Recombinant Human Granulocyte Colony-stimulating Factor Research Articles

Effect of human recombinant granulocyte colony-stimulating factor on rat busulfan-induced testis injury.

Granulocyte colony-stimulating factor (G-CSF) is known for proliferation and anti-apoptotic activities. We aimed to use this growth factor in busulfan-injured testis. 32 male Wistar rats were injected with a double dosage of 15 ml/kg busulfan with 14 days interval. Administration of human recombinant G-CSF (100 µg/kg) subcutaneously was performed in two different time periods: 3 days before and 2 days after receiving busulfan, G-CSF1; and at days 14-18 of busulfan injection, G-CSF2. Animals were sacrificed at the end of week five. Histological analysis, testis weight and sperm parameters (sperm count and viability) has been checked. Expressions of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4), deleted in azoospermia like (DAZL), transition protein 2 (TP2), proliferating cell nuclear antigen (PCNA) and 5-Bromo-20-deoxyuridine (BrdU) were assessed. Empty seminiferous tubules were apparent in the busulfan- and G-CSF2-injected rats, but not in the G-CSF1 group. The G-CSF1-treated animals showed an increase in testis weight and sperm count and viability along with high expressions of DDX4, DAZL, TP2, PCNA and BrdU; even so, the changes were reversed in the busulfan and G-CSF2 groups (for all p < 0.05). Our results revealed that G-CSF application prior to busulfan insult is a promising approach in fertility maintenance.

A Randomized Multicenter Phase III Study of Single Administration of Mecapegfilgrastim (HHPG-19K), a Pegfilgrastim Biosimilar, for Prophylaxis of Chemotherapy-Induced Neutropenia in Patients With Advanced Non–Small-Cell Lung Cancer (NSCLC)

BackgroundMecapegfilgrastim (code name HHPG-19K) is a biosimilar to pegylated recombinant human granulocyte-colony stimulating factor (PEG-rhG-CSF). The efficacy and safety of mecapegfilgrastim, using a regimen of once-per-cycle injection of 100-μg/kg or a fixed 6-mg dose, were evaluated for the prophylactic therapy for neutropenia in patients with advanced non–small-cell lung cancer (NSCLC) who were treated with myelosuppressive chemotherapy. Materials and MethodsPatients were randomized (1:1:1) blindly to 3 treatment arms to receive a single injection of mecapegfilgrastim 100 μg/kg, a 6-mg fixed dose of mecapegfilgrastim, or saline (control) in cycle 1. In cycles 2 to 4 following unblinding at the end of cycle 1, patients in the control arm received daily injections of short-acting rhG-CSF at a dose of 5 μg/kg, whereas patients in the 2 mecapegfilgrastim arms continued the same treatment as in cycle 1. All patients received 4 chemotherapy cycles of docetaxel combined with cisplatin or carboplatin every 21 days. The primary endpoint was the incidence of grade ≥ 3 neutropenia in cycle 1. ResultsA single dose of 100 μg/kg or a fixed 6-mg dose of mecapegfilgrastim per cycle effectively reduced chemotherapy-induced neutropenia and was comparable to daily rhG-CSF with regard to all efficacy endpoints, including incidence of grade ≥ 3 neutropenia, incidence of febrile neutropenia, duration of grade ≥ 3 neutropenia, and time to neutrophil recovery. No difference in efficacy parameters was observed between the 2-dose regimens of mecapegfilgrastim across all cycles. Mecapegfilgrastim was well-tolerated and was as safe as daily rhG-CSF. ConclusionOnce-per-cycle injection of mecapegfilgrastim is as effective and safe as daily rhG-CSF for prophylaxis of chemotherapy-induced neutropenia in patients with NSCLC. Mecapegfilgrastim (fixed 6-mg dose) is recommended in clinical practice for its convenient dose management.

G-CSF promotes autophagy and reduces neural tissue damage after spinal cord injury in mice

Granulocyte colony-stimulating factor (G-CSF) was investigated for its capacity to induce autophagy and related neuroprotective mechanisms in an acute spinal cord injury model. To accomplish this goal, we established a mouse spinal cord hemisection model to test the effects of recombinant human G-CSF. The results showed that autophagy was activated after spinal cord injury and G-CSF appears to induce a more rapid activation of autophagy within injured spinal cords as compared with that of non-treated animals. Apoptosis as induced in mechanically injured neurons with G-CSF treatment was enhanced after inhibiting autophagy by 3-methyladenine (3-MA), which partially blocked the neuroprotective effect of autophagy as induced by G-CSF. In addition, G-CSF inhibited the activity of the NF-κB signal pathway in neurons after mechanical injury. We conclude that G-CSF promotes autophagy by inhibiting the NF-κB signal pathway and protects neuronal structure after spinal cord injury. We therefore suggest that G-CSF, which rapidly induces autophagy after spinal cord injury to inhibit neuronal apoptosis, may thus provide an effective auxiliary therapeutic intervention for spinal cord injury.

High efficiency of peripheral blood hematopoietic stem cell mobilization in multiple myeloma: one case report and literature review

Objective To explore the influential factors of obtaining high proportion of CD34+ cells in patients with multiple myeloma (MM) peripheral blood hematopoietic stem cell (HSC) mobilization. Methods One patient with MM who was 64 years old and hospitalized in Department of Hematology, Hainan Branch of Chinese PLA General Hospital was enrolled into this study on January 2014. The patient achieved complete remission after four courses of bortezomib + doxorubicin hydrochloride liposome + dexamethasone induction regimens. Then peripheral blood HSC was mobilized with bortezomib + high dose cyclophosphamide + dexamethasone regimens in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human thrombopoietin (rhTPO). Mobilization effect was reported with its relevant literature reviewed. Results The proportion of CD34+ cells in HSC graft was 6.80%. The number of mononuclear cells and CD34+ cells in harvest of single collection was 3.17×108/kg and 19.20×106/kg, respectively. Engraftment time of neutrophil and platelet was 10 days and 12 days after HSC transplantation, respectively. Conclusions Chemotherapy in combination with rhG-CSF and rhTPO is effective and safety for autologous HSC mobilization in MM patient. As this research is just a case report, it still needs many large sample and multi-center randomized controlled trials to confirm whether it can be widely used in MM patients. Key words: Multiple myeloma; Hematopoietic stem cell transplantation; Hematopoietic stem cell mobilization

Pharmacokinetic and pharmacodynamic properties of a new biosimilar filgrastim TPI G-CSF in comparison to the marketed reference filgrastim Neupogen&amp;reg;: a double-blind, single-dose, two-period crossover trial

Pharmacokinetic and pharmacodynamic properties of a new biosimilar filgrastim TPI G-CSF in comparison to the marketed reference filgrastim Neupogen®: a double-blind, single-dose, two-period crossover trial Sarfaraz K Niazi Therapeutic Proteins International, LLC, Chicago, IL, USA Abstract: Biosimilar biological products are a safe and effective alternative to branded biological agents. One of the most common uses of the therapeutic protein filgrastim, a biological drug (recombinant human granulocyte-colony stimulating factor), is to reduce the occurrence and duration of severe neutropenia and its associated serious complications. TPI G-CSF, a filgrastim product under development by Therapeutic Proteins International, LLC, is a proposed biosimilar to Amgen's marketed filgrastim, Neupogen®. To evaluate bioequivalence, we conducted a double-blind, randomized, two-period crossover study that took place at a single center and had a washout period of at least 2 weeks. The pharmacokinetic endpoints (area under the concentration-time curve from time 0 to time of the last observed/measured non-zero concentration [AUC0-t], AUC from time 0 extrapolated to infinity [AUC0-inf], and maximum observed concentration [Cmax]) and the pharmacodynamic endpoints (baseline-corrected area under the effect curve from time zero to the last non-zero cell count data [AUEC0-t] and maximum observed effect [Emax]) for the absolute neutrophil count were compared after administration of a subcutaneous 5 µg/kg dose of TPI G-CSF or Neupogen® in 58 healthy adults. These 58 healthy subjects (72% male, 34.8±10.5 years, 77.1±14.1 kg) were randomly assigned to a treatment sequence group (TPI G-CSF-Neupogen® or Neupogen®-TPI G-CSF); subjects received a single subcutaneous injection of 5 µg/kg in each period. The 95% pharmacodynamic and 90% pharmacokinetic geometric confidence intervals of the ratio (TPI G-CSF/Neupogen®) of least-squares means from the analysis of variance of the natural log-transformed data for each parameter fell within the approved bioequivalence range of 80% to 125%. There were no serious adverse events in this study. Treatment-emergent adverse events were reported by 69% of the study subjects, with a similar incidence between treatments. The notable incidents were treatment-emergent injection site reactions. Injections of TPI G-CSF and Neupogen® appeared to be safe and equally well tolerated. Keywords: filgrastim, G-CSF, pharmacokinetic, pharmacodynamic, ANC, WBC

DEVELOPMENT OF AUTONOMOUSLY REPLICATING VIRAL RNA TO EXPRESS THE RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR

Recombinant human granulocyte colony-stimulating factor (G-CSF) produced in cultured mammalian cells undergoes proper posttranslational modifications and, thereby, possesses better pharmacological properties in comparison with the homologous protein expressed in bacteria. Biopharmaceuticals derived from cell culture tend to be expensive because of lower yields compared to bacterially expressed competitors and numerous issues with the scalability of production. Particular limitation of scalability pertains to delivery of expression vectors to the cell culture. Natural and efficient way to deliver foreign DNA or RNA to cell is a viral infection. We intended to develop the viral genome capable of G-CSF expression.Objectives: To develop autonomously replicating viral RNA capable of heterologous expression of the G-CSF in cultured mammalian cells.Methods: Genetic engineering, cell culture, and virology.Results: Viable genome of the Venezuelan equine encephalomyelitis virus (VEE) was constructed. The G-CSF gene was synthesized de novo. Gene cassette GFP-2A-G-CSF was constructed for simultaneous expression of the fluorescent marker of viral replication (GFP) and the product of interest. Recombinant viral genome VEE/GFP-2A-G-CSF was assembled; its viability was confirmed upon transfection to the BHK-21 cells.Conclusion: Genetic engineering of the cDNA copies of the genomes of RNA viruses provides excessive opportunities to develop mammalian expression systems. We achieved production of recombinant proteins in cultured mammalian cells transfected with the VEE viral RNA carrying the inserted genes of marker protein and G-CSF.

Biosimilar Filgrastim (Tevagrastim, XMO2) for AllogeneicHematopoietic Stem Cell Mobilization andTransplantation in Patients with Acute MyelogenousLeukemia/Myelodysplastic Syndromes.

Human recombinant granulocyte colony-stimulating factor (G-CSF), filgrastim (Neupogen; Amgen, Thousand Oaks, CA, USA), has been widely used for the mobilization of CD34(+) hematopoietic stem cells (HSC) from healthy donors. The experience with biosimilar G-CSF agents in this area is limited. We performed a prospective study assessing Tevagrastim (biosimilar filgrastim, XMO2; Teva, Israel) for mobilization of CD34(+) peripheral blood HSC in HLA-matched healthy sibling donors for transplantation in 24 patients with acute myelogenous leukemia (AML) and high-risk myelodysplastic syndromes (MDS) (NCT01542944). Results were compared to a historical control group of sibling donors who received filgrastim for stem cell mobilization for allogeneic stem cell transplantations in patients with AML and MDS. The healthy donors received Tevagrastim or filgrastim in a dose of 10 μg/kg body weight (BW) subcutaneously for 4 days. The target yields of CD34(+) cells was 5× 10(6) CD34(+) cells/kg BW of the recipient. A median 10.2× 10(6) (range, 2.52 to 35.4) and 9.35× 10(6) (range, 3.7 to 30.6) CD34(+) cells per kg BW were collected in the Tevagrastim and filgrastim groups, respectively. All patients promptly engrafted with a median day of absolute neutrophil count (ANC) of >.5× 10(9)/L and >1× 10(9)/L of 13 days (range, 10 to 21) and 13.5 days (range, 10 to 22), respectively in the Tevagrastim group and 12 days (range, 10 to 18) and 13 days (range, 10 to 18) in the filgrastim group, respectively. Platelets reached counts of >20× 10(9)/L and >50× 10(9)/L within a median of 14 days (range, 11 to 33) and 17 days (range, 12 to 33) in the Tevagrastim group and 13 (range, 10 to 29) and 15 (range, 10 to 32) days in the filgrastim group, respectively. The donors developed only mild and transient side effects, which were not different between the Tevagrastim study group and the filgrastim historical control group. Similarly, the transplantation-related toxicitiesand outcomes did not differ between the patients who underwent transplantation with Tevagrastim-mobilized grafts and the filgrastim historical controls. In summary, we observed a similar yield of CD34(+) stem cell mobilization in the Tevagrastim study group and the filgrastim historical control group with similar engraftment kinetic, hematopoietic reconstitution, and transplantation outcome. Tevagrastim administration was safe with minimal side effects and toxicity not different than historical controls. The lack of significant differences for all parameters of stem cell collection, engraftment, and safety with the biosimilar XMO2 Tevagrastim demonstrate the "similarity" of the biosimilar and recombinant human G-CSF inthis indication.

Neutrophil dynamics during concurrent chemotherapy and G-CSF administration: Mathematical modelling guides dose optimisation to minimise neutropenia

The choice of chemotherapy regimens is often constrained by the patient's tolerance to the side effects of chemotherapeutic agents. This dose-limiting issue is a major concern in dose regimen design, which is typically focused on maximising drug benefits. Chemotherapy-induced neutropenia is one of the most prevalent toxic effects patients experience and frequently threatens the efficient use of chemotherapy. In response, granulocyte colony-stimulating factor (G-CSF) is co-administered during chemotherapy to stimulate neutrophil production, increase neutrophil counts, and hopefully avoid neutropenia. Its clinical use is, however, largely dictated by trial and error processes. Based on up-to-date knowledge and rational considerations, we develop a physiologically realistic model to mathematically characterise the neutrophil production in the bone marrow which we then integrate with pharmacokinetic and pharmacodynamic (PKPD) models of a chemotherapeutic agent and an exogenous form of G-CSF (recombinant human G-CSF, or rhG-CSF). In this work, model parameters represent the average values for a general patient and are extracted from the literature or estimated from available data. The dose effect predicted by the model is confirmed through previously published data. Using our model, we were able to determine clinically relevant dosing regimens that advantageously reduce the number of rhG-CSF administrations compared to original studies while significantly improving the neutropenia status. More particularly, we determine that it could be beneficial to delay the first administration of rhG-CSF to day seven post-chemotherapy and reduce the number of administrations from ten to three or four for a patient undergoing 14-day periodic chemotherapy.

Site-specific characterization and absolute quantification of pegfilgrastim oxidation by top-down high-performance liquid chromatography-mass spectrometry.

The characterization and absolute quantification of protein biopharmaceuticals and their product-related impurities, e.g., oxidation variants, is essential due to their potential impact on biological activity and immunogenicity. Here, we present site assignment and absolute quantification of oxidation variants of pegfilgrastim, a poly(ethylene glycol) modified recombinant human granulocyte-colony stimulating factor. Pegfilgrastim stressed with 1.0% hydrogen peroxide served as a model protein for developing a top-down high-performance liquid chromatography-mass spectrometry (HPLC-MS) platform that allowed direct site assignment of Met122, Met127, and Met138 oxidation within a total analysis time of 30 min. Three different absolute quantification methods, namely, UV absorption spectroscopy, full-scan MS, and all-ion fragmentation (AIF) MS were compared. Additionally, the monitoring of all generated fragment ions or selected sets of fragment ions were evaluated for the AIF method. Linearity of calibration curves from 5.0 to 25 ng μL(-1), 25 to 250 ng μL(-1), and 100 to 1000 ng μL(-1) was confirmed. The AIF method achieved a lower limit of detection of 0.85 ng μL(-1) and a lower limit of quantification of 2.54 ng μL(-1). On the basis of the comparison of relative standard deviations of interday measurements, AIF was concluded to be the method of choice for concentrations up to 50 ng μL(-1), and UV measurements should be carried out above this concentration. Finally, an expired pegfilgrastim batch was analyzed as a a real biopharmaceutical sample to confirm the feasibility of our approach for monitoring low levels of oxidation variants.

Efficient Purification of rhG-CSF and its PEGylated Forms and Evaluation for In Vitro Activities.

Granulocyte-colony stimulating factor (G-CSF) has commonly been used to help the patients to recover from neutropenia inflicted due to radiotherapy, organ transplants and chemotherapy. As the number of people undergoing these therapies and procedures are increasing world-wide, the need for more economical ways of G-CSF production and improvement in its efficacy has become increasingly crucial. In the present study, recombinant human G-CSF (rhG-CSF) was expressed in E. coli and its purification process was optimized by demonstrating better efficiency and higher recoveries (upto 54%) in a multi-step chromatographic purification process, which is greater than the existing reports. Additionally, the efficacy of rhG-CSF was increased by derivatizing with polyethylene glycol (PEG; upto 85% PEGylation), which increases the plasma clearance time, reduces the immunogenicity and requires less frequent administration to the patient. Overall, the present study suggests a cost-effective purification process of rhG-CSF and also proposes its efficient conjugation with PEG for enhanced efficacy as compared to the existing commercially available forms.

The effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro maturation of porcine oocyte on subsequent embryonic development

Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus–oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus–oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF–treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF–treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF–treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF–treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF–treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF–treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF–treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.

Site-Specifically Modified Recombinant Human Granulocyte Colony-Stimulating Factor with Polyethylene Glycol

Site-Specifically Modified Recombinant Human Granulocyte Colony-Stimulating Factor with Polyethylene Glycol

Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR.

Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is marketed under the brand name Neupogen® by Amgen. Since this product has lost patent protection, many biosimilar versions have been approved or are in the process of filing for market authorization throughout the world. Here we show that NMR spectroscopy can be used to assess the three-dimensional structure of the active ingredient in the formulated approved product Neupogen®. Recombinant metHuG-CSF was prepared in E. coli and isotopically enriched with (13)C and (15) N isotopes. NMR spectroscopy was used to study the effects of excipients on the conformation. The effects of pH variation on the amide chemical shifts suggest the presence of cation-pi interactions between His-79 and Trp-118, and His-156-Trp-58-His-52 that stabilizes the conformation at low pH. This may be associated with a small local conformational change. The NMR data showed that polysorbate does not interact significantly with filgrastim thus allowing the collection of spectra in the presence of 20 times the formulation concentration in the sample. However, at higher detergent concentrations a reduction of signal intensity is observed. Conclusions The NMR fingerprint assay applied to filgrastim (Neupogen® and a CRS from the European Pharmacopeia (EP)) provided residue specific information of the structure of the drug substance. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate comparability exercises.

Assessment of the Effects of Glycosylation on the Pattern and Kinetics of Degradation of Lenograstim in Comparison to Filgrastim Using a Stability-Indicating Orthogonal Testing Protocol

In the biopharmaceutical industry, protein aggregation and/or degradation has profound pathological implications and is encountered routinely during production, shipping, storage and administration. Lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) was subjected to stress conditions, namely, oxidation, pH, temperature, agitation and repeated freeze–thaw to generate all possible degradation products. An orthogonal stability-indicating testing protocol (RP-HPLC, SE-HPLC, ELISA and SDS-PAGE) was developed and validated for assessment of the pattern and kinetics of aggregation/degradation, under the studied experimental conditions. Results indicated clearly that Lenograstim is susceptible to degradation induced by the studied stress conditions. However, Lenograstim was found relatively more stable than Filgrastim (non-glycosylated recombinant human granulocyte colony-stimulating factor) which was attributed to the effect of glycosylation. Oxidized forms and high molecular weight aggregates of Lenograstim and Filgrastim were detected in all samples subjected to stress conditions to different degrees. ELISA assay and SDS-PAGE results were generally in agreement to those obtained using SE-HPLC assay which confirmed its selectivity to the intact drug. However, formation of soluble aggregates of both drugs was found to occur via physical adsorption and formation of intermolecular disulfide bonds. Results confirmed the need for an orthogonal testing protocol since it was impossible to reveal all types of degradation products using a single technique. Results raised a concern about the efficacy and safety of such sensitive products and highlighted the need for simple tools to inspect biologics for soluble aggregates and sub-visible particles before administration.

Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats

Spinal cord injury (SCI) can cause neuropathic pain (NeP), often reducing a patient's quality of life. We recently reported that granulocyte colony-stimulating factor (G-CSF) could attenuate NeP in several SCI patients. However, the mechanism of action underlying G-CSF-mediated attenuation of SCI-NeP remains to be elucidated.The purpose of the present study was to elucidate the therapeutic effect and mechanism of action of granulocyte colony-stimulating factor for SCI-induced NeP.T9 level contusive SCI was introduced to adult male Sprague Dawley rats. Three weeks after injury, rats received intraperitoneal recombinant human G-CSF (15.0μg/kg) for 5days. Mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry and western blot analysis were performed in spinal cord lumbar enlargement samples.Testing with von Frey filaments showed significant increase in the paw withdrawal threshold in the G-CSF group compared with the vehicle group 4weeks, 5weeks, 6weeks and 7weeks after injury. Immunohistochemistry for CD11b (clone OX-42) revealed that the number of OX-42-positive activated microglia was significantly smaller in the G-CSF group than that in the vehicle rats. Western blot analysis indicated that phosphorylated-p38 mitogen-activated protein kinase (p38MAPK) and interleukin-1β expression in spinal cord lumbar enlargement were attenuated in the G-CSF-treated rats compared with that in the vehicle-treated rats.The present results demonstrate a therapeutic effect of G-CSF treatment for SCI-induced NeP, possibly through the inhibition of microglial activation and the suppression of p38MAPK phosphorylation and the upregulation of interleukin-1β.

Design Rationale and Development Approach for Pegfilgrastim as a Long-Acting Granulocyte Colony-Stimulating Factor.

Filgrastim, a recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF), is efficacious in stimulating neutrophil production and maturation to prevent febrile neutropenia (FN) in response to chemotherapy. Because of its relatively short circulating half-life, daily filgrastim injections are required to stimulate neutrophil recovery. In an effort to develop a long-acting form of filgrastim that was as safe and efficacious as filgrastim but had a longer in vivo residence time, a number of strategies were considered. Ultimately, fusion of filgrastim to polyethylene glycol (PEG) was selected. Following extensive analysis of conjugation chemistries as well as in vitro and in vivo characterization of a panel of PEGylated proteins, a construct containing a 20 kDa PEG moiety covalently conjugated to the N-terminus of filgrastim was chosen for advancement as pegfilgrastim. Pegfilgrastim is primarily cleared by neutrophils and neutrophil precursors (rather than the kidneys), meaning that clearance from the circulation is self-regulating and pegfilgrastim is eliminated only after neutrophils start to recover. Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim. Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy. This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim. Approaches that may provide advances for therapeutic agonists of G-CSF receptor are also discussed.

Granulocyte Colony-Stimulating Factor Induces Osteoblast Inhibition by B Lymphocytes and Osteoclast Activation by T Lymphocytes during Hematopoietic Stem/Progenitor Cell Mobilization

In the bone marrow (BM), hematopoietic stem and progenitor cells (HSPCs) reside in specialized niches near osteoblast cells at the endosteum. HSPCs that egress to peripheral blood are widely used for transplant, and mobilization is most commonly performed with recombinant human granulocyte colony-stimulating factor (G-CSF). However, the cellular targets of G-CSF that initiate the mobilization cascade and bone remodeling are not completely understood. Here, we examined whether T and B lymphocytes modulate the bone niche and influence HSPC mobilization. We used T and B defective mice to show that G-CSF–induced mobilization of HSPCs correlated with B lymphocytes but poorly with T lymphocytes. In addition, we found that defective B lymphocytes prevent G-CSF–mediated osteoblast disruption, and further study showed BM osteoblasts were reduced coincident with mobilization, induced by elevated expression of dickkopf1 of BM B lymphocytes. BM T cells were also involved in G-CSF–induced osteoclast activation by regulating the Receptor Activator of Nuclear Factor-κ B Ligand/Osteoprotegerin (RANKL/OPG) axis. These data provide evidence that BM B and T lymphocytes play a role in G-CSF–induced HSPC mobilization by regulating bone remodeling.

Abstract P3-12-07: A multicenter randomized double-blind phase III clinical trial to evaluate the efficacy and safety of GCPGC, a novel pegylated G-CSF in patients receiving DA or TAC chemotherapy for breast cancer compared to peg-filgrastim

Abstract Introduction Treatment with cytotoxic chemotherapy is known to be associated with a significant risk of febrile neutropenia. GCPGC(Green Cross Co., Korea) is a novel long acting recombinant human granulocyte colony-stimulating factor (G-CSF) analog that reduces the severity and duration of neutropenia. We conducted a phase III trial to evaluate the efficacy and safety of GCPGC compared to pegfilgrastim. Methods A total of 117 patients were enrolled in this multicenter, phase III, double-blind randomized trial between Feb 2012 and May 2013. They were randomly assigned to receive either GCPGC or pegfilgrastim during the maximal 6 cycles of chemotherapy which consisted of DA (docetaxel and doxorubicin) or TAC (docetaxel, doxorubicin, and cyclophosphamide). Based on the results of the phase II dose-finding study for GCPGC, the dose of 6 mg was selected. Both medications were administered 24 hours after the completion of each cycle of chemotherapy. The primary efficacy endpoint of this study was the duration of grade 4 neutropenia (Absolute Neutrophil Count (ANC) &amp;lt;500/mm3) during the first cycle of chemotherapy. Secondary endpoints included the time to ANC recovery during the first cycle of chemotherapy, which was defined as the number of days required for neutrophil counts to exceed 2,000/mm3, the rate of febrile neutropenia, the rate of severe neutropenia which persisted for more than three days during first cycle of chemotherapy, the depth of ANC nadir, the ANC level on day 7 after each cycle of chemotherapy, the frequency of dose reduction or delay, the number of hospitalization related to febrile neutropenia after the first cycle of chemotherapy, and the number of treatment with intravenous antibiotics. Results A total of 116 patients were evaluable for safety and 115 patients were evaluable for efficacy. The intention-to-treat analysis showed that there was no statistical difference between GCPGC and pegfilgrastim in terms of the duration (days) of grade 4 neutropenia in chemotherapy cycle 1 (1.64±1.18 vs 1.80±1.05; difference -0.15±1.11 [97.5% C.I. -, 0.26]; the pre-specified non-inferiority margin of 1.0). Notably, patients treated with GCPGC had a statistically significant reduction in the time to recovery from neutropenia (ANC &amp;gt;2,000/mm3), one of the secondary endpoints of the study, compared to those treated with pegfilgrastim (8.85 ± 1.45 vs. 9.83 ± 1.20 days; p &amp;lt;0.0001). There was no difference between groups regarding the other secondary endpoints. In addition, no significant differences were observed between the safety profiles of the two groups. Conclusion Collectively, GCPGC is not inferior to peg-filgrastim and represents an effective alternative for reducing the duration of neutropenia in breast cancer patients receiving TAC or DA chemotherapy. Citation Format: Joo Han Lim, Ki-Hyeong Lee, Keon-Uk Park, In-Hae Park, Eun Kyung Cho, Moon Hee Lee, So-Young Yoon, Jee-Hyun Kim, In-Sil Choi, Jae-Hoo Park, Young-Jin Choi, Hee-Jun Kim, Kyung Hae Jung, Si-Young Kim, Do-Youn Oh, Seock-Ah Im. A multicenter randomized double-blind phase III clinical trial to evaluate the efficacy and safety of GCPGC, a novel pegylated G-CSF in patients receiving DA or TAC chemotherapy for breast cancer compared to peg-filgrastim [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-12-07.

Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli.

Background:The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. Methods: An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h-1 at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. Results:Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l-1 and 32.1 g.l-1. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g-1 DCW and 1.83 g.l-1.h-1, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen® and PD-Grastim showed that all proteins have a similar secondary structure. Conclusion:Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity.

Effect of recombinant human granulocyte colony-stimulating factor on Nogo receptor expression in the brain tissues of neonatal rats after hypoxic-ischemic brain damage

Objective To observe the different effects of 2 doses recombinant human granulocyte colony-stimulating factors(rhG-CSF)on Nogo receptor(NgR)expression in the brain tissue of neonatal rats after hypoxic-ischemic brain damage(HIBD)at different times in order to reveal the neuroprotective effects of rhG-CSF. Methods Seven-day neonatal Sprague-Dawley(SD)rats were randomly divided into 4 groups by drawing method: sham operation group, model group, low-dose rhG-CSF group and high-dose rhG-CSF group, 24 rats in each group.Then each group was divided into 4 subgroups(6 rats in each subgroup)and all rats were exterminated at different times after HIBD(1 d, 3 d, 7 d and 14 d). In the low-dose rhG-CSF group and high-dose rhG-CSF group, the rats were given daily doses of rhG-CSF 50 μg/kg, 100 μg/kg respectively for 7 days by subcutaneous injection immediately after the molding(total 7 injections). In model group, rats received an injection of same amount of 9 g/L saline.In sham operation group, rats received no special treatment.Brain tissues of rats from each group were collected at different time points.The expressions of NgR protein and NgR mRNA in the left brain tissue were detected by immunohistochemistry and real-time fluorescent quantitative polymerase chain reaction(PCR). Results Immunohistochemistry: NgR proteins were constitutively expressed in the cerebral cortex in sham operation group at each time point; compared with sham operation group, the expressions of NgR in model group were increased markedly at each time point (135.67±16.63, 173.98±17.82, 234.00±14.70, 319.59±25.22), and the differences were statistically significant(all P<0.01); compared with model group, the expressions of NgR in the cerebral cortex in low-dose rhG-CSF group (134.35±8.89, 109.04±12.62, 75.99±13.39)and high-dose rhG-CSF group (81.38±12.25, 80.14±10.50, 72.58±13.66) on the 3rd, 7th, 14th day were reduced significantly(all P<0.01). Compared with low-dose rhG-CSF group, the protein expressions of NgR in the high-does rhG-CSF group were decreased faster, and had the marked difference on the 3rd, 7th day (P<0.05). Real-time fluorescent quantitative PCR: compared with the sham operation group, the expressions of NgR mRNA increased gradually in the cerebral cortex in the model group (1.34±0.24, 1.88±0.27, 2.88±0.84, 4.26±0.86), the differences in NgR mRNA expression were statistically significant at different times(all P<0.05); compared with model group, the expressions of NgR mRNA in low-dose rhG-CSF group on the 7th (1.08±0.30), 14th day (0.93±0.26) and high-dose rhG-CSF group on the 3rd(0.61±0.10), 7th(0.56±0.28), 14th day (0.47±0.12) were significantly different(all P<0.05). The expressions of low-dose group and high-dose group were reduced gradually.The NgR mRNA expression reduced more quickly in the high-dose group than in the low-dose rhG-CSF group and had substantial difference between two groups in 3 days (P<0.05). Conclusions The findings suggest that rhG-CSF intervention can reduce the expressions of NgR in the brain tissues of neonatal rats after HIBD, and low-dose rhG-CSF also has neuroprotective effect, but it could be weaker than high-dose rhG-CSF. Key words: Recombinant human granulocyte colony-stimulating factor; Hypoxic ischemic brain damage; Nogo receptor