Recombinant Granulocyte Colony-stimulating Factor Research Articles

Analytical characterization of in vitro refolding in the quality by design paradigm: Refolding of recombinant human granulocyte colony stimulating factor

Protein based therapeutics dominate most pharmaceutical pipelines today. For a therapeutic product to be effective, it is important that it is in its native form as slight modifications have been known to result in significantly different performance in the clinic. When expressed in hosts such as Escherichia coli, formation of inactive insoluble aggregates of proteins popularly known as inclusion bodies occurs in most cases. This necessitates the need for in vitro refolding to generate the native (and active) form of the therapeutic protein. This paper aims to provide an approach to generate a deeper understanding of refolding of a therapeutic protein and then to use it for its optimal production commercially. Recombinant human granulocyte colony stimulating factor has been chosen as the model protein. Seven orthogonal analytical tools have been used to elucidate the refolding process. By strategically using these tools protein refolding has been segregated into a series of well-defined sequence of events, starting from the unfolded random coil and ending with the uniquely folded metastable state. The study also suggests the choice of tools that can be used to monitor each event. We believe that this paper successfully demonstrates an approach to generate deeper understanding of the protein refolding process as per the expectations laid out in the Quality by Design paradigm.

Phase II Trial of Intravenous Low-Dose Granulocyte Colony-Stimulating Factor in Acute Ischemic Stroke

Granulocyte colony-stimulating factor (G-CSF) has shown neuroprotective and neurogenerative activities in experimental studies, and our previous phase I clinical study suggested the safety and potential efficacy of low-dose G-CSF in acute ischemic stroke patients. The present phase II trial is aimed to evaluate the effect of G-CSF administration on neurological function and infarct volume, compared with a placebo group. Forty-nine acute ischemic stroke patients (29 males, 20 females; 71 ± 10 years) within 24 hours after onset were recruited. Eligible patients were randomized 2:2:1 to receive G-CSF 150 µg/body/day, G-CSF 300 µg/body/day, and placebo, respectively. We evaluated clinical outcome in terms of the National Institutes of Health Stroke Scale, the modified Rankin Scale, and the Barthel Index at 90 days after onset, together with changes in infarct volume on magnetic resonance imaging. We found no serious adverse event, including change in leukocyte levels, which remained below 31,000/µL, at 150 and 300 µg G-CSF/body/day. Clinical outcome scores did not show any significant difference among the 3 groups. Chronological changes in infarct volume also showed no significant difference. G-CSF was well-tolerated at 150 and 300 µg/body/day in patients with acute ischemic stroke. However, administration of G-CSF at both 150 and 300 µg/body/day neither contributed to functional recovery nor reduced infarct volume at 3 months after onset, compared with the control group. The apparent lack of effectiveness may have been due to the small sample size. A trial of combination therapy with recombinant tissue plasminogen activator and G-CSF is planned.

Immunogenicity Assessment of Lipegfilgrastim in Patients with Breast Cancer Receiving Chemotherapy.

Lipegfilgrastim is a long-acting, once-per-cycle, glycopegylated recombinant granulocyte colony-stimulating factor (G-CSF) used to prevent neutropenia in patients receiving myelosuppressive chemotherapy. This integrated analysis examined the immunogenicity of lipegfilgrastim and its potential clinical impact in two double-blind randomized studies (phases II and III) of patients with breast cancer receiving chemotherapy. Serum samples were analyzed using sequential assays for screening, confirmation, antibody titer, and characterization of antidrug antibodies (ADA). Neutropenia-related efficacy measures were reviewed for each ADA-positive patient. Among 255 patients receiving lipegfilgrastim (154 in phase II, 101 in phase III) and 155 patients receiving pegfilgrastim (54 in phase II, 101 in phase III), the incidence of treatment-emergent ADA was low and similar between the lipegfilgrastim (phase II: 1.3%; phase III: 1.0%) and pegfilgrastim (phase II: 1.9%; phase III: 1.0%) arms. None of the treatment-emergent ADA-positive samples exhibited neutralizing activity against lipegfilgrastim, pegfilgrastim, or glycosylated G-CSF in a cell-based neutralizing antibody assay. No changes were observed in neutropenia-related efficacy measures among ADA-positive patients, and no treatment-related hypersensitivity or anaphylaxis occurred. These results indicate that there is no apparent impact of ADA on lipegfilgrastim efficacy and safety.

Four-Week Repeated Intravenous Dose Toxicity and Toxicokinetic Study of TS-DP2, a Novel Human Granulocyte Colony Stimulating Factor in Rats.

TS-DP2 is a recombinant human granulocyte colony stimulating factor (rhG-CSF) manufactured by TS Corporation. We conducted a four-week study of TS-DP2 (test article) in repeated intravenous doses in male and female Sprague-Dawley (SD) rats. Lenograstim was used as a reference article and was administered intravenously at a dose of 1000 μg/kg/day. Rats received TS-DP2 intravenously at doses of 250, 500, and 1000 μg/kg/day once daily for 4 weeks, and evaluated following a 2-week recovery period. Edema in the hind limbs and loss of mean body weight and body weight gain were observed in both the highest dose group of TS-DP2 and the lenograstim group in male rats. Fibro-osseous lesions were observed in the lenograstim group in both sexes, and at all groups of TS-DP2 in males, and at doses of TS-DP2 500 μg/kg/day and higher in females. The lesion was considered a toxicological change. Therefore, bone is the primary toxicological target of TS-DP2. The lowest observed adverse effect level (LOAEL) in males was 250 μg/kg/day, and no observed adverse effect level (NOAEL) in females was 250 μg/kg/day in this study. In the toxicokinetic study, the serum concentrations of G-CSF were maintained until 8 hr after administration. The systemic exposures (AUC0-24h and C0) were not markedly different between male and female rats, between the administration periods, or between TS-DP2 and lenograstim. In conclusion, TS-DP2 shows toxicological similarity to lenograstim over 4-weeks of repeated doses in rats.

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.

Inclusion of Plerixafor Increases the Efficacy of Stem Cell Harvesting in Poorly Mobilizing Patients with Multiple Myeloma and Lymphoma

Introduction: Up to 30% of patients (pts) undergoing stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) with or without prior chemotherapy mobilize poorly. Plerixafor is a potent CXCR4 receptor antagonist that is licensed for stem cell mobilization in autologous stem cell donors with multiple myeloma (MM) or lymphoma, who show poor mobilization after stimulation with recombinant G-CSF (r-metHuG-CSF, Filgastrim). Here we present our experience with plerixafor in poorly mobilizing autologous donors.Patients and Methods: We performed 35 stem cell collections between January 2011 and December 2014 in 28 poorly mobilizing autologous donors with MM (n=14), non-Hodgkin lymphoma (NHL, n=11), Hodgkin lymphoma (n=1) or both MM & NHL (n=2), adding plerixafor to the stimulation regimen with recombinant G-CSF. Known risk factors for poor mobilization included: diagnosis of lymphoma (50%); older age (61% >60y at apheresis); >1 chemotherapy lines (86%) & pretreatment with melphalan (18%). Median age of patients (pts) was 61 years (y, range 37-72y) at stem cell collection and 15 of the 28 pts were females. All pts received previous treatment with G-CSF (2x5µg/kg body weight [BW]) followed by a large volume leukapheresis (maximal 4 x total blood volume). All pts received 0.24 mg/kg BW plerixafor following insufficient mobilization of CD34+ cells into the peripheral blood (PB, <10/µl CD34+ cells) - either after peripheral reconstitution of white blood cells (WBC, >1 Gpt/l) following chemotherapy (n=22, 79%) or after 5 days of stimulation with G-CSF in steady state (n=6, 21%). Six pts treated with chemotherapy prior to mobilization received a second dose of plerixafor in response to insufficient CD34+ cell harvesting on the first collection day.As a control cohort, we evaluated the data of 151 collections from MM, NHL or Hodgkin lymphoma pts (106 males, 45 females, median age 56y, range 22-74y) with efficient mobilization in response to G-CSF treatment over the same time period.Results: The median concentration of CD34+ cells in PB before & after the first application of plerixafor was 8.4/µl (range 2.9-20.5 /µl) & 23.1/µl (range 10.2-54.1 /µl) respectively, corresponding to a median 3.3-fold increase (range 1.4-5.3-fold, P<0.001, Figure 1). In comparison, pts in the control group had a median concentration of 101.9/µl (range 13-1190/µl) CD34+ cells in PB on the day of stem cell collection (Figure 1).The median CD34+ cell yield for pts receiving plerixafor was 3.7x10^6/kg BW (range 1.6-12.8x10^6/kg BW) compared to 10.5x10^6/kg BW (range 2.1-38.8x10^6/kg BW) in the control cohort (Figure 2).As expected, the WBC count in the product was higher in the plerixafor group (median 899.9x10^8, range 446-2214x10^8) compared to the efficiently mobilizing control cohort (median 470.2x10^8, range 99.9 -1537.1x10^8, P<0.001). The vitality of the cells after thawing was tested with methan blue staining & was comparable in both groups: median 85.3% (range 73.3-92.8%) in the plerixafor group & 84.4% (range 50.6-96.8%) in the control cohort (P=0.242). Furthermore, all harvests were tested for their proliferation capacity by quantifying colony forming units (CFU). Pts receiving plerixafor had a median CFU number of 22.5 (range 1-199), which did not differ from the control cohort with a median of 200.2 CFUs (range 1-1341, P=0.568). To date, 22 (78.6%) of the poorly mobilizing pts have received an autologous stem cell transplantation. All showed good peripheral WBC (median day 11, range 10-15) & platelet (median day 16, range 11-46) recovery.Discussion: Our data shows that addition of plerixafor can increase the efficacy of stem cell harvesting in poor mobilizers. The collected stem cells fulfilled the appropriate quality control criteria including vitality after thawing (>50%) & proliferation capacity. We identified a median 3.3-fold increase in CD34+ cell concentration in PB & a median CD34+ cell yield of 3.7x10^6/kg BW after the application of plerixafor in poor mobilizers. Despite the fact that the CD34+ cell yield in pts receiving plerixafor remained significantly lower than the control cohort, we were able to harvest >2x10^6/kg BW CD34+ cells in 93% (n=26) of poor mobilizers. Thus, the mobilization outcome of pts receiving plerixafor is acceptable and within the expected range. [Display omitted] [Display omitted] DisclosuresFranke:BMS: Honoraria; MSD: Other: Travel Costs; Novartis: Other: Travel Costs. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Effects of HS 6101 and rhG-CSF on Hematopoiesis Recovery of ICR Mice Injured by Cyclophosphamide

To investigate the effects of HS 6101 and recombinant human granulocyte colony stimulating factor (rhG-CSF) on hematopoiesis recovery of ICR mice injured by cyclophosphamide (CTX). A total of 103 ICR mice were divided into 4 groups, including CTX control (46 mice), HS 6101 (21 mice), rhG-CSF (18 mice) and HS 6101+rhG-CSF (18 mice), respectively. The mouse model of chemotherapy-induced haematopoietic injury was established by CTX intraperitoneal injection at a dose of 100 mg/kg once a day for 3 consecutive days. Single dose of HS 6101 (27 µg/mouse) was injected subcutaneously at 1 hour before the first administration of CTX. One day after the last CTX treatment, 2 µg/mouse of rhG-CSF was injected subcutaneously once a day for 5 consecutive days. The peripheral blood cell counts of the mice were observed once every 2 days. Hematopoietic progenitor cell colony counting and histopathological assessment of bone marrow cells were evaluated in the mice at days 4 and 9 after the first administration of CTX. Both HS 6101 and rhG-CSF alone or in combination, significantly elevated the nadirs of peripheral blood leukocytes and neutrophils, increased the number of bone marrow hematopoietic progenitor cells, and stimulated the hematopoietic cell hyperplasia of bone marrow in the mice treated with CTX. The effect of HS 6101 combined with rhG-CSF was better than that of the drugs used alone. The HS 6101 at 27 µg/mouse can significantly promote the recovery of hematopoiesis in ICR mice treated with CTX chemotherapy, and its combination with rhG-CSF shows synergistic effects.

G-CSF Predicts Cardiovascular Events in Patients with Stable Coronary Artery Disease.

Granulocyte-colony-stimulating-factor (G-CSF) induces mobilization of progenitor cells but may also exert pro-inflammatory and pro-thrombotic effects. Treatment with recombinant G-CSF after acute myocardial infarction is currently under examination and has been associated with in-stent restenosis. However, it is not known whether plasma levels of endogenous G-CSF are also associated with an increased cardiovascular risk. Therefore we included 280 patients with angiographically proven stable coronary artery disease. G-CSF was measured by specific ELISA and patients were followed for a median of 30 months for the occurrence of major adverse cardiovascular events (MACE: death, myocardial infarction, re-hospitalization). Those with cardiac events during follow-up showed significant higher G-CSF levels (32.3 pg/mL IQR 21.4–40.5 pg/mL vs. 24.6 pg/mL IQR 16.4–34.9 pg/mL; p<0.05) at baseline. Patients with G-CSF plasma levels above the median had a 2-fold increased risk for MACE (p<0.05). This was independent from established cardiovascular risk factors. In addition, G-CSF above the median was a predictor of clinical in-stent restenosis after implantation of bare-metal stents (6.6% vs. 19.4%; p<0.05) but not of drug-eluting stents (7.7% vs. 7.6%; p = 0.98). This data suggests that endogenous plasma levels of G-CSF predict cardiovascular events independently from established cardiac risk factors and are associated with increased in-stent restenosis rates after implantation of bare metal stents.

STAT5 plays a critical role in regulating the 5'-flanking region of the porcine whey acidic protein gene in transgenic mice.

The mammary gland serves as a valuable bioreactor system for the production of recombinant proteins in lactating animals. Pharmaceutical-grade recombinant protein can be harvested from the milk of transgenic animals that carry a protein of interest under the control of promoter regions genes encoding milk proteins. Whey acidic protein (WAP), for example, is predominantly expressed in the mammary gland and is regulated by lactating hormones during pregnancy. We cloned the 5'-flanking region of the porcine WAP gene (pWAP) to confirm the sequence elements in its promoter that are required for gene-expression activity. In the present study, we investigated how lactogenic hormones--including prolactin, hydrocortisone, and insulin--contribute to the transcriptional activation of the pWAP promoter region in mammalian cells, finding that these hormones activate STAT5 signaling, which in turn induce gene expression via STAT5 binding sites in its 5'-flanking region. To confirm the expression and hormonal regulation of the 5'-flanking region of pWAP in vivo, we generated transgenic mice expressing human recombinant granulocyte colony stimulating factor (hCSF2) in the mammary gland under the control of the pWAP promoter. These mice secreted hCSF2 protein in their milk at levels ranging from 242 to 1,274.8 ng/ml. Collectively, our findings show that the pWAP promoter may be useful for confining the expression of foreign proteins to the mammary gland, where they can be secreted along with milk.

Early-Onset Neutropenia in Small-for-Gestational-Age Infants

Early neutropenia is more common in small for gestational age (SGA) neonates (birth weight <10th percentile) than in appropriately grown neonates. However, several aspects of this variety of neutropenia are unknown, including the duration, kinetic mechanism, and outcomes. Using 10 years of multihospital records, we studied SGA neonates who, during the first week after birth, had neutrophil counts <1000/μL. This degree of neutropenia was more common in SGA neonates (6%, 207/3650) than in non-SGA matched controls (1%, 46/3650; P < .001). Neutrophil counts stayed below the lower reference interval for 7 days. Ratios of immature to total neutrophils were within the reference interval, suggesting reduced neutrophil production, not accelerated neutrophil use or destruction. Increased nucleated red cells at birth correlated with decreased neutrophils (P < .001). Neutropenia was not independently associated with maternal hypertensive disorders, over and above the effect of SGA. Of 201 neutropenic SGA neonates, 129 (64%) also had thrombocytopenia. Sixteen percent of neutropenic neonates were treated with recombinant granulocyte colony-stimulating factor (rG-CSF) or intravenous immunoglobulin (IVIG), with no reduction in late-onset sepsis or necrotizing enterocolitis (NEC). Regression analysis showed that neutropenia (but not thrombocytopenia in the absence of neutropenia) was independently associated with increased odds of developing necrotizing enterocolitis (odds ratio 4.01, 90% confidence interval 2.08 to 7.35, P < .001). Neutropenia of SGA is a condition of 1-week duration. It is more closely associated with SGA than maternal hypertension (likely owing to neutrophil hypoproduction associated with intrauterine hypoxia), often accompanied by thrombocytopenia, not obviously improved by rG-CSF or IVIG, and associated with an increased risk for NEC.

Pegfilgrastim for the prevention of chemotherapy-induced febrile neutropenia in patients with solid tumors

Introduction: Neutropenia and febrile neutropenia are the most common and most severe bone marrow toxicities of chemotherapy. Recombinant granulocyte-colony stimulating factors (G-CSFs), both daily (filgrastim and biosimilars, and lenograstim) and long-acting (pegfilgrastim and lipegfilgrastim) formulations, are currently available to counteract the negative consequences of these side effects.Areas covered: The purpose of this article is to review the physiopathology of chemotherapy-induced febrile neutropenia and its consequences, and the current evidence regarding the pharmacological properties, clinical efficacy and cost-effectiveness of pegfilgrastim as a strategy to prevent chemotherapy-induced febrile neutropenia in patients with solid tumors.Expert opinion: Chemotherapy-induced febrile neutropenia and its complications are still a major health-care concern, and the inappropriate employment of G-CSFs in clinical practice can partially explain its burden. Pegfilgrastim has pharmacological advantages over daily G-CSFs that makes it easily administrable, thus reducing the chance of incorrect delivery. The once-per-cycle administration might explain the findings derived from observational studies suggesting a possible superior efficacy of pegfilgrastim over daily G-CSFs. For patients at higher risk of failure with daily G-CSF prophylaxis (e.g. risk of non-compliance, difficulties on performing regular hemograms, high risk of developing febrile neutropenia), pegfilgrastim might be the most appropriate option.

Downregulation of P-gp, Ras and p-ERK1/2 contributes to the arsenic trioxide-induced reduction in drug resistance towards doxorubicin in gastric cancer cell lines.

Multidrug resistance (MDR) to doxorubicin (DOX) limits its effectiveness against tumor cells. Arsenic trioxide (As2O3) has been reported to reduce MDR in various types of cancer, but the mechanisms involving Ras and p-glycoprotein (P-gp) remain to be fully elucidated. The objectives of the present study were to evaluate As2O3 in reversing MDR to DOX, and to identify the association in antitumor activities between the effectiveness of DOX and Ras/phosphorylated (p-) extracellular signal-regulated kinase (ERK)1/2 signaling in SGC7901/ADM and SGC7901/S human gastric cancer cell lines. Cytotoxicity and sensitivity towards As2O3 were assessed using non-toxic and mildly-toxic concentrations (0.1 and 0.5 µM, respectively). The reversing effect of As2O3 on MDR was investigated prior to and following treatment with a cytokine activation of the recombinant human granulocyte colony stimulating factor ERK pathway. The SGC7901/ADM and SGC7901/S cells had the same sensitivity to As2O3. The SGC7901/ADM cells were resistant to DOX and As2O3 treatment reduced the level of resistance to DOX (P<0.01). The expression of P-glycoprotein (P-gp) in the SGC7901/ADM cells was higher than in the SGC7901/S cells (P<0.001). As2O3 treatment decreased the levels of P-gp in a time- and dose-dependent manner (P<0.01). The expression of Ras was higher in the SGC7901/ADM cells than in the SGC7901/S cells, while the expression of p-ERK1/2 remained the same. As2O3 decreased the levels of Ras and p-ERK1/2 (P<0.01). Following pretreatment with rhG-CSF, the levels of Ras and p-ERK1/2 were further decreased (P<0.01). Drug-resistant gastric cancer cells had higher expression levels of P-gp and Ras, but not of p-ERK1/2. Non- and mildly-toxic doses of As2O3 reduced MDR to DOX through Ras/p-ERK1/2 signaling.

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.

Clinical analysis on malignant clonal hematopoiesis in severe aplastic anemia patients with immunosuppressive therapy

To investigate the clinical characteristics and risk factors of monosomy 7 malignant clonal evolution in patients with severe aplastic anemia (SAA) treated with combined immunosuppressive therapy (IST). The clinical data of SAA patients treated with IST who had monosomy 7 malignant clonal evolution from October 2004 to January 2012 were analyzed respectively. Six patients (4.2%) had monosomy 7 clonal evolutions. The median time to monosomy 7 was 36 (12-75) months after IST. All 6 patients were diagnosed myelodysplastic syndromes (MDS). Among them, 3 patients transformed to acute myeloid leukemia following MDS. The time was 24, 45 and 51 months after IST. The median following time was 42 (17-84) months. Four patients died during the following time. The median time from MDS to death was 9 (5-17) months. Among them, three patients died with infection, one died with cerebral hemorrhage. Six patients had the clinical characteristics that they had no response to IST after 6 months, high monocyte percentage in one month after IST combined with recombinant human granulocyte colony stimulating factor (rHu-GCSF) and agranulocytosis in 3 months after IST. Poor myeloid response to IST suggests malignant clonal hematopoiesis and poor prognosis in SAA patients.

Acute myeloid leukemia developing in a granulocyte colony-stimulating factor-mobilized stem cell donor: A case report and review of the literature

We describe the first case of a FLT-3 mutated AML in a healthy donor, 3years after recombinant human granulocyte colony stimulating factor (rhG-CSF)-mobilized peripheral blood stem cell (PBSC) harvest. The patient had a myeloablative (MA) matched unrelated donor (MUD) stem cell transplant (SCT) for refractory AML. However, he experienced a secondary graft failure. He had a second non myeloablative (NMA) on day +75 from a second MUD. He achieved a complete neutrophil and platelet engraftment. After 4years of follow up, he is alive in complete remission with full second donor chimerism.

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.

Tbo-Filgrastim versus Filgrastim during Mobilization and Neutrophil Engraftment for Autologous Stem Cell Transplantation

There are limited data available supporting the use of the recombinant granulocyte colony–stimulating factor (G-CSF), tbo-filgrastim, rather than traditionally used filgrastim to mobilize peripheral blood stem cells (PBSC) or to accelerate engraftment after autologous stem cell transplantation (ASCT). We sought to compare the efficacy and cost of tbo-filgrastim to filgrastim in these settings. Patients diagnosed with lymphoma or plasma cell disorders undergoing G-CSF mobilization, with or without plerixafor, were included in this retrospective analysis. The primary outcome was total collected CD34+ cells/kg. Secondary mobilization endpoints included peripheral CD34+ cells/μL on days 4 and 5 of mobilization, adjunctive use of plerixafor, CD34+ cells/kg collected on day 5, number of collection days and volumes processed, number of collections reaching 5 million CD34+ cells/kg, and percent reaching target collection goal in 1 day. Secondary engraftment endpoints included time to neutrophil and platelet engraftment, number of blood product transfusions required before engraftment, events of febrile neutropenia, and length of stay. A total of 185 patients were included in the final analysis. Patients receiving filgrastim (n = 86) collected a median of 5.56 × 106 CD34+ cells/kg, compared with a median of 5.85 × 106 CD34+ cells/kg in the tbo-filgrastim group (n = 99; P = .58). There were no statistically significant differences in all secondary endpoints with the exception of apheresis volumes processed (tbo-filgrastim, 17.0 liters versus filgrastim, 19.7 liters; P < .01) and mean platelet transfusions (tbo-filgrastim, 1.7 units versus filgrastim, 1.4 units; P = .04). In conclusion, tbo-filgrastim demonstrated similar CD34+ yield compared with filgrastim in mobilization and post-transplantation settings, with no clinically meaningful differences in secondary efficacy and safety endpoints. Furthermore, tbo-filgrastim utilization was associated with cost savings of approximately $1406 per patient utilizing average wholesale price.

Incidence of bone pain in patients with breast cancer treated with lipegfilgrastim or pegfilgrastim: an integrated analysis from phase II and III studies

Lipegfilgrastim is a once-per-cycle, fixed-dose, glycoPEGylated recombinant granulocyte colony-stimulating factor (G-CSF) recently approved in Europe to reduce the duration of chemotherapy-induced neutropenia and incidence of febrile neutropenia in patients with cancer receiving chemotherapy. Bone pain-related (BPR) adverse events are commonly associated with G-CSF therapy. This post hoc analysis examined BPR treatment-emergent adverse events (TEAEs) in two comparative studies of lipegfilgrastim or pegfilgrastim in patients receiving chemotherapy. A post hoc analysis was conducted using integrated data from two double-blind randomized studies in patients with breast cancer receiving docetaxel and doxorubicin and treated prophylactically with subcutaneous lipegfilgrastim 6 mg or pegfilgrastim 6 mg once per cycle. BPR TEAEs were defined as arthralgia, back pain, bone pain, musculoskeletal chest pain, musculoskeletal discomfort, musculoskeletal pain, myalgia, neck pain, noncardiac chest pain, and pain in extremity. Relationship of BPR TEAEs to study treatment or chemotherapy was also reported by the investigators. The analysis included 306 patients (lipegfilgrastim: n = 151; pegfilgrastim: n = 155). The proportion of patients experiencing BPR TEAEs was similar with lipegfilgrastim and pegfilgrastim (25.2 vs 21.9%, respectively), as was the proportion of patients experiencing BPR treatment-emergent adverse drug reactions (TEADRs) (18.5 vs 16.8%, respectively). No BPR TEADRs were serious, and none led to discontinuation. Nonsevere BPR TEAEs and TEADRs were observed in patients with breast cancer receiving chemotherapy and G-CSF; rates of BPR events were similar between lipegfilgrastim and pegfilgrastim. The similar BPR safety profile of lipegfilgrastim and pegfilgrastim provides support for use in patients with breast cancer receiving chemotherapy.

Effects of Sucrose and Benzyl Alcohol on GCSF Conformational Dynamics Revealed by Hydrogen Deuterium Exchange Mass Spectrometry

Protein stability, one of the major concerns for therapeutic protein development, can be optimized during process development by evaluating multiple formulation conditions. This can be a costly and lengthy procedure where different excipients and storage conditions are tested for their impact on protein stability. A better understanding of the effects of different formulation conditions at the molecular level will provide information on the local interactions within the protein leading to a more rational design of stable and efficacious formulations. In this study, we examined the roles of the excipients, sucrose and benzyl alcohol, on the conformational dynamics of recombinant human granulocyte colony stimulating factor using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS). Under physiological pH and temperature, sucrose globally protects the whole molecule from deuterium uptake, whereas benzyl alcohol induces increased deuterium uptake of the regions within the α-helical bundle, with even larger extent. The HDX experiments described were incorporated a set of internal peptides (Zhang et al., 2012. Anal Chem 84:4942-4949) to monitor the differences in intrinsic exchange rates in different formulations. In addition, we discussed the feasibility of implementing HDX-MS with these peptide probes in protein formulation development.

Myeloprotective Effect of Combined Application of Ukrainian-Made Recombinant Granulocyte Colony Stimulating Factor (R-GCSF) and С2 Enterosorbent in Rats with Malignant Guerin Carcinoma

Myeloprotective Effect of Combined Application of Ukrainian-Made Recombinant Granulocyte Colony Stimulating Factor (R-GCSF) and С2 Enterosorbent in Rats with Malignant Guerin Carcinoma