RESUMEN
Background: Dasiglucagon is a novel glucagon analog that is stable in aqueous formulation and approved for use in severe hypoglycemia. Concentration QTc analyses are critical for assessing risk of drug-induced QTc prolongation and potential for fatal cardiac arrhythmias such as torsades de pointes. Objective: The aim of this study was to determine whether dasiglucagon treatment resulted in any clinically relevant effect on cardiac repolarization in healthy volunteers. Methods: This double-blind, placebo-controlled, dose-escalation Phase I trial was conducted at a single center in Germany between November 2018 and June 2019. Sixty healthy volunteers aged 18 to 45 years were randomized within dose cohorts to receive intravenous dasiglucagon, intravenous placebo, or subcutaneous dasiglucagon. In the intravenous administration cohorts, doses ranged from 0.03 mg to 1.5 mg. The subcutaneous administration cohort received the approved 0.6 mg dose. In the intravenous administration cohorts, serial electrocardiograms were extracted from continuous Holter monitors at prespecified time points beginning the day before dosing and through 24 hours postdose. Heart rate, PR interval, and QRS duration were evaluated. Concentration-QT analyses corrected by Fridericia's formula (QTcF) were performed using both a linear mixed-effects and a maximum estimated effect (Emax) model. Results: At the doses studied, dasiglucagon did not have any clinically relevant effect on heart rate, PR interval, or QRS duration. A minor prolongation of the QTcF interval was observed without any clear dose or concentration dependency. Both the linear and Emax models predicted mean and 90% CIs of placebo-corrected change in QTcF remained below 10 ms (the threshold of regulatory concern), although the linear model did not fit the data well at low dasiglucagon plasma concentrations. In the Emax model, the Emax of dasiglucagon was 3.6 ms (90% CI, 1.23-5.95 ms), and the amount to produce half the effect of Emax) was 426.0 pmol/L (90% CI, -48.8 to 900.71 pmol/L). The treatment effect-specific intercept was -0.44 ms (90% CI, -2.37 to 1.49 ms). The most frequently observed treatment-emergent adverse events reported in the trial were gastrointestinal disorders such as nausea and vomiting. Conclusions: Dasiglucagon does not cause clinically relevant QTc prolongation in concentrations up to ≈30,000 pmol/L, a level 5-fold higher than the highest observed plasma concentrations in clinical trials investigating use of the approved 0.6 mg SC dose. ClinicalTrials.gov Identifier: NCT03735225; EudraCT identifier: 2018-002025-32. (Curr Ther Res Clin Exp. 2022; 83:XXX-XXX).
RESUMEN
AIM: To confirm the efficacy and safety of dasiglucagon when administered via an autoinjector device. MATERIALS AND METHODS: In this double-blind trial, 45 participants with type 1 diabetes were randomized 3:1 to receive a single subcutaneous dose of dasiglucagon 0.6 mg or placebo following controlled induction of hypoglycaemia. The primary endpoint was time to plasma glucose recovery, defined as a plasma glucose increase of 20 mg/dL or higher from baseline without rescue intravenous glucose. RESULTS: Median (95% CI) observed time to recovery was 10.0 (8.0; 12.0) minutes for dasiglucagon and 35.0 (20.0; -) minutes for placebo (P < .001). Plasma glucose recovery was achieved within 15 minutes by 88% of participants receiving dasiglucagon versus none receiving placebo (P < .01). Site of injection (buttock or deltoid) was not shown to have any effect on time to recovery (P = .84). No serious adverse events occurred. As expected for glucagon treatment, nausea and vomiting were common adverse events in dasiglucagon-treated participants. CONCLUSIONS: Dasiglucagon provided rapid reversal of hypoglycaemia in adults with type 1 diabetes. Dasiglucagon administration was well tolerated. The aqueous formulation of dasiglucagon in a ready-to-use autoinjector device that can be carried at room temperature may provide a reliable treatment for severe hypoglycaemia.
Asunto(s)
Diabetes Mellitus Tipo 1 , Hipoglucemia , Adulto , Glucemia , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Método Doble Ciego , Glucagón/análogos & derivados , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemia/tratamiento farmacológico , Hipoglucemia/prevención & control , Hipoglucemiantes/efectos adversosRESUMEN
BACKGROUND: Dasiglucagon, a next-generation, ready-to-use aqueous glucagon analog formulation, has been developed to treat severe hypoglycemia in individuals with diabetes. OBJECTIVE: The aim of this trial was to evaluate the safety and efficacy of dasiglucagon in pediatric individuals with type 1 diabetes (T1DM). Participants were children and adolescents (6-17 years) with T1DM. METHODS: In this randomized double-blind trial, 42 participants were randomly allocated (2:1:1) to a single subcutaneous (SC) injection of dasiglucagon (0.6 mg), placebo, or reconstituted glucagon (GlucaGen; dosed per label) during insulin-induced hypoglycemia. The primary endpoint was time to plasma glucose (PG) recovery (first PG increase ≥20 mg/dL after treatment initiation without rescue intravenous glucose). The primary comparison was dasiglucagon vs. placebo; glucagon acted as a reference. RESULTS: The median time (95% confidence interval) to PG recovery following SC injection was 10 min (8-12) for dasiglucagon vs. 30 min (20 to -) for placebo (P < .001); the median time for glucagon was 10 min (8-12), which did not include the time taken to reconstitute the lyophilized powder. PG recovery was achieved in all participants in the dasiglucagon and glucagon groups within 20 min of dosing compared to 2 out of 11 patients (18%) with placebo. The most frequent adverse events were nausea and vomiting, as expected with glucagon treatment. CONCLUSIONS: Consistent with adult phase 3 trials, dasiglucagon rapidly and effectively restored PG levels following insulin-induced hypoglycemia in children and adolescents with T1DM, with an overall safety profile similar to glucagon.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Glucagón/análogos & derivados , Hipoglucemia/tratamiento farmacológico , Adolescente , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Niño , Diabetes Mellitus Tipo 1/sangre , Método Doble Ciego , Femenino , Alemania , Glucagón/administración & dosificación , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemia/patología , Inyecciones Subcutáneas , Insulina/uso terapéutico , Masculino , Gravedad del Paciente , Eslovenia , Resultado del Tratamiento , Estados UnidosRESUMEN
Congenital factor XIII (FXIII) deficiency is a rare, autosomal-recessive disorder, with most patients having an A-subunit (FXIII-A) deficiency. Patients experience life-threatening bleeds, impaired wound healing, and spontaneous abortions. In many countries, only plasma or cryoprecipitate treatments are available, but these carry a risk for allergic reactions and infection with blood-borne pathogens. The present study was a multinational, open-label, single-arm, phase 3 prophylaxis trial evaluating the efficacy and safety of a novel recombinant FXIII (rFXIII) in congenital FXIII-A subunit deficiency. Forty-one patients ≥ 6 years of age (mean, 26.4; range, 7-60) with congenital FXIII-A subunit deficiency were enrolled. Throughout the rFXIII prophylaxis, only 5 bleeding episodes (all trauma induced) in 4 patients were treated with FXIII-containing products. The crude mean bleeding rate was significantly lower than the historic bleeding rate (0.138 vs 2.91 bleeds/patient/year, respectively) for on-demand treatment. Transient, non-neutralizing, low-titer anti-rFXIII Abs developed in 4 patients, none of whom experienced allergic reactions, any bleeds requiring treatment, or changes in FXIII pharmacokinetics during the trial or follow-up. These non-neutralizing Abs declined below detection limits in all 4 patients despite further exposure to rFXIII or other FXIII-containing products. We conclude that rFXIII is safe and effective in preventing bleeding episodes in patients with congenital FXIII-A subunit deficiency. This study is registered at http://www..clinicaltrials.gov as number NCT00713648.
Asunto(s)
Factor VIII/administración & dosificación , Hemofilia A/tratamiento farmacológico , Hemorragia/tratamiento farmacológico , Adolescente , Adulto , Anticuerpos/sangre , Niño , Hipersensibilidad a las Drogas/etiología , Factor VIII/efectos adversos , Femenino , Hemofilia A/sangre , Hemorragia/sangre , Humanos , Masculino , Persona de Mediana Edad , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Factores de Riesgo , SeguridadRESUMEN
BACKGROUND: The in vivo clinical significance of malignant stem cells remains unclear. METHODS: Patients who have the 5q deletion (del[5q]) myelodysplastic syndrome (interstitial deletions involving the long arm of chromosome 5) have complete clinical and cytogenetic remissions in response to lenalidomide treatment, but they often have relapse. To determine whether the persistence of rare but distinct malignant stem cells accounts for such relapses, we examined bone marrow specimens obtained from seven patients with the del(5q) myelodysplastic syndrome who became transfusion-independent while receiving lenalidomide treatment and entered cytogenetic remission. RESULTS: Virtually all CD34+, CD38+ progenitor cells and stem cells that were positive for CD34 and CD90, with undetectable or low CD38 (CD38−/low), had the 5q deletion before treatment. Although lenalidomide efficiently reduced these progenitors in patients in complete remission, a larger fraction of the minor, quiescent, CD34+,CD38-/low, CD90+ del(5q) stem cells as well as functionally defined del(5q) stem cells remained distinctly resistant to lenalidomide. Over time, lenalidomide resistance developed in most of the patients in partial and complete remission, with recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. CONCLUSIONS: In these patients with the del(5q) myelodysplastic syndrome, we identified rare and phenotypically distinct del(5q) myelodysplastic syndrome stem cells that were also selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission. (Funded by the EuroCancerStemCell Consortium and others.)
Asunto(s)
Antineoplásicos/farmacología , Deleción Cromosómica , Cromosomas Humanos Par 5/genética , Resistencia a Medicamentos/genética , Síndromes Mielodisplásicos/patología , Células Madre Neoplásicas/efectos de los fármacos , Talidomida/análogos & derivados , ADP-Ribosil Ciclasa 1/análisis , Anciano , Anciano de 80 o más Años , Antígenos CD34/análisis , Antineoplásicos/uso terapéutico , Femenino , Expresión Génica , Humanos , Lenalidomida , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Células Madre Neoplásicas/inmunología , Fenotipo , Inducción de Remisión , Talidomida/farmacología , Talidomida/uso terapéutico , Antígenos Thy-1/análisisRESUMEN
Dasiglucagon is a next-generation glucagon analogue that is stable in aqueous formulation. This dedicated immunogenicity trial to support use as rescue treatment for severe hypoglycemia was conducted to evaluate the immunogenicity of repeated subcutaneous doses of dasiglucagon in subjects with type 1 diabetes. A total of 112 subjects were randomized 1:1 to receive three subcutaneous weekly doses of either 0.6 mg dasiglucagon or 1.0 mg recombinant glucagon (GlucaGen®) according to a double-blind parallel-group trial design. Subjects were followed for 15 weeks, with a multitiered testing approach planned for assessment of antidrug antibody (ADA) formation. For the primary immunogenicity endpoint, the overall ADA incidence was zero, as no subject demonstrated any treatment-induced or treatment-boosted ADA response at any time point in this trial involving three consecutive weekly doses of trial drug. No injection site reactions were reported for subjects receiving dasiglucagon. There were no unexpected safety findings for the trial.
Asunto(s)
Diabetes Mellitus Tipo 1 , Hipoglucemia , Glucemia , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Método Doble Ciego , Glucagón/análogos & derivados , Humanos , Hipoglucemia/inducido químicamenteRESUMEN
OBJECTIVE: To evaluate the efficacy and safety of dasiglucagon, a ready-to-use, next-generation glucagon analog in aqueous formulation for subcutaneous dosing, for treatment of severe hypoglycemia in adults with type 1 diabetes. RESEARCH DESIGN AND METHODS: This randomized, double-blind trial included 170 adult participants with type 1 diabetes, each randomly assigned to receive a single subcutaneous dose of 0.6 mg dasiglucagon, placebo, or 1 mg reconstituted glucagon (2:1:1 randomization) during controlled insulin-induced hypoglycemia. The primary end point was time to plasma glucose recovery, defined as an increase of ≥20 mg/dL from baseline without rescue intravenous glucose. The primary comparison was dasiglucagon versus placebo; reconstituted lyophilized glucagon was included as reference. RESULTS: Median (95% CI) time to recovery was 10 (10, 10) minutes for dasiglucagon compared with 40 (30, 40) minutes for placebo (P < 0.001); the corresponding result for reconstituted glucagon was 12 (10, 12) minutes. In the dasiglucagon group, plasma glucose recovery was achieved within 15 min in all but one participant (99%), superior to placebo (2%; P < 0.001) and similar to glucagon (95%). Similar outcomes were observed for the other investigated time points at 10, 20, and 30 min after dosing. The most frequent adverse effects were nausea and vomiting, as expected with glucagon treatment. CONCLUSIONS: Dasiglucagon provided rapid and effective reversal of hypoglycemia in adults with type 1 diabetes, with safety and tolerability similar to those reported for reconstituted glucagon injection. The ready-to-use, aqueous formulation of dasiglucagon offers the potential to provide rapid and reliable treatment of severe hypoglycemia.
Asunto(s)
Diabetes Mellitus Tipo 2 , Hipoglucemia , Adulto , Glucemia , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Método Doble Ciego , Glucagón/análogos & derivados , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemia/tratamiento farmacológico , Hipoglucemiantes/efectos adversos , Insulina/uso terapéuticoRESUMEN
Myelosuppression is one the most frequent side effects of chemotherapy. New agents that more selectively target cancer cells have been developed in attempt to improve the effects and to decrease the side effects of cancer treatment. Roscovitine is a purine analogue and cyclin-dependent kinase inhibitor. Several studies have shown its cytotoxic effect in cancer cell lines in vitro and in xenograft models in vivo. In this study, we investigated the effect of roscovitine on hematopoietic progenitors in vitro and in vivo in mice. The clonogenic capacity of hematopoietic progenitors was studied using burst-forming unit-erythroid (BFU-E), colony-forming unit granulocyte, macrophage (CFU-GM) and colony-forming unit granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). In vitro, bone marrow cells were exposed to roscovitine (25-250 microM) in Iscove's modified Dulbecco's media for 4 h or to roscovitine (1-100 microM) in MethoCult media for 12 days. No effect on colony formation was observed after exposure to roscovitine for 4 h; however, concentration- and cell type-dependent effects were observed after 12 days. Roscovitine in concentration of 100 microM inhibited the growth of all types of colonies, while lower concentrations have shown differential effect on hematopoietic progenitors. The most sensitive were CFU-GEMM, followed by BFU-E and then CFU-GM. In vivo, mice were treated with single dose of roscovitine (50, 100 or 250 mg/kg) and the effect on bone marrow was studied on day 1, 3, 6, 9 or 12 after the treatment. In the second part of experiment, the mice were treated with roscovitine 350 mg/kg/day divided into two daily doses for 4 days. The bone marrow was examined on day 1 and 5 after the last dose of roscovitine. On day 1, BFU-E decreased to less than 50% of the controls (P = 0.019). No decrease in BFU-E formation was observed on day 5. No significant effect was observed on CFU-GM and CFU-GEMM growth after the treatment with multiple doses of roscovitine. Single doses of roscovitine or dimethylsulfoxide did not affect the colony formation. We also studied the distribution of roscovitine to the bone marrow after a dose of 50 mg/kg was administered intraperitoneally. Only 1.5% of the drug was detected in the bone marrow. Thus, the roscovitine effect on hematopoietic progenitors in bone marrow in vivo is only transient. One reason may be that only a small fraction of roscovitine reaches the bone marrow. Another explanation may be the short half-life observed for roscovitine that might not allow enough cell exposure to the drug. However, the toxicity of roscovitine to hematopoietic progenitors in vitro is within the same exposure range as cytotoxicity to cancer cells. Thus, precaution should be taken in clinical trials, especially when combinations with myelosuppressive cytostatics are used.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Células Madre Hematopoyéticas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/efectos adversos , Purinas/efectos adversos , Animales , Área Bajo la Curva , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Supervivencia Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Femenino , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Semivida , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos BALB C , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Purinas/administración & dosificación , Purinas/farmacocinética , Roscovitina , Factores de Tiempo , Distribución TisularRESUMEN
PURPOSE: Erythroid apoptosis in low-risk myelodysplastic syndrome (MDS) maybe mediated via mitochondrial release of cytochrome c and subsequent caspase activation. In the present study, we compared the in vitro and in vivo effects of proerythroid treatment with erythropoietin + granulocyte colony-stimulating factor (G-CSF) on myelodysplastic erythropoiesis regarding apoptosis and preferential growth of clones with cytogenetic abnormalities. EXPERIMENTAL DESIGN: We enrolled 15 refractory anemia (RA) and 11 refractory anemia with ringed sideroblasts (RARS), including 5q- aberration, monosomy 7, and trisomy 8, before initiation of treatment and followed nine patients after successful treatment. The effects of G-CSF and erythropoietin were assessed. The expression of G-CSF receptor (G-CSFR) was explored during erythroid maturation. The relative growth of erythroid progenitors with cytogenetic aberrations in presence of erythropoietin was investigated. RESULTS: Significant redistribution of cytochrome c was seen before treatment at all stages of erythroid differentiation. This release was blocked by G-CSF during the whole culture period and by erythropoietin during the latter phase. Both freshly isolated glycophorin A+ bone marrow cells and intermediate erythroblasts during cultivation retained their expression of G-CSFR. Cytochrome c release and caspase activation were significantly less pronounced in progenitors obtained from successfully treated nonanemic patients and showed no further response to G-CSF in vitro. Moreover, erythropoietin significantly promoted growth of cytogenetically normal cells from 5q- patients, whereas no such effect was observed on erythroblasts from monosomy 7 or trisomy 8 patients. CONCLUSION: We conclude that growth factors such as erythropoietin and G-CSF can act both via inhibition of apoptosis of myelodysplastic erythroid precursors and via selection of cytogenetically normal progenitors.
Asunto(s)
Anemia Refractaria/tratamiento farmacológico , Anemia Sideroblástica/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Células Precursoras Eritroides/efectos de los fármacos , Adulto , Anciano , Anciano de 80 o más Años , Anemia Refractaria/patología , Anemia Sideroblástica/patología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Caspasas/metabolismo , Citocromos c/metabolismo , Activación Enzimática/efectos de los fármacos , Células Precursoras Eritroides/patología , Eritropoyetina/farmacología , Glicoforinas/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Técnicas In Vitro , Persona de Mediana Edad , Monosomía , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Proteínas Recombinantes , TrisomíaRESUMEN
INTRODUCTION: Paradigm™4 was an international extension trial investigating the safety and efficacy of nonacog beta pegol, a recombinant glycoPEGylated factor IX (FIX) with extended half-life, in haemophilia B patients (FIX activity ≤2%; aged 13-70years) who had previously participated in phase III pivotal (paradigm™2) or surgery (paradigm™3) trials. METHODS: Patients chose to continue treatment with nonacog beta pegol in either one of two once-weekly prophylaxis arms (10IU/kg or 40IU/kg), or an on-demand arm (40IU/kg for mild/moderate bleeds; 80IU/kg for severe bleeds). The primary objective was to evaluate immunogenicity; key secondary objectives included assessing safety and haemostatic efficacy in the treatment and prevention of bleeds. RESULTS: Seventy-one patients received prophylaxis or on-demand treatment. No patient developed an inhibitor and no safety concerns were identified. The success rate for the treatment of reported bleeds was 94.6%; most (87.9%) resolved with one injection. The median annualised bleeding rate for patients on prophylaxis was 1.36 (interquartile range [IQR] 0.00-2.23) and 1.00 (IQR 0.00-2.03) for the 10 and 40IU/kg treatment arms, respectively. The mean FIX activity trough achieved for 10 and 40IU once weekly was 9.8% and 21.3%, respectively. Fourteen patients on prophylaxis underwent 23 minor surgical procedures; haemostatic perioperative outcomes for all of those evaluated were 'excellent' or 'good'. CONCLUSIONS: Nonacog beta pegol showed a favourable tolerability profile (with no safety issues identified) with good prophylactic protection and control of bleeding in previously treated adult and adolescent haemophilia B patients.
Asunto(s)
Factor IX/uso terapéutico , Hemofilia B/tratamiento farmacológico , Hemorragia/tratamiento farmacológico , Hemostáticos/uso terapéutico , Polietilenglicoles/uso terapéutico , Adolescente , Adulto , Anciano , Factor IX/administración & dosificación , Factor IX/efectos adversos , Hemofilia B/complicaciones , Hemofilia B/prevención & control , Hemorragia/complicaciones , Hemorragia/prevención & control , Hemostáticos/administración & dosificación , Hemostáticos/efectos adversos , Humanos , Persona de Mediana Edad , Polietilenglicoles/administración & dosificación , Polietilenglicoles/efectos adversos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Adulto JovenRESUMEN
OBJECTIVE: We aimed to establish the ideal injection techniques using 5-mm needles to reliably inject insulin into the subcutaneous fat in both children and adults and to quantify the associated pain and leakage of the test medium. RESEARCH DESIGN AND METHODS: A total of 259 subjects (122 children/adolescents and 137 adults) were injected with sterile air corresponding to 20 IU insulin (200 microl) with 32-G 5-mm needles at 90 degrees or 45 degrees , in the abdomen and thigh, and with or without a pinched skin fold. Injection depth was assessed via ultrasonography. Subjects rated pain on a visual analog scale. Test medium injections into the abdomen and thigh (0.2-0.6 ml) were also administered to assess injection leakage. RESULTS: Among children, 5.5% of injections were intramuscular (IM) and 0.5% were intradermal, while in adults, the incidence was 1.3 and 0.6%, respectively. The frequency of IM injections was greater in boys and negligible among adult women. Subcutaneous fat thickness was the primary predictor of the likelihood of IM injections (P < 0.001). A third of all patients reported experiencing no pain during insulin injection, with children/adolescents experiencing considerably more discomfort than adults. Some leakage of medium was observed, but was unrelated to injection volume and was generally minimal. CONCLUSIONS: 5-mm needles are reliably inserted into subcutaneous fat in both adults and children. These needles were associated with reduced pain and minimal leakage. We recommend an angled injection with a pinched skin fold for children, while in adults, the technique should be left to patient preference.
Asunto(s)
Inyecciones/efectos adversos , Agujas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Diabetes Mellitus Tipo 1 , Femenino , Humanos , Inyecciones Intramusculares/efectos adversos , Inyecciones Subcutáneas/efectos adversos , Modelos Lineales , Masculino , Persona de Mediana Edad , Dolor/etiología , Adulto JovenRESUMEN
Early erythroblasts from patients with refractory anemia (RA) and RA with ringed sideroblasts (RARS) show constitutive mitochondrial release of cytochrome c. Moreover, mature erythroblasts in RARS, but not in RA, display aberrant accumulation of mitochondrial ferritin (MtF). We analyzed cytochrome c release, MtF expression, and gene expression during erythroid differentiation in bone marrow cells from myelodysplastic syndrome (MDS) patients and healthy controls. Whereas none or few cultured erythroid cells from healthy individuals and RA patients expressed MtF, those from RARS patients showed MtF expression at an early stage, when cells were CD34+ and without morphologic signs of erythroid differentiation. The proportion of RARS erythroblasts that were MtF+ increased further upon in vitro maturation. Moreover, a significant overexpression of mRNA encoding cytochrome c, and proapoptotic Bid and Bax, was seen in freshly isolated cells from MDS patients. Genes involved in erythroid differentiation were also dysregulated in MDS cells. Importantly, GATA-1 expression increased during normal erythroid maturation, but remained low in MDS cultures, indicating a block of erythroid maturation at the transcriptional level. In conclusion, aberrant MtF expression in RARS erythroblasts occurs at a very early stage of erythroid differentiation and is paralleled by an up-regulation of genes involved in this process.
Asunto(s)
Células Madre Hematopoyéticas/patología , Hierro/metabolismo , Mitocondrias/metabolismo , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , Adenosina Trifosfato/metabolismo , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patología , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/genética , Citocromos c/genética , Citocromos c/metabolismo , Proteínas de Unión al ADN/genética , Eritroblastos/metabolismo , Eritroblastos/patología , Factores de Unión al ADN Específico de las Células Eritroides , Ferritinas/metabolismo , Factor de Transcripción GATA1 , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Glicoproteínas/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Hematopoyéticas/metabolismo , Hemoglobinas/genética , Humanos , Síndromes Mielodisplásicos/epidemiología , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/análisis , Factores de Riesgo , Factores de Transcripción/genética , Proteína X Asociada a bcl-2RESUMEN
The purpose of this study was to determine the facility and reliability of the World Health Organization (WHO) classification of myelodysplastic syndromes (MDSs) with several observers reviewing the same diagnostic specimens. We also wanted to determine if the WHO classification provided additional information about predictability of clinical response outcome. To accomplish these goals we reviewed 103 previously diagnosed cases of low-risk MDS. We found 92% interobserver agreement (P <.001). Sixty-four of these patients had been entered into clinical trials using growth factors by the Nordic MDS Study Group. The WHO classification reliably predicted therapeutic response to the combination of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (Epo). The response rate differed significantly between refractory anemia with ringed sideroblasts (RARS) and refractory anemia with multilineage dysplasia and ringed sideroblasts (RCMD/RS) with regard to therapeutic response (75% versus 9%; P =.003). Also, in the group of patients with less than 5% marrow blasts, there was a difference in median survival between patients with unilineage dysplasia (51% surviving at 67 months) and those with multilineage dysplasia (median survival, 28.5 months; P =.03).
Asunto(s)
Síndromes Mielodisplásicos/clasificación , Organización Mundial de la Salud , Clasificación/métodos , Quimioterapia Combinada , Eritropoyetina/uso terapéutico , Estudios de Seguimiento , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Humanos , Síndromes Mielodisplásicos/epidemiología , Síndromes Mielodisplásicos/mortalidad , Variaciones Dependientes del Observador , Valor Predictivo de las Pruebas , Pronóstico , Estudios Retrospectivos , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Low-risk myelodysplastic syndromes (MDS), including refractory anemia and sideroblastic anemia, are characterized by increased apoptotic death of erythroid progenitors. The signaling pathways that elicit this pathologic cell death in MDS have, however, remained unclear. Treatment with erythropoietin in combination with granulocyte colony-stimulating factor (G-CSF) may synergistically improve the anemia in patients with MDS, with a concomitant decrease in the number of apoptotic bone marrow precursors. Moreover, we have previously reported that G-CSF inhibits Fas-induced caspase activation in sideroblastic anemia (RARS). The present data demonstrate that almost 50% of erythroid progenitor cells derived from patients with MDS exhibit spontaneous release of cytochrome c from mitochondria with ensuing activation of caspase-9, whereas normal erythroid progenitors display neither of these features. G-CSF significantly inhibited cytochrome c release and suppressed apoptosis, most noticeably in cells from patients with sideroblastic anemia. Furthermore, inhibition of caspase-9 suppressed both spontaneous and Fas-mediated apoptosis of erythroid progenitors in all low-risk MDS cases studied. We propose that the increased sensitivity of MDS progenitor cells to death receptor stimulation is due to a constitutive activation of the mitochondrial axis of the apoptotic signaling pathway in these cells. These studies yield a mechanistic explanation for the beneficial clinical effects of growth factor administration in patients with MDS, and provide a model for the study of growth factor-mediated suppression of apoptosis in other bone marrow disorders.
Asunto(s)
Apoptosis/efectos de los fármacos , Grupo Citocromo c/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Mitocondrias/fisiología , Síndromes Mielodisplásicos/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Inhibidores de Caspasas , Grupo Citocromo c/efectos de los fármacos , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/patología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Persona de Mediana Edad , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor fas/farmacologíaRESUMEN
Kostmann syndrome, or severe congenital neutropenia (SCN), is an autosomal recessive disorder of neutrophil production. To investigate the potential role of apoptosis in SCN, bone marrow aspirates and biopsies were obtained from 4 patients belonging to the kindred originally described by Kostmann and 1 patient with SCN of unknown inheritance. An elevated degree of apoptosis was observed in the bone marrow of these patients, and a selective decrease in B-cell lymphoma-2 (Bcl-2) expression was seen in myeloid progenitor cells. Furthermore, in vitro apoptosis of bone marrow-derived Kostmann progenitor cells was increased, and mitochondrial release of cytochrome c was detected in CD34(+) and CD33(+) progenitors from patients, but not in controls. Administration of granulocyte colony-stimulating factor (G-CSF) restored Bcl-2 expression and improved survival of myeloid progenitor cells. In addition, cytochrome c release was partially reversed upon incubation of progenitor cells with G-CSF. In sum, these studies establish a role for mitochondria-dependent apoptosis in the pathogenesis of Kostmann syndrome and yield a tentative explanation for the beneficial effect of growth factor administration in these patients.