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Granulocyte-colony stimulating factor (G-CSF) in stroke patients with concomitant vascular disease--a randomized controlled trial.

ABSTRACT: G-CSF has been shown in animal models of stroke to promote functional and structural regeneration of the central nervous system. It thus might present a therapy to promote recovery in the chronic stage after stroke.Here, we assessed the safety and tolerability of G-CSF in chronic stroke patients with concomitant vascular disease, and explored efficacy data. 41 patients were studied in a double-blind, randomized approach to either receive 10 days of G-CSF (10 µg/kg body weight/day), or placebo. Main inclusion criteria were an ischemic infarct >4 months prior to inclusion, and white matter hyperintensities on MRI. Primary endpoint was number of adverse events. We also explored changes in hand motor function for activities of daily living, motor and verbal learning, and finger tapping speed, over the course of the study.Adverse events (AEs) were more frequent in the G-CSF group, but were generally graded mild or moderate and from the known side-effect spectrum of G-CSF. Leukocyte count rose after day 2 of G-CSF dosing, reached a maximum on day 8 (mean 42/nl), and returned to baseline 1 week after treatment cessation. No significant effect of treatment was detected for the primary efficacy endpoint, the test of hand motor function.These results demonstrate the feasibility, safety and reasonable tolerability of subcutaneous G-CSF in chronic stroke patients. This study thus provides the basis to explore the efficacy of G-CSF in improving chronic stroke-related deficits.ClinicalTrials.gov NCT00298597.

SUBMITTER: Floel A

PROVIDER: S-EPMC3100298 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Granulocyte-colony stimulating factor (G-CSF) in stroke patients with concomitant vascular disease--a randomized controlled trial.

Floel Agnes A Warnecke Tobias T Duning Thomas T Lating Yvonne Y Uhlenbrock Jan J Schneider Armin A Vogt Gerhard G Laage Rico R Koch Winfried W Knecht Stefan S Schäbitz Wolf-Rüdiger WR

PloS one 20110523 5

<h4>Background</h4>G-CSF has been shown in animal models of stroke to promote functional and structural regeneration of the central nervous system. It thus might present a therapy to promote recovery in the chronic stage after stroke.<h4>Methods</h4>Here, we assessed the safety and tolerability of G-CSF in chronic stroke patients with concomitant vascular disease, and explored efficacy data. 41 patients were studied in a double-blind, randomized approach to either receive 10 days of G-CSF (10 µg ...[more]

PMID: 21625426

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Project description:BACKGROUND:Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. RESULTS:Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. CONCLUSION:The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Project description:BACKGROUND:The FDA approved drug granulocyte-colony stimulating factor (G-CSF) displays anti-apoptotic and immunomodulatory properties with neurogenesis and angiogenic functions. It is known to demonstrate neuroprotective mechanisms against ischemic global stroke. Autophagy is a method for the degradation of intracellular components and in particular, unrestrained autophagy may lead to uncontrolled digestion of affected neurons as well as neuronal death in cerebral ischemia. Mitochondrial dynamics is vital for the regulation of cell survival and death after cerebral ischemia and an early upstream event in neuronal death is mitochondrial fission. We examined the pro-survival mechanisms of G-CSF against apoptosis resulting from autophagy, mitochondrial stress and endoplasmic reticulum (ER) stress. METHODS:Male Swiss Webster mice (20 weeks of age) were subjected to bilateral common carotid artery occlusion (BCAO) for 30 min. After occlusion, mice were injected with G-CSF (50 μg/kg) subcutaneously for 4 days. Behavioral analysis was carried out using the corner test and locomotor activity test before animals were sacrificed on day 4 or day 7. Key proteins in ER stress, autophagy and mitochondrial stress induced apoptosis were analyzed by immunoblotting. RESULTS:G-CSF improved neurological deficits and improved behavioral performance on corner and locomotor test. G-CSF binds to G-CSF receptors and its activation leads to upregulation of Akt phosphorylation (P-Akt) which in turn decreases levels of the ER stress sensor, GRP 78 and expression of proteins involved in ER stress apoptosis pathway; ATF6, ATF4, eIF2α, XBP1, Caspase 12 and CHOP. G-CSF treatment significantly decreased Beclin-1, an autophagy marker, and decreased mitochondrial stress biomarkers DRP1 and P53. G-CSF also up-regulated the mitochondrial fusion protein, OPA1 and anti-apoptotic protein Bcl-2 while down-regulating the pro-apoptotic proteins Bax, Bak and PUMA. CONCLUSIONS:G-CSF is an endogenous ligand in the CNS that has a dual activity that is beneficial both in reducing acute neuronal degeneration and adding to long-term plasticity after cerebral ischemia. G-CSF treatment exerts neuroprotective effects on damaged neurons through the suppression of the ER stress and mitochondrial stress and maintains cellular homeostasis by decreasing pro-apoptotic proteins and increasing of anti-apoptotic proteins.

Project description:Attenuation of the secondary injury of spinal cord injury (SCI) can suppress the spread of spinal cord tissue damage, possibly resulting in spinal cord sparing that can improve functional prognoses. Granulocyte colony-stimulating factor (G-CSF) is a haematological cytokine commonly used to treat neutropenia. Previous reports have shown that G-CSF promotes functional recovery in rodent models of SCI. Based on preclinical results, we conducted early phase clinical trials, showing safety/feasibility and suggestive efficacy. These lines of evidence demonstrate that G-CSF might have therapeutic benefits for acute SCI in humans. To confirm this efficacy and to obtain strong evidence for pharmaceutical approval of G-CSF therapy for SCI, we conducted a phase 3 clinical trial designed as a prospective, randomized, double-blinded and placebo-controlled comparative trial. The current trial included cervical SCI [severity of American Spinal Injury Association (ASIA) Impairment Scale (AIS) B or C] within 48 h after injury. Patients are randomly assigned to G-CSF and placebo groups. The G-CSF group was administered 400 μg/m2/day × 5 days of G-CSF in normal saline via intravenous infusion for five consecutive days. The placebo group was similarly administered a placebo. Allocation was concealed between blinded evaluators of efficacy/safety and those for laboratory data, as G-CSF markedly increases white blood cell counts that can reveal patient treatment. Efficacy and safety were evaluated by blinded observer. Our primary end point was changes in ASIA motor scores from baseline to 3 months after drug administration. Each group includes 44 patients (88 total patients). Our protocol was approved by the Pharmaceuticals and Medical Device Agency in Japan and this trial is funded by the Center for Clinical Trials, Japan Medical Association. There was no significant difference in the primary end point between the G-CSF and the placebo control groups. In contrast, one of the secondary end points showed that the ASIA motor score 6 months (P = 0.062) and 1 year (P = 0.073) after drug administration tend to be higher in the G-CSF group compared with the placebo control group. Moreover, in patients aged over 65 years old, motor recovery 6 months after drug administration showed a strong trend towards a better recovery in the G-CSF treated group (P = 0.056) compared with the control group. The present trial failed to show a significant effect of G-CSF in primary end point although the subanalyses of the present trial suggested potential G-CSF benefits for specific population.

Dataset Information

Granulocyte-colony stimulating factor (G-CSF) in stroke patients with concomitant vascular disease--a randomized controlled trial.

Publications

Granulocyte-colony stimulating factor (G-CSF) in stroke patients with concomitant vascular disease--a randomized controlled trial.

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