Maintenance of viability and proliferation of 3T3 cell aggregates incorporating fibroin microspheres into cultures.

This study investigated whether micron-sized microspheres can be used as dispersed scaffolds where anchorage-dependent cells can proliferate and survive in suspension culture. Aggregates of murine 3T3 cells in a non-adherent plate cultured remained viable for more than 2 weeks by the presence of 0.5 mg/ml fibroin microspheres. A nucleoside incorporation assay confirmed the proliferation of 3T3 cells in the aggregates only when cultured with microspheres. Under these conditions, the glucose consumption rate of 3T3 cells increased to 66.5 nmol day-1 cell-1. Histological analysis demonstrated that the intercellular space of cell aggregates was larger in cultures supplemented with 0.5 mg/ml microspheres than in non-supplemented cultures. The cell aggregates with microspheres also exhibited a reduced arrest in G1 phase. Transmission electron microscopy verified the presence of microspheres in the space between cells in aggregates. Fibroin microspheres maintained the viability and proliferability of 3T3 cells cultured under non-adherent conditions and thus can be used to develop viable suspensions of anchorage-dependent cells.

Pharmacokinetic and dose-finding studies on efpeglenatide in patients with type 2 diabetes.

AIM: To assess the efficacy, safety and pharmacokinetic/pharmacodynamic properties of efpeglenatide, a long-acting glucagon-like peptide-1 receptor agonist, in patients with type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: Two randomized, double-blind, placebo-controlled phase 2 trials were conducted. The single-dose study (n = 48) was a first-in-patient, sequential dose-escalation study. Patients received a single subcutaneous injection of efpeglenatide (2-100 µg/kg) or placebo. The repeated-dose study (n = 71) was a multiple-ascending-dose trial. Patients received weekly (1, 2 or 4 mg once weekly; 8-week period) or monthly (8, 12 or 16 mg once monthly; 9-week period) subcutaneous injections of efpeglenatide or placebo (without titration). RESULTS: Both studies demonstrated dose-proportional increases in efpeglenatide serum concentrations. The median time to attain maximum serum concentration (tmax ) for efpeglenatide ranged from 72 to 144 hours in the single-dose study and from 48 to 120 hours in the repeated-dose study (following final dose). Geometric mean t1/2 ranged from 135 to 180 hours across studies. Peak-to-trough ratios in the repeated-dose study ranged from 1.3 to 1.4 with once-weekly dosing and from 5.9 to 12.9 with once-monthly dosing. Following a single dose of efpeglenatide 14-100 µg/kg, fasting plasma glucose and postprandial plasma glucose levels were decreased at week 1 and remained below baseline levels for ≥3 weeks post-dosing. Repeated doses of efpeglenatide led to significant reductions in glycated haemoglobin vs placebo. In both studies, efpeglenatide was generally well tolerated. Gastrointestinal disorders were the most frequently reported treatment-emergent adverse events in efpeglenatide-treated patients. CONCLUSIONS: The delayed tmax, long half-life, and low peak-to-trough ratios observed demonstrate potential for improved efficacy and dosing flexibility, with good tolerability of efpeglenatide in patients with T2D.

LAPS Insulin115: A novel ultra-long-acting basal insulin with a unique action profile.

AIMS: To conduct a comprehensive pre-clinical study of the novel ultra-long acting insulin analogue LAPS Insulin115. METHODS: Pharmacokinetic/pharmacodynamic studies comparing LAPS Insulin115 with other basal insulins were conducted in genetically diabetic (db/db) mice. Insulin signalling in the major target organs was analysed using Western blot after single subcutaneous injection in wild-type male Wistar rats. Using in vitro assays we analysed transendothelial transport, insulin receptor (IR) interaction, and the mitogenic and metabolic properties of LAPS Insulin115. Furthermore, IR downregulation after long-term exposure to high concentrations of LAPS Insulin115 was analysed using an in vitro desensitization/resensitization model. RESULTS: The novel Fc-conjugated insulin derivative LAPS Insulin115 showed an extensively prolonged pharmacokinetic and pharmacodynamic profile in rodents. Despite its size of 59 kDa, LAPS Insulin115 passes the vascular endothelial barrier and induces insulin signalling in all major target tissues in rats. In vitro, LAPS Insulin115 showed a very slow onset of action because of its reduced IR affinity; however, after long-term stimulation it was equipotent in respect to its metabolic potency and showed no increased mitogenic action when compared with regular insulin. Remarkably, under conditions of chronic exposure, LAPS Insulin115 does not induce irreversible desensitization of target cells, which is probably attributable to much less prominent IR downregulation. CONCLUSION: Thus, LAPS Insulin115 exhibits a unique in vivo and in vitro profile and thereby represents an excellent candidate for a once-weekly insulin analogue.

Cobalt(III)-induced hexamerization of PEGylated insulin.

Insulin conjugates in which the B1Phe residue has been chemically modified often exhibit a reduced tendency to associate into hexamers due to weakened interactions between subunits. The purpose of this study was to prepare a hexamer formulation for such insulin conjugates by using Co(III) as a coordinating metal ion. PEGylated insulin in which monomethoxypoly(ethylene glycol) (mPEG, M(r) 5000 or 20,000) had been site-specifically attached to B1Phe was chosen as a model conjugate. Hexamerization of mPEG-insulin upon H(2)O(2)-mediated oxidation of Co(II) was kinetically and quantitatively analysed by visible spectrometry and size-exclusion HPLC. Co(III) mPEG-insulin hexamers thus obtained were extremely stable, existing mostly as a hexameric form even at nanomolar concentrations. A remarkable increase in hydrodynamic volumes was observed for Co(III) mPEG(20k)-insulin hexamers (1600 kDa), as well as Co(III) mPEG(5k)-insulin hexamers (300 kDa). Our results demonstrate the potential benefits of Co(III) hexamer formulation for weakly associating insulin conjugates in the treatment of diabetes.

Production of biofunctional recombinant human interleukin 1 receptor antagonist (rhIL1RN) from transgenic quail egg white.

Oviduct-specific expression of heterologous recombinant proteins in transgenic birds is a promising technology for the large-scale production of therapeutic proteins in eggs. We describe the production of recombinant human interleukin 1 receptor antagonist (rhIL1RN) in the eggs of transgenic quails. To drive tissue-specific expression of rhIL1RN, a 1.35-kb fragment of the chicken ovalbumin promoter, which contains both the steroid-dependent regulatory element and the negative regulatory element, was used. A transgenic quail was generated by microinjection of a concentrated stock of lentivirus into stage X blastodermal cells. A single copy of the transgene was integrated into the seventh intron of the gene for conserved oligomeric golgi complex protein 5 (COG5) on chromosome 1. As expected, rhIL1RN expression was restricted to oviductal tissue, and the amount of protein deposited in the eggs of homozygous transgenic quails ranged from 88.7 to 233.8 ng/ml. Transgene expression was conserved from the G(1) generation to the G(4) generation, and there was no evidence of transgene silencing. In a bioassay using the EL4.NOB-1/CTLL-2 coculture system, no significant difference was observed between the egg-produced rhIL1RN and a commercially available rhIL1RN (anakinra).

Genotoxicity studies on HM10760A, recombinant human erythropoietin conjugated to globin fragment.

HM10760A is a recombinant human erythropoietin chemically conjugated to the N-terminus of human immunoglobulin Fc fragment through a polyethylene glycol linker. HM10760A was shown to have a relatively long half-life, compared with unconjugated recombinant erythropoietin. In this study, the genotoxicity of HM10760A was investigated by using a test battery of three different methods. In the Ames assay, five strains (TA100, TA1535, TA98, TA1537, and Escherichia coli WP2 uvrA) were tested at six concentrations of 3.13, 6.25, 12.5, 25, 50, and 100microg/plate. HM10760A did not increase the number of revertant colonies in any tester strains with and without metabolic activation by rat-liver S9 mix. Subsequently, in vitro chromosomal aberration test, using Chinese hamster lung cells, were conducted at the concentrations of 25, 50, and 100microg/mL. HM10760A did not induce chromosomal aberrations either in the short-period (6 hours) test with or without rat-liver S9 mix or in the continuous-treatment (24 hours) test. In the in vivo bone marrow micronucleus assay using the male ICR (imprinting control region) mouse, HM10760A was subcutaneously administered twice at 24-hour intervals at doses of 0, 150, 300, and 600microg/kg. HM10760A produced a slight, but statistically significant, increase in the frequency of micronucleated polychromatic erythrocytes at 600microg/kg. However, no biological significance was assumed, because this value was within the historical control range. From these findings obtained from the genotoxicity assays performed in this study, it appears unlikely that HM10760A acts as a genotoxic agent in vitro and in vivo.

Serum immunoglobulin fused interferon-alpha inhibited tumor growth in athymic mice bearing colon 26 adenocarcinoma cells.

Interferon (IFN) has therapeutic potential for a wide range of infectious and proliferative disorders. However, the half-life of IFN is too short to have a stable therapeutic effect. To overcome this problem, serum immunoglobulin has been fused to IFN. In this study, the efficacy of serum immunoglobulin fused INFs (si-IFN1 and si-IFN2) was evaluated on athymic mice bearing colon 26 adenocarcinoma cells. Seven days after the implantation of tumor cells, each group of mice was injected once a week with si-IFN1 and si-IFN2 at two different concentrations (10 x : 30 microg/kg and 50 x : 150 microg/kg). A slight anti-tumoral effect was observed in all 10 x groups compared to the control. In the 50 x groups, however, si-IFN1 and si-IFN2 showed significant anti- tumoral effects compared to the control. To gain more information on the mechanisms associated with the decrease of tumor size, a Western blot assay of apoptosis-related molecules was performed. The protein expression of cytochrome c, caspase 9, 6, and 3 were increased by si-IFN1 and si-IFN2. These 2 IFNs also increased the expressions of p53, p21, Bax and Bad. Interestingly, si-IFN1 and si-IFN2 decreased the expression of VEGF-beta. Taken together, serum immunoglobulin fused IFNs increased therapeutic efficacy under current experimental condition.

Generation of transgenic quail through germ cell-mediated germline transmission.

Here, we describe the production of transgenic quail via a germline transmission system using postmigratory gonadal primordial germ cells (gPGCs). gPGCs retrieved from the embryonic gonads of 5-day-old birds were transduced with a lentiviral vector and subsequently transferred into recipient embryos. Testcross and genetic analyses revealed that among three germline chimeric G0 quail, one male produced transgenic offspring; of 310 hatchlings from the transgenic germline chimera, 24 were identified as donor-derived offspring, and 6 were transgenic (6/310, 1.9%). Conventional transgenesis using stage X blastodermal embryos was also conducted, but the efficiency of transgenesis was similar between the two systems (<1.6 vs. 1.9% for the conventional and gPGC-mediated systems, respectively). However, substantial advantages can be gained from gPGC-mediated method in that it enables an induced germline modification, whereas direct retroviral transfer to stage X embryos causes mosaic integration. The use of gonadal PGCs for transgenesis may lead to the production of bioreactors.

Genotoxicity assessment of HM10620 containing recombinant human interferon-alpha.

HM10620 is a recombinant human interferon-alpha (rhIFN-alpha) linked to immunoglobulin via N-terminal-specific non-peptidyl polyethylene glycol linker to improve the in vivo stability of interferon. Potential genotoxic effects of HM10620 in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro chromosomal aberration assay, and the in vivo micronucleus assay. HM10620 did not cause any mutation in the Ames assay tested using five tester strains at six concentrations of 6.25, 12.5, 25, 50, 100, and 200 microg/plate. To assess clastogenic effect, the in vitro chromosomal aberration assay and the in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Chromosomal aberration was not induced at the concentrations of 10, 20, and 40 microg/mL. Also, there was no difference in the incidence of micronucleated polychromatic erythrocytes at doses of 10, 20, and 40 mg/kg in male mice compared with the vehicle control group. Therefore, based on the results obtained from the three studies, it is concluded that HM10620 is not a mutagenic agent in bacterial cells and causes no chromosomal damage in mammalian cells both in vitro and in vivo.

Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos.

This study reports for the first time the production of chicken germline chimeras by transfer of embryonic germ (EG) cells into recipient embryos of different strain. EG cells were established by the subculture of gonadal tissue cells retrieved from stage 28 White Leghorn (WL) embryos with I/I gene. During primary culture (P(0)), gonadal primordial germ cells (gPGCs) in the stromal cells began to form colonies after 7 days in culture with significant (P < 0.0001) increase in cell population. Colonized gPGCs were then subcultured with chicken embryonic fibroblast monolayer for EG cell preparation. Prepared EG cells or gPGCs at P(0) were transferred to stage 17 Korean Ogol chicken (KOC) embryos with i/i gene. The recipient chickens were raised for 6 months to sexual maturity, then a testcross analysis by artificial insemination was conducted for evaluating germline chimerism. As results, transfer of EG cells and gPGCs yielded total 17 germline chimeras; 2 out of 15 (13.3%) and 15 of 176 sexually matured chickens (8.5%), respectively. The efficiency of germline transmission in the chimeras was 1.5-14.6% in EG cells, while 1.3-27.6% in gPGCs. In conclusion, chicken germline chimeras could be produced by the transfer of EG cells, as well as gPGCs, which might enormously contribute to establishing various innovative technologies in the field of avian transgenic research for bioreactor production.