangre , Hipoxia/fisiopatología , Cinética , Agregación Plaquetaria/efectos de los fármacos , Proteínas Recombinantes/farmacología , Porcinos , Trombina/efectos de los fármacos , Trombosis/patología , Trombosis/fisiopatología
18.
Circulation ; 90(6): 2671-8, 1994 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-7994807

RESUMEN

BACKGROUND: The degree to which antithrombotic drugs suppress thrombin generation is unknown. Because hirudin, unlike antithrombin III, binds intravascular thrombin rapidly and selectively to yield a circulating inactive complex of 3- to 4-hour half-life, we used intravenous hirudin in humans to investigate the course of thrombin generation during and early after anticoagulation with this potent, direct antithrombin. METHODS AND RESULTS: Intravascular thrombin was measured with an ELISA for the thrombin-hirudin complex formed during and for 18 hours after stopping a 6-hour infusion of hirudin at 0.1, 0.2, and 0.3 mg.kg-1.h-1 in three groups of six patients each. With free hirudin in 20- to 10,000-fold molar excess of thrombin and peak activated partial thromboplastin times of 2.3 to 3.0 times baseline, mean plasma thrombin-hirudin complex increased from 794 +/- 85 pg/mL (mean +/- SEM) 15 minutes after the start of the infusion to 1617 +/- 151 pg/mL at 6 hours of infusion to 2667 +/- 654 pg/mL at 24 hours. During the 24-hour observation period, plasma concentration of fragment 1.2 (the peptide released during conversion of prothrombin to thrombin) never fell below baseline but rather increased transiently during the hirudin infusion. Plasma concentrations of thrombin-antithrombin III complex (in ng/mL) decreased from 4.34 +/- 0.40 at baseline to 1.64 +/- 0.13 at 6 hours (P < .001) and gradually increased after stopping the infusion to 5.7 +/- 0.87 at 24 hours (nonsignificant compared with baseline). CONCLUSIONS: Measurement of thrombin-hirudin complex may be used as a marker of thrombin generation in humans. Persistent accumulation of thrombin-hirudin complex and generation of fragment 1.2 during and after completion of potent anticoagulation with hirudin suggest thrombin generation is not blocked by high-affinity thrombin inhibition. The persistent formation of thrombin during declining plasma levels of hirudin may contribute to the pathogenesis of rethrombosis early after antithrombin therapy or during inadequate anticoagulation.


Asunto(s)
Hirudinas/farmacología , Trombina/antagonistas & inhibidores , Trombina/biosíntesis , Anciano , Antitrombina III/análisis , Ensayo de Inmunoadsorción Enzimática , Hirudinas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Concentración Osmolar , Fragmentos de Péptidos/análisis , Péptido Hidrolasas/análisis , Protrombina/análisis , Trombina/metabolismo
19.
Coron Artery Dis ; 5(11): 943-9, 1994 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-7719527

RESUMEN

BACKGROUND: As a result of their polypeptidic nature, hirudins could theoretically elicit an immunologic response in humans. METHODS: The present open survey evaluates the allergenic potential of recombinant hirudin CGP 39393 (REVASC) after repeated intravenous or subcutaneous exposures in immunocompetent volunteers with no known previous exposure to hirudins. Clinical signs and symptoms of allergic manifestations and surrogate markers of allergy (i.e. response to skin tests) were collected before and after each administration of CGP 39393. Hirudin-specific immunoglobulin (Ig)G or IgE antibodies were measured. RESULTS: Two hundred and sixty-three healthy volunteers were eligible, of whom 12.2% had a history of allergy and 18.3% had a high level of total IgE. No signs or symptoms of allergy directly attributable to CGP 39393 were reported, either during or immediately after a first challenge. Irritative skin reactions, to either the prick or the intradermal skin test, were observed in eight volunteers 28-56 days after the challenge. Three out of 200 volunteers exposed to a second course of CGP 39393 showed signs and symptoms of an allergic reaction. In all but one subject with a pruritic erythema it was possible to rule out a causative role for CGP 39393 (0.50%, 95% confidence limits [CL] 0.01-2.75). Three other volunteers displayed a suspect or a positive immediate-type skin reaction to the follow-up intradermal skin test without any signs or symptoms of allergy. Another asymptomatic volunteer (0.80%, 95% CL: 0.02-4.41) developed a low [i.e. 1+ on the radio-allergosorbent test (RAST) scale] but measurable titre of hirudin-specific IgE antibodies after the second exposure to CGP 39393. A third exposure in five volunteers was clinically uneventful. A positive reaction to the prick test and to the intradermal skin test was observed in one individual. CONCLUSIONS: Recombinant hirudin CGP 39393 appears to be a weak allergen. Repeated exposures are safe in fully immunocompetent subjects, including those with a history of previous allergies and high levels of total IgE. Type I allergic reactions are rare (i.e. less than 1%) after a second exposure and are limited to the skin. Routine skin tests are not needed to identify patients at risk of developing type I allergic reactions. Hirudin-specific IgE antibodies are rarely seen and then at very low titres.


Asunto(s)
Erupciones por Medicamentos/inmunología , Fibrinolíticos/inmunología , Hirudinas/análogos & derivados , Hipersensibilidad Inmediata/inducido químicamente , Adulto , Relación Dosis-Respuesta Inmunológica , Erupciones por Medicamentos/sangre , Erupciones por Medicamentos/diagnóstico , Europa (Continente) , Femenino , Fibrinolíticos/administración & dosificación , Fibrinolíticos/efectos adversos , Hirudinas/administración & dosificación , Hirudinas/efectos adversos , Hirudinas/inmunología , Humanos , Hipersensibilidad Inmediata/sangre , Hipersensibilidad Inmediata/diagnóstico , Hipersensibilidad Inmediata/inmunología , Inmunocompetencia , Inmunoglobulina E/sangre , Inmunoglobulina E/efectos de los fármacos , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/efectos de los fármacos , Inmunoglobulina G/inmunología , Infusiones Intravenosas , Inyecciones Intradérmicas , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/inmunología , Índice de Severidad de la Enfermedad , Pruebas Cutáneas
20.
Biochem J ; 296 ( Pt 3): 771-6, 1993 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-8280076

RESUMEN

The metabolic fate of the anticoagulant protein, hirudin, and its complex with thrombin are presently unknown. Therefore we have labelled hirudin and human thrombin-hirudin complex with the residualizing label dilactitol-125I-tyramine (*I-DLT) in order to identify their tissue sites of catabolism in the rat. The rapid plasma clearance of hirudin after intravenous injection was unaffected by *I-DLT labelling, and by 2 h 6% or less of the injected dose remained in the blood. The majority (80.3 +/- 4.0%, n = 2) of *I-DLT-hirudin radioactivity recovered in tissues was found in kidney, and kidney was also at least 150 times more active in taking up hirudin, on a weight basis, than any other tissue examined (liver, spleen, skin, muscle, intestine, fat, lung). *I-DLT-hirudin which bound to thrombin was isolated by chromatography on concanavalin A-Sepharose; hirudin itself does not bind to concanavalin A. Radioactivity from thrombin-*I-DLT-hirudin was precipitable by anti-thrombin antibody and *I-DLT-thrombin-hirudin was precipitable by anti-hirudin antibody. By 1 h after injection of labelled thrombin-hirudin complexes, the recoveries of radioactivity from hirudin and thrombin in liver were comparable (38.6 +/- 3.0 and 36.4 +/- 4.1%, n = 3), whereas more radioactivity was recovered in kidney from hirudin than from thrombin (27.6 +/- 8.7 compared with 13.6 +/- 4.5%) and less was recovered in lung (0.4 +/- 0.2 compared with 17.7 +/- 2.9%). We conclude that hirudin is catabolized predominantly in kidney, whereas the thrombin-hirudin complex is catabolized by both liver and kidney.


Asunto(s)
Hirudinas/metabolismo , Trombina/metabolismo , Animales , Cromatografía de Afinidad , Concanavalina A , Inhibidores Enzimáticos , Femenino , Hirudinas/aislamiento & purificación , Humanos , Cinética , Pruebas de Precipitina , Ratas , Ratas Sprague-Dawley , Trombina/aislamiento & purificación , Tiramina/análogos & derivados
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