Methionyl Granulocyte Colony-stimulating Factor Research Articles

Filgrastim, a Recombinant Form of Granulocyte Colony-stimulating Factor, Ameliorates 3-nitropropionic Acid and Haloperidol-induced Striatal Neurotoxicity in Rats.

Striatal neurotoxicity is the pathological hallmark for a heterogeneous group of movement disorders like Tardive dyskinesia (TD) and Huntington's disease (HD). Both diseases are characterized by progressive impairment in motor function. TD and HD share common features at both cellular and subcellular levels. Filgrastim, a recombinant methionyl granulocyte colony-stimulating factor (GCSF), shows neuroprotective properties in in-vivo models of movement disorders. This study seeks to evaluate the neuroprotective effect of filgrastim in haloperidol and 3-NP-induced neurotoxicity in rats. The study was divided into two: in study one, rats were administered with haloperidol for 21days, filgrastim at the dose of (20, 40, 60µg/kg,s.c.) was administered once a day before haloperidol treatment and the following parameters (orofacial movements, rotarod, actophotometer) were performed to assess TD. Similarly, in the second study, rats were administered with 3-NP for 21days, filgrastim at a dose of (20 and 40µg/kg, s.c.) was administered, and the following parameters (rotarod, narrow beam walk, and open field test) were assessed for HD. On the 22nd day, animals were sacrificed and cortex and striatum isolated for oxidative stress (LPO, GSH, SOD, catalase, and nitrate) marker assessment. Results revealed that haloperidol and 3-NP treatment significantly impaired motor coordination, and oxidative defense inducing TD and HD-like symptoms. Treatment with filgrastim significantly averted haloperidol and 3-NP-induced behavioral and biochemical alterations. Conclusively, the neuroprotective effect of filgrastim is credited to its antioxidant properties. Hence, filgrastim might be a novel therapeutic candidate for the management of TD and HD.

Analytical Method Development and Validation of Filgrastim by UV and RP-UFLC Methods

The research work was carried out for establishing a new Ultra Violet (UV)— Visible spectroscopy and Reverse phase-Ultra Fast Liquid Chromatography (RP-UFLC) method for the analysis and quantification of a biosimilar drug, Filgrastim. Filgrastim or recombinant methionyl granulocyte colony stimulating factor (rGCSF) is a glycoprotein. It has a biological action essential for proliferation and differentiation of hematopoetic and progenitor cells. The UV and RP-UFLC work was carried on a Shimadzu UV1800 Spectrophotometer and Shimadzu Prominence LC-20AD UFLC systems, respectively. The λmax of filgrastim was found to be 215 nm. The correlation coefficient by UV spectroscopy was found to be 0.9994 for the concentration range of 1 to 3 μg/ml in double distilled water. The Reverse phase UFLC was done by using Phenomenex C4 (25 cm × 0.46 cm internal diameter) 15 μ, 300 A° analytical column. The optimized mobile phase for binary elution was Acetonitrile and double distilled water (80:20) with a flow rate of 1 ml/min. The retention time of drug was at 3.2 min. It was observed that the response of the detector was linear in the range of 5 - 15 μg/ml with correlation coefficient value of 0.999. After developing the methods, it was assured for the intended use by validation of the analytical parameters like linearity, accuracy, precision, limit of detection, limit of quantitation, ruggedness and robustness. The results of all the parameters for both the methods were found to be within the acceptance criteria as per the International Council for Harmonisation (ICH) guidelines.

Neuroprotective Effects of Filgrastim in Rotenone-Induced Parkinson's Disease in Rats: Insights into its Anti-Inflammatory, Neurotrophic, and Antiapoptotic Effects.

All current treatments of Parkinson's disease (PD) focus on enhancing the dopaminergic effects and providing symptomatic relief; however, they cannot delay the disease progression. Filgrastim, a recombinant methionyl granulocyte colony-stimulating factor, demonstrated neuroprotection in many neurodegenerative and neurological diseases. This study aimed to assess the neuroprotective effects of filgrastim in rotenone-induced rat model of PD and investigate the potential underlying mechanisms of filgrastim actions. The effects of two doses of filgrastim (20 and 40μg/kg) on spontaneous locomotion, catalepsy, body weight, histology, and striatal dopamine (DA) content, as well as tyrosine hydroxylase (TH) and α-synuclein expression, were evaluated. Then, the effective dose was further tested for its potential anti-inflammatory, neurotrophic, and antiapoptotic effects. Filgrastim (40μg/kg) prevented rotenone-induced motor deficits, weight reduction, striatal DA depletion, and histological damage. Besides, it significantly inhibited rotenone-induced decrease in TH expression and increase in α-synuclein immunoreactivity in the midbrains and striata of the rats. These effects were associated with reduction of rotenone-induced neuroinflammation, apoptosis, and brain-derived neurotrophic factor depletion. Collectively, these results suggest that filgrastim might be a good candidate for management of PD.

Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay

A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins.

Preclinical and clinical phase I studies of a new recombinant Filgrastim (BK0023) in comparison with Neupogen®

BackgroundFilgrastim or methionyl-granulocyte colony-stimulating factor (Met-G-CSF), is a recombinant therapeutic protein widely used to treat severe neutropenia caused by myelosuppressive drugs in patients with nonmyeloid malignancies. In addition to its role in the regulation of granulopoiesis, treatment with G-CSF is considered the standard approach to mobilize CD34 positive (CD34+) mononuclear cells for reconstituting hemopoietic ability for bone marrow transplantation. An intended biosimilar filgrastim (coded BK0023) was produced in GMP conditions by E.coli fermentation according to an original recombinant process and showed physico-chemical properties and purity profile similar to Neupogen®, a commercial preparation of filgrastim. The aim of the present study was to demonstrate the comparability of BK0023 to Neupogen® in terms of both in vitro biological activities and in vivo toxicology, pharmacokinetics and pharmacodynamics.MethodsCell proliferation and radioligand binding assays were conducted in NFS-60 cells to compare the biological activity and functional interaction with the G-CSF receptor in vitro, while preclinical in vivo studies, including pharmacokinetics and pharmacodynamics after repeated dose were performed in normal and neutropenic rats. A phase I study was carried out in healthy male volunteers treated by multiple-dose subcutaneous administration of BK0023 and Neupogen® to evaluate their pharmacodynamic effects as well as their pharmacokinetic and safety profile and to demonstrate their pharmacodynamic equivalence and pharmacokinetic bioequivalence.ResultsThe results reported in this work demonstrate that BK0023 is comparable in terms of biological activity, efficacy and safety to Neupogen®.ConclusionsBK0023 has the same pharmacokinetic profile, efficacy and safety as the reference commercial filgrastim Neupogen® and therefore could be further developed to become a convenient option to treat neutropenia in oncological patients.Trial registrationTrial registration number (TRN): NCT01933971. Date of registration: Sept 6th 2013.

Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.

A Phase 3, Randomized, Placebo-Controlled Trial of Filgrastim in Patients (Pts) with Hematologic Malignancies (HM) Treated with a Myeloablative Conditioning Regimen and Allogenic Bone Marrow Transplantation (allo BMT).

Background. Recombinant methionyl granulocyte colony-stimulating factor (methuG-CSF; Filgrastim) is commonly used to reduce the duration of neutropenia that follows allo BMT performed after high-dose chemo-/radiotherapy (HDT) in pts with HM. Recently, a retrospective chart review suggested that leukemic pts treated with G-CSF after allo BMT had faster neutrophil recovery, but slower platelet engraftment, increased risk of graft vs host disease (GvHD), and reduced survival relative to pts who did not receive G-CSF (Ringdén et al, 2004). The present double-blind, randomized, placebo-controlled, multicenter phase 3 study was designed to evaluate the effects of Filgrastim treatment on neutrophil recovery. Pts were to be followed for 2 years.Methods. Eligible pts were aged 12–55 and scheduled for allo BMT from a sibling donor preceded by high-dose chemotherapy +/− total body irradiation for treatment of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL), high-risk non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML). Pts were randomized to receive either Filgrastim (5μg/kg/day) or placebo from transplant until recovery of an absolute neutrophil count (ANC) of ≥0.5x109/L or for a maximum of 42 days. Randomization was stratified by age (<18 and ≥18 years) and remission status (1st complete remission/subsequent remissions) within each center. The primary endpoint was the time to ANC of ≥0.5x109/L. Assuming a standard deviation of 3.8 days, the study was powered to have a probability of at least 80% of detecting a difference of at least 3.6 days between the groups for the endpoint of time to ANC ≥0.5x109/L using a two-tailed significance of 5%.Results. The first pt was enrolled in 1993; however, the planned sample size of 100 pts (50 per arm) was not reached due to slow enrollment and the study was terminated in 1996. A total of 66 pts were enrolled; of these, 51 were analysed (25 Filgrastim, 26 placebo). Filgrastim-treated pts achieved an ANC of ≥0.5x109/L at a median (quartiles) time of 15 (13, 16) days compared with 19 (17, 22) days for placebo; ANC ≥1.0x109/L was reached by 16 (15, 17) and 22 (21, 28) days, respectively. Median (quartiles) time to discharge from hospital was 30 (26, 43) days for Filgrastim vs 36 (28, 42) days for placebo. The number of pts with platelet count ≥25x109/L within 56 days was 16 for Filgrastim vs 23 for placebo, and 12 vs 18, respectively, for platelet count ≥50x109/L within 56 days. The incidence, maximum severity and number of days to onset of acute GvHD were similar across the groups. No difference in adverse events was reported; 4 Filgrastim pts died (0 of disease progression) and 8 placebo pts died (3 of disease progression).Conclusion. The results of this phase 3, double-blind, placebo-controlled, randomized trial in pts with HM receiving allo BMT may suggest that Filgrastim treatment enhances neutrophil engraftment with no negative impact on platelet recovery or GvHD.

Neoadjuvant Etoposide, Ifosfamide, and Cisplatin Followed by Concomitant Thoracic Radiotherapy and Continuous Cisplatin Infusion in Stage IIIb Non-small Cell Lung Cancer

To determine the applicability and safety of an ifosfamide, cisplatin, and etoposide (VIP) regimen as a neoadjuvant chemotherapy to a concomitant thoracic radiotherapy and cisplatin continuous infusion in locally advanced non-small cell lung cancer (NSCLC). Forty-four patients (stage IIIb in 43 and stage IIIa in 1) entered a study of VIP, followed by concomitant thoracic radiotherapy and cisplatin continuous infusion. Chemotherapy consisted of three courses of cisplatin 25 mg/m2, ifosfamide 1.5 g/m2 (with uroprotection), and etoposide 100 mg/m2 given on days 1 to 4 of a 21-day cycle with hematopoietic support using recombinant human methionyl granulocyte colony stimulating factor. Patients who achieved a response or a stabilization were planned to receive a split-course normofractionated thoracic radiotherapy (first course: 30 Gy/10; 4-week rest period; second course: 25 Gy/10). A continuous cisplatin infusion of 6 mg/m2 daily was administered using an autonomous chemotherapy delivery device. Total plasma platinum titration was performed daily during the two courses in five of the patients. Analyses were done on an intent-to-treat basis. Thirty-nine of the 44 patients received the three-cycle chemotherapy program. Received dose intensity was 82%. Thirty-eight patients received the radiotherapy and, among them, 35 received the complete concomitant continuous cisplatin infusion. Objective (complete) response rates were 48% (7%) at the end of chemotherapy and increased up to 61% (16%) by the end of radiotherapy. At the end of the first radiotherapy cycle, the mean total plasma platinum concentration was twice as high as that of the residual postinduction chemotherapy concentration. During induction chemotherapy, myelosuppression was the limiting toxicity requiring hospital readmission in 23 patients. During radiotherapy, the main toxicity was acute esophagitis. A relatively high rate of pulmonary fibrosis was observed using the subjective objective management analytic--late effects of normal tissue score without life-threatening pulmonary function impairment. None of the patients died from toxic reactions. Probability of survival at 1, 2, and 3 years was 49%, 19%, and 5%, respectively. Primary cause of failure was a local relapse in 63% of the patients, brain metastases in 24%, and hematogeneous metastases to other sites in 13%. Neoadjuvant VIP followed by concomitant radiotherapy-chemotherapy is feasible, but the split-course radiotherapy did not prevent a high rate of local recurrences. The high rate of toxic reactions requiring hospital readmission limits further development of such an aggressive regimen in NSCLC.

Affinity chromatography with immunochemical detection applied to the analysis of human methionyl granulocyte colony stimulating factor in serum.

An on-line, automated, HPLC method was developed for the separation and detection of recombinant human methionyl granulocyte colony stimulating factor (GCSF) and GCSF modified with poly(ethylene glycol) (PEG-GCSF) in rat serum. The automated method consists of an initial immunoaffinity chromatography step, a microbore reverse phase separation, and postcolumn immunochemical detection. Calibration curves were constructed with a lower limit of 1.5 ng of GCSF (80 fmol) and 7.5 ng of PEG-GCSF in 120 microL of rat serum. In vivo serum samples from rats dosed with PEG-GCSF were also analyzed. The development of the method is discussed as well as its general application to the detection of metabolites of recombinant proteins in body fluids.

Increasing and planned dose intensity of doxorubicin, cyclophosphamide and etoposide (ACE) by adding recombinant human methionyl granulocyte colony-stimulating factor (G-CSF; filgrastim) in the treatment of small cell lung cancer (SCLC)

The aim of this Phase II study was to test the feasibility of intensifying standard chemotherapy in the treatment of small cell lung cancer (SCLC) by reducing the interval between cycles from 3 to 2 weeks by adding recombinant human methionyl granulocyte colony-stimulating factor (G-CSF; filgrastim) to shorten the duration of neutropenia following each cycle. Thirty-two patients with SCLC were prescribed six cycles of 2-weekly doxorubicin 50 mg/m 2 and cyclophosphamide 1 g/m 2 on day 1, and etoposide 120 mg/m 2 i.v. on days 1, 2 and 3 (ACE), plus filgrastim in a fixed dose of 300 μg s.c. on days 4–14 of each cycle. Three patients died during the treatment period and a further nine had chemotherapy terminated before the sixth cycle, all nine because of toxicity. All 32 patients have been followed up for at least 21 months; 14 (44%) were alive at 12 months and the median survival period was 356 days. Of the 127 intervals between cycles of chemotherapy, 74 (58%) were of the prescribed 14 days, 18 (14%) of 15–20 days, 25 (20%) of 21 days, and 10 (8%) were longer. The results were best during the first four cycles, during which 71% of the 83 intervals were of 14 days and a further 10% were less than 21 days. The main reason for delay was haematological toxicity in 37 of the 53 instances. Symptoms of myelosuppression occurred in 23 patients, but, at 14 days after a cycle of chemotherapy, all 127 available neutrophil granulocyte counts were normal. Twenty-one patients received blood transfusion and five platelet transfusion. The only adverse effects attributed to filgrastim were episodes of rash, throat swelling, anorexia and shivering, affecting one patient. We conclude that the policy of adding filgrastim allows the dose intensity of ACE chemotherapy to be increased by reducing the intervals between cycles. The findings reinforce those of a parallel study involving lenograstim.

Reduction by Granulocyte Colony-Stimulating Factor of Fever and Neutropenia Induced by Chemotherapy in Patients with Small-Cell Lung Cancer

Background. Neutropenia and infection are major dose-limiting side effects of chemotherapy. Previous studies have suggested that recombinant methionyl granulocyte colony-stimulating factor (G-CSF) can reduce chemotherapy-related neutropenia in patients with cancer. We conducted a randomized clinical trial to test this hypothesis and the clinical implications. Methods. Patients with small-cell lung cancer were enrolled in a multicenter, randomized, double-blind, placebo-controlled trial of recombinant methionyl G-CSF to study the incidence of infection as manifested by fever with neutropenia (absolute neutrophil count, <1.0×10 9 per liter, with a temperature ≥38.2 o C) resulting from up to six cycles of chemotherapy with cyclophosphamide, doxorubicin, and etoposide

Effective stimulation of donors for granulocyte transfusions with recombinant methionyl granulocyte colony-stimulating factor [see comments

Effective granulocyte transfusion (GT) therapy has been hampered by the low yield of neutrophil granulocytes (PMN) obtainable from normal donors even by use of corticosteroid prestimulation, hydroxyethyl starch (HES), and modern leukapheresis (LA) techniques. To increase the PMN yield we performed LA in 22 healthy volunteer donors after a single subcutaneous administration of 300 micrograms of granulocyte colony- stimulating factor (G-CSF) 12 to 16 hours before LA. Five to 7 L of blood was processed within 1.9 to 3 hours using the standard CS- 3000Plus (Baxter, Deerfield, IL) LA protocol including HES. The mean number of PMN harvested was 44.32 +/- 15.5 x 10(9), corresponding to 6.88 +/- 2.1 x 10(9)/L of blood processed. In the final product PMN functions (in vitro: chemotaxis, phagocytosis, chemiluminescence, superoxide anion production; in vivo: chemiluminescence, half-life) were at least normal. In all donors G-CSF induced a consistent increase of white blood cell (mean 16.46 +/- 3.8 x 10(9)/L) and PMN counts (15.94 +/- 3.6 x 10(9)/L). No G-CSF-related side effects were observed and LA was well tolerated. G-CSF prestimulation allows to harvest three to five times higher numbers of functionally normal PMN by LA compared with corticosteroid pretreatment. This may help to overcome one of the major limitations of an effective PMN support.

Reduction by Granulocyte Colony-Stimulating Factor of Fever and Neutropenia Induced by Chemotherapy in Patients with Small-Cell Lung Cancer

Neutropenia and infection are major dose-limiting side effects of chemotherapy. Previous studies have suggested that recombinant methionyl granulocyte colony-stimulating factor (G-CSF) can reduce chemotherapy-related neutropenia in patients with cancer. We conducted a randomized clinical trial to test this hypothesis and the clinical implications. Patients with small-cell lung cancer were enrolled in a multicenter, randomized, double-blind, placebo-controlled trial of recombinant methionyl G-CSF to study the incidence of infection as manifested by fever with neutropenia (absolute neutrophil count, less than 1.0 x 10(9) per liter, with a temperature greater than or equal to 38.2 degrees C) resulting from up to six cycles of chemotherapy with cyclophosphamide, doxorubicin, and etoposide. The patients were randomly assigned to receive either placebo or G-CSF, with treatment beginning on day 4 and continuing through day 17 of a 21-day cycle. The safety of the study treatment could be evaluated in 207 of the 211 patients assigned to either drug, and its efficacy in 199. At least one episode of fever with neutropenia occurred in 77 percent of the placebo group, as compared with 40 percent of the G-CSF group (P less than 0.001). Over all cycles of chemotherapy, the median duration of grade IV neutropenia (absolute neutrophil count, less than 0.5 x 10(9) per liter) was six days with placebo as compared with one day with G-CSF. During cycles of blinded treatment, the number of days of treatment with intravenous antibiotics, the number of days of hospitalization, and the incidence of confirmed infections were reduced by approximately 50 percent when G-CSF was given, as compared with placebo. Mild-to-moderate medullary bone pain occurred in 20 percent of the patients receiving G-CSF. The use of G-CSF as an adjunct to chemotherapy in patients with small-cell cancer of the lung was well tolerated and led to reductions in the incidence of fever with neutropenia and culture-confirmed infections; in the incidence, duration, and severity of grade IV neutropenia; and in the total number of days of treatment with intravenous antibiotics and days of hospitalization.