First-in-human, randomized, double-blind, placebo-controlled, dose escalation trial of the anti-herpes simplex virus monoclonal antibody HDIT101 in healthy volunteers.

HDIT101 is a first-in-class humanized monoclonal antibody recognizing a conserved epitope in glycoprotein B, a target present on the surface of herpes simplex virus 1 (HSV-1) and HSV-2 particles as well as on virus-infected cells. This was a first-in-human, single-center, double-blind, placebo-controlled trial in 24 healthy volunteers, randomized 3:1 (placebo:active) in each of the six dose levels with escalating doses up to 12,150 mg HDIT101. HDIT101 was administered intravenously, to study safety, pharmacokinetics (PKs), and immunogenicity. HDIT101 was well-tolerated in all recipients and no serious or severe adverse events, no infusion-related reactions, and no events suggestive of dose limiting off-target toxicity occurred. The mean serum exposure (area under the curve from zero to infinity [AUC0-∞ ]) of HDIT101 showed a linear increase from 4340 h*µg/ml at a dose of 50 mg to 1,122,247 h*µg/ml at a dose of 12,150 mg. No immunogenic effects following HDIT101 exposure were observed at any of the applied doses. HDIT101 demonstrated the expected PK properties of a monoclonal antibody was well-tolerated, and could be safely administered even at excessively high doses that may be required for treatment of patients with septical HSV spread.

Reversible Edema in the Penumbra Correlates With Severity of Hypoperfusion.

Background and Purpose: We aimed to investigate fluid-attenuated inversion recovery changes in the penumbra. Methods: We determined core and perfusion lesions in subjects from the WAKE-UP trial (Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke) and AXIS 2 trial (Granulocyte Colony-Stimulating Factor in Patients With Acute Ischemic Stroke) with perfusion- and diffusion-weighted imaging at baseline. Only subjects with a mismatch volume >15 mL and ratio >1.2 were included. We created voxel-based relative fluid-attenuated inversion recovery signal intensity (rFLAIR SI) maps at baseline and follow-up. We studied rFLAIR SI in 2 regions of interest: baseline penumbra (baseline perfusion lesion−[core lesion+voxels with apparent diffusion coefficient <620 10−6 mm2/s]) and noninfarcted penumbra (baseline perfusion lesion−follow-up fluid-attenuated inversion recovery lesion) at 24 hours (WAKE-UP) or 30 days (AXIS 2). We analyzed the association between rFLAIR SI and severity of hypoperfusion, defined as time to maximum of the residue function. Results: In the baseline penumbra, rFLAIR SI was elevated (ratio, 1.04; P=1.7×10−13; n=126) and correlated with severity of hypoperfusion (Pearson r, 0.03; P<1.0×10−4; n=126). In WAKE-UP, imaging at 24 hours revealed a further increase of rFLAIR SI in the noninfarcted penumbra (ratio, 1.05 at 24 hours versus 1.03 at baseline; P=7.1×10−3; n=43). In AXIS 2, imaging at 30 days identified reversibility of the rFLAIR SI (ratio, 1.02 at 30 days versus 1.04 at baseline; P=1.5×10−3; n=26) since it was no longer different from 1 (ratio, 1.01 at 30 days; P=0.099; n=26). Conclusions: Penumbral rFLAIR SI increases appear early after stroke onset, correlate with severity of hypoperfusion, further increase at 24 hours, and are reversible by 30 days. Registration: URL: https://clinicaltrials.gov; Unique identifier: NCT01525290. URL: https://clinicaltrials.gov; Unique identifier: NCT00927836.

Imaging Markers of Brain Frailty and Outcome in Patients With Acute Ischemic Stroke.

BACKGROUND AND PURPOSE: Functional outcome after stroke may be related to preexisting brain health. Several imaging markers of brain frailty have been described including brain atrophy and markers of small vessel disease. We investigated the association of these imaging markers with functional outcome after acute ischemic stroke. METHODS: We retrospectively studied patients with acute ischemic stroke enrolled in the AXIS-2 trial (AX200 in Ischemic Stroke Trial), a randomized controlled clinical trial of granulocyte colony-stimulating factor versus placebo. We assessed the ratio of brain parenchymal volume to total intracerebral volumes (ie, the brain parenchymal fraction) and total brain volumes from routine baseline magnetic resonance imaging data obtained within 9 hours of symptom onset using the unified segmentation algorithm in SPM12. Enlarged perivascular spaces, white matter hyperintensities, lacunes, as well as a small vessel disease burden, were rated visually. Functional outcomes (modified Rankin Scale score) at day 90 were determined. Logistic regression was used to test associations between brain imaging features and functional outcomes. RESULTS: We enrolled 259 patients with a mean age of 69±12 years and 46 % were female. Increased brain parenchymal fraction was associated with higher odds of excellent outcome (odds ratio per percent increase, 1.078 [95% CI, 1.008-1.153]). Total brain volumes and small vessel disease burden were not associated with functional outcome. An interaction between brain parenchymal fraction and large vessel occlusion on excellent outcome was not observed. CONCLUSIONS: Global brain health, as assessed by brain parenchymal fraction on magnetic resonance imaging, is associated with excellent functional outcome after ischemic stroke. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00927836.

Early Brain Volume Changes After Stroke: Subgroup Analysis From the AXIS-2 Trial.

BACKGROUND AND PURPOSE: The evolution of total brain volume early after stroke is not well understood. We investigated the associations between age and imaging features and brain volume change in the first month after stroke. METHODS: We retrospectively studied patients with acute ischemic stroke enrolled in the AXIS-2 trial. Total brain volume change from hyperacute MRI data to the first month after stroke was assessed using unified segmentation in SPM12. We hypothesized that age, ischemic brain lesion size, and white matter (WM) changes were associated with larger brain volume change. Enlarged perivascular spaces (EPVSs) and white matter hyperintensities (WMHs) were rated visually and the presence of lacunes was assessed. RESULTS: We enrolled 173 patients with a mean age of 67 ± 11 years, 44% were women. There was a median 6 ml decrease in volume (25th percentile -1 ml to 75th percentile 21 ml) over time, equivalent to a median 0.5% (interquartile range [IQR], -0.07%-1.4%), decrease in brain volume. Age was associated with larger brain volume loss (per 10 years of age, 5 ml 95% CI 2-8 ml). Baseline diffusion weighted imaging (DWI) lesion volume was not associated with greater volume loss per 10 ml of lesion volume, change by 0 ml (95% CI -0.1 to 0.1 ml). Increasing Fazekas scores of deep WMH were associated with greater tissue loss (5 ml, 95% CI 1-10 ml). CONCLUSIONS: Total brain volume changes in a heterogenous fashion after stroke. Volume loss occurs over 1 month after stroke and is associated with age and deep WM disease. We did not find evidence that more severe strokes lead to increased early tissue loss.

Automated DWI analysis can identify patients within the thrombolysis time window of 4.5 hours.

OBJECTIVE: To develop an automated model based on diffusion-weighted imaging (DWI) to detect patients within 4.5 hours after stroke onset and compare this method to the visual DWI-FLAIR (fluid-attenuated inversion recovery) mismatch. METHODS: We performed a subanalysis of the "DWI-FLAIR mismatch for the identification of patients with acute ischemic stroke within 4.5 hours of symptom onset" (PRE-FLAIR) and the "AX200 for ischemic stroke" (AXIS 2) trials. We developed a prediction model with data from the PRE-FLAIR study by backward logistic regression with the 4.5-hour time window as dependent variable and the following explanatory variables: age and median relative DWI (rDWI) signal intensity, interquartile range (IQR) rDWI signal intensity, and volume of the core. We obtained the accuracy of the model to predict the 4.5-hour time window and validated our findings in an independent cohort from the AXIS 2 trial. We compared the receiver operating characteristic curve to the visual DWI-FLAIR mismatch. RESULTS: In the derivation cohort of 118 patients, we retained the IQR rDWI as explanatory variable. A threshold of 0.39 was most optimal in selecting patients within 4.5 hours after stroke onset resulting in a sensitivity of 76% and specificity of 63%. The accuracy was validated in an independent cohort of 200 patients. The predictive value of the area under the curve of 0.72 (95% confidence interval 0.64-0.80) was similar to the visual DWI-FLAIR mismatch (area under the curve = 0.65; 95% confidence interval 0.58-0.72; p for difference = 0.18). CONCLUSIONS: An automated analysis of DWI performs at least as good as the visual DWI-FLAIR mismatch in selecting patients within the 4.5-hour time window.

Multimodal magnetic resonance imaging to identify stroke onset within 6 h in patients with large vessel occlusions.

INTRODUCTION: Mechanical thrombectomy within 6 h after stroke onset improves the outcome in patients with large vessel occlusions. The aim of our study was to establish a model based on diffusion weighted and perfusion weighted imaging to provide an accurate prediction for the 6 h time-window in patients with unknown time of stroke onset. PATIENTS AND METHODS: A predictive model was designed based on data from the DEFUSE 2 study and validated in a subgroup of patients with large vessel occlusions from the AXIS 2 trial. RESULTS: We constructed the model in 91 patients from DEFUSE 2. The following parameters were independently associated with <6 h time-window and included in the model: interquartile range and median relative diffusion weighted imaging, hypoperfusion intensity ratio, core volume and the interaction between median relative diffusion weighted imaging and hypoperfusion intensity ratio as predictors of the 6 h time-window. The area under the curve was 0.80 with a positive predictive value of 0.90 (95%CI 0.79-0.96). In the validation cohort (N = 90), the area under the curve was 0.73 (P for difference = 0.4) with a positive predictive value of 0.85 (95%CI 0.69-0.95). DISCUSSION: After validation in a larger independent dataset the model can be considered to select patients for endovascular treatment in whom stroke onset is unknown. CONCLUSION: In patients with large vessel occlusion and unknown time of stroke onset an automated multivariate imaging model is able to select patients who are likely within the 6 h time-window.

Prediction of Stroke Onset Is Improved by Relative Fluid-Attenuated Inversion Recovery and Perfusion Imaging Compared to the Visual Diffusion-Weighted Imaging/Fluid-Attenuated Inversion Recovery Mismatch.

BACKGROUND AND PURPOSE: Acute stroke patients with unknown time of symptom onset are ineligible for thrombolysis. The diffusion-weighted imaging and fluid-attenuated inversion recovery (FLAIR) mismatch is a reasonable predictor of stroke within 4.5 hours of symptom onset, and its clinical usefulness in selecting patients for thrombolysis is currently being investigated. The accuracy of the visual mismatch rating is moderate, and we hypothesized that the predictive value of stroke onset within 4.5 hours could be improved by including various clinical and imaging parameters. METHODS: In this study, 141 patients in whom magnetic resonance imaging was obtained within 9 hours after symptom onset were included. Relative FLAIR signal intensity was calculated in the region of nonreperfused core. Mean Tmax was calculated in the total region with Tmax >6 s. Mean relative FLAIR, mean Tmax, lesion volume with Tmax >6 s, age, site of arterial stenosis, core volume, and location of infarct were analyzed by logistic regression to predict stroke onset time before or after 4.5 hours. RESULTS: Receiver-operating characteristic curve analysis revealed an area under the curve of 0.68 (95% confidence interval 0.59-0.78) for the visual diffusion-weighted imaging/FLAIR mismatch, thereby correctly classifying 69% of patients with an onset time before or after 4.5 hours. Age, relative FLAIR, and Tmax increased the accuracy significantly (P<0.01) to an area under the curve of 0.82 (95% confidence interval 0.74-0.89). This new predictive model correctly categorized 77% of patients according to stroke onset before versus after 4.5 hours. CONCLUSIONS: In patients with unknown stroke onset, the accuracy of predicting time from symptom onset within 4.5 hours is improved by obtaining relative FLAIR and perfusion imaging.

Association Between Time From Stroke Onset and Fluid-Attenuated Inversion Recovery Lesion Intensity Is Modified by Status of Collateral Circulation.

BACKGROUND AND PURPOSE: In patients with acute stroke, the intensity of a fluid-attenuated inversion recovery (FLAIR) lesion in the region of diffusion restriction is associated with time from symptom onset. We hypothesized that collateral status as assessed by the hypoperfusion intensity ratio could modify the association between time from stroke onset and FLAIR lesion intensity. METHODS: From the AX200 for ischemic stroke trial, 141 patients had appropriate FLAIR, diffusion-weighted imaging, and perfusion-weighted imaging. In the region of nonreperfused core, we calculated voxel-based relative FLAIR (rFLAIR) signal intensity. The hypoperfusion intensity ratio was defined as the ratio of the Tmax >10 s lesion over the Tmax >6 s lesion volume. A hypoperfusion intensity ratio threshold of ≤0.4 was used to dichotomize good versus poor collaterals. We studied the interaction between collateral status on the association between time from symptom onset and FLAIR intensity. RESULTS: Time from symptom onset was associated with the rFLAIR intensity in the region of nonreperfused core (B=1.05; 95% confidence interval, 1.0-1.1). We identified an interaction between this association and collateral status; an association was present between time and rFLAIR intensity in patients with poor collaterals (r=0.53), but absent in patients with good collaterals (r=0.17; P=0.04). CONCLUSIONS: Our findings show that the relationship between time from symptom onset and rFLAIR lesion intensity depends on collateral status. In patients with good collaterals, the development of an rFLAIR-positive lesion is less dependent on time from symptom onset compared with patients with poor collaterals.

Magnetic resonance imaging-based endovascular versus medical stroke treatment for symptom onset up to 12 h.

BACKGROUND: Recent trials have shown a clear benefit of endovascular therapy for stroke patients presenting within 6 h after stroke onset. Imaging-based selection may identify a cohort with a favorable response to endovascular therapy, in an even later time window. AIMS: We performed an indirect comparison between outcomes seen in DEFUSE 2, a prospective cohort study of patients who received a baseline MRI before endovascular therapy, and a control group from AXIS 2 receiving standard medical care up to 12 h after symptom onset. METHODS: Patients from AXIS 2 with a confirmed large vessel occlusion were selected as a control group for DEFUSE 2-patients. The primary endpoint was good functional outcome at day 90 (Modified Rankin Score 0-2). We performed a stratified analysis based on the presence of the target mismatch for both studies and reperfusion status in DEFUSE 2. RESULTS: We compared good functional outcome in 108 patients from AXIS 2 and 99 patients from DEFUSE 2. In DEFUSE 2-patients with the target mismatch profile in whom reperfusion was achieved, the rate of good functional outcome was increased compared to target mismatch patients in AXIS 2, 54% versus 29% (OR 3.2, 95% CI 1.1-9.4). In target mismatch patients treated between 6 and 12 h after stroke onset, this association between study and good functional outcome remained present (OR 9.0, 95% CI 1.1-75.8). CONCLUSIONS: This indirect comparison suggests that endovascular treatment resulting in substantial reperfusion is associated with improved outcome in target mismatch patients even beyond 6 h after stroke onset. Confirmation is needed from future clinical trials that randomize patients beyond the 6 h time window.

Association between the perfusion/diffusion and diffusion/FLAIR mismatch: data from the AXIS2 trial.

The perfusion-/diffusion-weighted imaging (PWI/DWI) mismatch and the diffusion/fluid attenuated inversion recovery (DWI/FLAIR) mismatch are magnetic resonance imaging (MRI) markers of evolving brain ischemia. We examined whether the DWI/FLAIR mismatch was independently associated with the PWI/DWI mismatch. Furthermore, we determined whether the presence of the DWI/FLAIR mismatch in patients with the PWI/DWI mismatch would provide additional information regarding last seen normal time (LTM). We used data from the 'AX200 for ischemic stroke' trial (AXIS 2 study NCT00927836). We studied the association between the presence of the DWI/FLAIR and PWI/DWI mismatch, baseline National Institute of Health Stroke Scale (NIHSS), age, ischemic-core volume, gender, intravenous (IV) tissue plasminogen activator (tPA), and perfusion-mismatch volume in univariate analysis. Significant variables (P<0.05) were added into the final multivariate model. We analyzed 197 patients. Seventy-two (37%) had both the PWI/DWI and the DWI/FLAIR mismatch. Patients with the double mismatch pattern had a shorter LTM than patients with the PWI/DWI mismatch alone (Median difference 90 minutes, P<0.01). Multivariate analysis confirmed the independent association between the two mismatch patterns (odds ratio (OR) 2.6, 95% confidence interval (CI) 1.2 to 5.4). Our study implies that the DWI/FLAIR mismatch and PWI/DWI mismatch are strongly associated, independent from LTM. Furthermore, in the presence of the PWI/DWI mismatch, the DWI/FLAIR pattern indicates a shorter LTM. This could have implications in selecting patients for reperfusion therapy.

Infarct volume-based subgroup selection in acute ischemic stroke trials.

BACKGROUND AND PURPOSE: We investigated whether hyperintensities with a diameter of at least 3, 3.5, and 4 cm and visible on at least 3 slices on diffusion-weighted imaging enables patient selection with an infarct volume of ≥15 mL. METHODS: Consecutive acute stroke patients were screened for the AXIS2 trial and examined according to a standardized magnetic resonance imaging protocol in 65 sites. Diffusion-weighted lesion diameters were measured and compared with volumetric assessments. RESULTS: Out of 238 patients, 86.2% (N=206) had infarct diameter of at least 3 cm. Volumetric assessments showed infarct volume of ≥15 mL in 157 patients. A cut-off value of 3 cm led to 96.8% sensitivity and 33.3% specificity for predicting lesion volume of ≥15 mL. Analogously, a 3.5 cm cut-off led to 96.8% sensitivity and 50.6% specificity and a 4 cm cut-off led to 91.7% sensitivity and 61.7% specificity. CONCLUSIONS: Lesion diameter measures may enable multicentric patient recruitment with a prespecified minimal infarct volume. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00927836.

Forced arm use is superior to voluntary training for motor recovery and brain plasticity after cortical ischemia in rats.

BACKGROUND AND PURPOSE: Both the immobilization of the unaffected arm combined with physical therapy (forced arm use, FAU) and voluntary exercise (VE) as model for enriched environment are promising approaches to enhance recovery after stroke. The genomic mechanisms involved in long-term plasticity changes after different means of rehabilitative training post-stroke are largely unexplored. The present investigation explored the effects of these physical therapies on behavioral recovery and molecular markers of regeneration after experimental ischemia. METHODS: 42 Wistar rats were randomly treated with either forced arm use (FAU, 1-sleeve plaster cast onto unaffected limb at 8/10 days), voluntary exercise (VE, connection of a freely accessible running wheel to cage), or controls with no access to a running wheel for 10 days starting at 48 hours after photothrombotic stroke of the sensorimotor cortex. Functional outcome was measured using sensorimotor test before ischemia, after ischemia, after the training period of 10 days, at 3 and 4 weeks after ischemia. Global gene expression changes were assessed from the ipsi- and contralateral cortex and the hippocampus. RESULTS: FAU-treated animals demonstrated significantly improved functional recovery compared to the VE-treated group. Both were superior to cage control. A large number of genes are altered by both training paradigms in the ipsi- and contralateral cortex and the hippocampus. Overall, the extent of changes observed correlated well with the functional recovery obtained. One category of genes overrepresented in the gene set is linked to neuronal plasticity processes, containing marker genes such as the NMDA 2a receptor, PKC ζ, NTRK2, or MAP 1b. CONCLUSIONS: We show that physical training after photothrombotic stroke significantly and permanently improves functional recovery after stroke, and that forced arm training is clearly superior to voluntary running training. The behavioral outcomes seen correlate with patterns and extent of gene expression changes in all brain areas examined. We propose that physical training induces a fundamental change in plasticity-relevant gene expression in several brain regions that enables recovery processes. These results contribute to the debate on optimal rehabilitation strategies, and provide a valuable source of molecular entry points for future pharmacological enhancement of recovery.

Granulocyte colony-stimulating factor in patients with acute ischemic stroke: results of the AX200 for Ischemic Stroke trial.

BACKGROUND AND PURPOSE: Granulocyte colony-stimulating factor (G-CSF; AX200; Filgrastim) is a stroke drug candidate with excellent preclinical evidence for efficacy. A previous phase IIa dose-escalation study suggested potential efficacy in humans. The present large phase IIb trial was powered to detect clinical efficacy in acute ischemic stroke patients. METHODS: G-CSF (135 µg/kg body weight intravenous over 72 hours) was tested against placebo in 328 patients in a multinational, multicenter, randomized, and placebo-controlled trial (NCT00927836; www.clinicaltrial.gov). Main inclusion criteria were ≤9-hour time window after stroke onset, infarct localization in the middle cerebral artery territory, baseline National Institutes of Health Stroke Scale score range of 6 to 22, and baseline diffusion-weighted imaging lesion size ≥15 mL. Primary and secondary end points were the modified Rankin scale score and the National Institutes of Health Stroke Scale score at day 90, respectively. Data were analyzed using a prespecified model that adjusted for age, National Institutes of Health Stroke Scale score at baseline, and initial infarct volume (diffusion-weighted imaging). RESULTS: G-CSF treatment failed to meet the primary and secondary end points of the trial. For additional end points such as mortality, Barthel index, or infarct size at day 30, G-CSF did not show efficacy either. There was, however, a trend for reduced infarct growth in the G-CSF group. G-CSF showed the expected peripheral pharmacokinetic and pharmacodynamic profiles, with a strong increase in leukocytes and monocytes. In parallel, the cytokine profile showed a significant decrease of interleukin-1. CONCLUSIONS: G-CSF, a novel and promising drug candidate with a comprehensive preclinical and clinical package, did not provide any significant benefit with respect to either clinical outcome or imaging biomarkers. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00927836.

Initial lesion volume is an independent predictor of clinical stroke outcome at day 90: an analysis of the Virtual International Stroke Trials Archive (VISTA) database.

BACKGROUND AND PURPOSE: Age and National Institutes of Health Stroke Scale early after stroke onset have been identified as important determinants of final stroke outcome. We analyzed the Virtual International Stroke Trials Archive (VISTA) database to define the influence of infarct or hemorrhagic volume on clinical outcome after stroke. METHODS: All patients were extracted from VISTA where infarct or hemorrhage volume information was available (n=2538; most images obtained by CT within 72 hours after stroke onset with a subset of MRI data included, volumes calculated by the ABC/2 approximation method). We used multivariate regression models to study the influence of age, National Institutes of Health Stroke Scale at baseline, and initial infarct/hemorrhage volume on clinical outcome (modified Rankin Scale, National Institutes of Health Stroke Scale, mortality) at day 90. RESULTS: We find that in a large cohort of >1800 patients with ischemic stroke, initial lesion size is a strong and independent predictor of stroke outcome in a statistical regression model that also accounts for age and National Institutes of Health Stroke Scale at baseline (P<0.0001). The use of infarct/hemorrhage volume as an additional predictive factor further reduces the fraction of unexplained variance in outcome by approximately 15% (R(2) of 0.41 versus 0.26 in a model without lesion volume). The predictive strength of initial lesion size is only marginally influenced by image modality or time point of image acquisition within the first 72 hours. The model was equally valid for both ischemic and hemorrhagic strokes. CONCLUSIONS: Infarct/hemorrhage volume at baseline together with age and National Institutes of Health Stroke Scale at baseline should be used in the effect analysis of future therapeutic stroke trials to improve power.

Pegylated granulocyte colony-stimulating factor conveys long-term neuroprotection and improves functional outcome in a model of Parkinson's disease.

Recent proof-of-principle data showed that the haematopoietic growth factor granulocyte colony-stimulating factor (filgrastim) mediates neuroprotection in rodent models of Parkinson's disease. In preparation for future clinical trials, we performed a preclinical characterization of a pegylated derivative of granulocyte colony-stimulating factor (pegfilgrastim) in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. We determined serum and cerebrospinal fluid drug levels after subcutaneous injection. A single injection of pegfilgrastim was shown to achieve stable levels of granulocyte colony-stimulating factor in both serum and cerebrospinal fluid with substantially higher levels compared to repetitive filgrastim injections. Leucocyte blood counts were only transiently increased after repeated injections. We demonstrated substantial dose-dependent long-term neuroprotection by pegfilgrastim in both young and aged mice, using bodyweight-adjusted doses that are applicable in clinical settings. Importantly, we found evidence for the functionally relevant preservation of nigrostriatal projections by pegfilgrastim in our model of Parkinson's disease, which resulted in improved motor performance. The more stable levels of pegylated neuroprotective proteins in serum and cerebrospinal fluid may represent a general advantage in the treatment of chronic neurodegenerative diseases and the resulting longer injection intervals are likely to improve patient compliance. In summary, we found that pegylation of a neuroprotective growth factor improved its pharmacokinetic profile over its non-modified counterpart in an in vivo model of Parkinson's disease. As the clinical safety profile of pegfilgrastim is already established, these data suggest that evaluation of pegfilgrastim in further Parkinson's disease models and ultimately clinical feasibility studies are warranted.

Granulocyte colony-stimulating factor improves cerebrovascular reserve capacity by enhancing collateral growth in the circle of Willis.

BACKGROUND AND PURPOSE: Restoration of cerebrovascular reserve capacity (CVRC) depends on the recruitment and positive outward remodeling of preexistent collaterals (arteriogenesis). With this study, we provide functional evidence that granulocyte colony-stimulating factor (G-CSF) augments therapeutic arteriogenesis in two animal models of cerebral hypoperfusion. We identified an effective dosing regimen that improved CVRC and stimulated collateral growth, thereby improving the outcome after experimentally induced stroke. METHODS: We used two established animal models of (a) cerebral hypoperfusion (mouse, common carotid artery ligation) and (b) cerebral arteriogenesis (rat, 3-vessel occlusion). Following therapeutic dose determination, both models received either G-CSF, 40 µg/kg every other day, or vehicle for 1 week. Collateral vessel diameters were measured following latex angiography. Cerebrovascular reserve capacities were assessed after acetazolamide stimulation. Mice with left common carotid artery occlusion (CCAO) were additionally subjected to middle cerebral artery occlusion, and stroke volumes were assessed after triphenyltetrazolium chloride staining. Given the vital role of monocytes in arteriogenesis, we assessed (a) the influence of G-CSF on monocyte migration in vitro and (b) monocyte counts in the adventitial tissues of the growing collaterals in vivo. RESULTS: CVRC was impaired in both animal models 1 week after induction of hypoperfusion. While G-CSF, 40 µg/kg every other day, significantly augmented cerebral arteriogenesis in the rat model, 50 or 150 µg/kg every day did not show any noticeable therapeutic impact. G-CSF restored CVRC in mice (5 ± 2 to 12 ± 6%) and rats (3 ± 4 to 19 ± 12%). Vessel diameters changed accordingly: in rats, the diameters of posterior cerebral arteries (ipsilateral: 209 ± 7-271 ± 57 µm; contralateral: 208 ± 11-252 ± 28 µm) and in mice the diameter of anterior cerebral arteries (185 ± 15-222 ± 12 µm) significantly increased in the G-CSF groups compared to controls. Stroke volume in mice (10 ± 2%) was diminished following CCAO (7 ± 4%) and G-CSF treatment (4 ± 2%). G-CSF significantly increased monocyte migration in vitro and perivascular monocyte numbers in vivo. CONCLUSION: G-CSF augments cerebral collateral artery growth, increases CVRC and protects from experimentally induced ischemic stroke. When comparing three different dosing regimens, a relatively low dosage of G-CSF was most effective, indicating that the common side effects of this cytokine might be significantly reduced or possibly even avoided in this indication.

Granulocyte-colony stimulating factor (G-CSF) improves motor recovery in the rat impactor model for spinal cord injury.

Granulocyte-colony stimulating factor (G-CSF) improves outcome after experimental SCI by counteracting apoptosis, and enhancing connectivity in the injured spinal cord. Previously we have employed the mouse hemisection SCI model and studied motor function after subcutaneous or transgenic delivery of the protein. To further broaden confidence in animal efficacy data we sought to determine efficacy in a different model and a different species. Here we investigated the effects of G-CSF in Wistar rats using the New York University Impactor. In this model, corroborating our previous data, rats treated subcutaneously with G-CSF over 2 weeks show significant improvement of motor function.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. TRIAL REGISTRATION: ClinicalTrials.gov NCT00298597.

AXIS: a trial of intravenous granulocyte colony-stimulating factor in acute ischemic stroke.

BACKGROUND AND PURPOSE: Granulocyte colony-stimulating factor (G-CSF) is a promising stroke drug candidate. The present phase IIa study assessed safety and tolerability over a broad dose range of G-CSF doses in acute ischemic stroke patients and explored outcome data. METHODS: Four intravenous dose regimens (total cumulative doses of 30-180 µg/kg over the course of 3 days) of G-CSF were tested in 44 patients in a national, multicenter, randomized, placebo-controlled dose escalation study (NCT00132470; www.clinicaltrial.gov). Main inclusion criteria were a 12-hour time window after stroke onset, infarct localization to the middle cerebral artery territory, a baseline National Institutes of Health Stroke Scale range of 4 to 22, and presence of diffusion-weighted imaging/perfusion-weighted imaging mismatch. RESULTS: Concerning the primary safety end points, we observed no increase of thromboembolic events in the active treatment groups, and no increase in related serious adverse events. G-CSF led to expected increases in neutrophils and monocytes that resolved rapidly after end of treatment. We observed a clinically insignificant drug-related decrease of platelets. As expected from the low number of patients, we did not observe significant differences in clinical outcome in treatment vs. placebo. In exploratory analyses, we observed an interesting dose-dependent beneficial effect of treatment in patients with DWI lesions > 14-17 cm³. CONCLUSIONS: We conclude that G-CSF was well-tolerated even at high dosages in patients with acute ischemic stroke, and that a substantial increase in leukocytes appears not problematic in stroke patients. In addition, exploratory analyses suggest treatment effects in patients with larger baseline diffusion-weighted imaging lesions. The obtained data provide the basis for a second trial aimed to demonstrate safety and efficacy of G-CSF on clinical end points.

The hematopoietic factor granulocyte-colony stimulating factor improves outcome in experimental spinal cord injury.

Granulocyte-colony stimulating factor (G-CSF) is a potent hematopoietic factor that drives differentiation of neutrophilic granulocytes. We have recently shown that G-CSF also acts as a neuronal growth factor, protects neurons in vitro and in vivo, and has regenerative potential in various neurological disease models. Spinal cord injury (SCI) following trauma or secondary to skeletal instability is a terrible condition with no effective therapies available at present. In this study, we show that the G-CSF receptor is up-regulated upon experimental SCI and that G-CSF improves functional outcome in a partial dissection model of SCI. G-CSF significantly decreases apoptosis in an experimental partial spinal transsection model in the mouse and increases expression of the anti-apoptotic G-CSF target gene Bcl-X(L). In vitro, G-CSF enhances neurite outgrowth and branching capacity of hippocampal neurons. In vivo, G-CSF treatment results in improved functional connectivity of the injured spinal cord as measured by Mn(2+)-enhanced MRI. G-CSF also increased length of the dorsal corticospinal tract and density of serotonergic fibers cranial to the lesion center. Mice treated systemically with G-CSF as well as transgenic mice over-expressing G-CSF in the CNS exhibit a strong improvement in functional outcome as measured by the BBB score and gridwalk analysis. We show that G-CSF improves outcome after experimental SCI by counteracting apoptosis, and enhancing connectivity in the injured spinal cord. We conclude that G-CSF constitutes a promising and feasible new therapy option for SCI.