Pre-clinical pharmacological profile of QF-036, a potent HIV-1 maturation inhibitor.

QF-036 is an HIV-1 maturation inhibitor in pre-clinical development, and its antiviral activity against a laboratory HIV-1 strain and two drug-resistant strains was determined in the C8166 line. QF-036 was also subjected to absorption, distribution and metabolism (ADM) assessment in vitro, and pharmacokinetic profiles were evaluated in rats and monkeys. The 50% effective concentrations (EC50 ) of QF-036 against the three strains were 20.36 nM, 0.39 µM and 2.11 nM, respectively, demonstrating better antiviral potential than the first-generation antiviral maturation inhibitor bevirimat. QF-036 demonstrated moderate cell permeability, high plasma protein binding ability and good metabolic stability in vitro. After oral QF-036 administration to rats and monkeys, both species exhibited moderate bioavailability, and the plasma drug exposure increased in an approximately dose-proportional manner. When administered orally (30 mg/kg) to monkeys, the QF-036 plasma concentration (Cmax ) peaked at 3671 ng/mL (4.82 µM), 12 to 2410 times higher than the EC50 of laboratory or resistant HIV-1 strains. Moreover, the plasma concentration of QF-036 at 12 hours after administration was 263 ng/mL (0.35 µM), which approximately matched the highest EC50 value of the three test strains. The favourable viral inhibitory activity and pharmacokinetic properties provide critical support for QF-036 as a promising anti-HIV therapeutic candidate.

Single- and repeated-dose toxicity studies on the novel HIV maturation inhibitor QF-036 in Sprague-Dawley rats.

QF-036 is a novel human immunodeficiency virus (HIV) maturation inhibitor that is a lupine triterpenoid derivative. The objective of this study was to evaluate the safety of QF-036. A single oral toxicity and a 4-week repeated oral toxicity were investigated in Sprague-Dawley (SD) rats. The single oral toxicity study of QF-036 in SD rats showed that no mortality or visible pathological changes were noted at doses of 100, 300, and 1000 mg/kg. QF-036 exhibited a non-linear toxicokinetic profile over the dose range of 100-1000 mg/kg in the single dose study, and a saturation trend appeared at doses of 100 and 300 mg/kg. In the 4-week oral toxicity and toxicokinetic study, SD rats were given 0, 50, 100, and 200 mg/kg QF-036 once daily for 4 weeks, followed by a 4-week recovery period. No mortality or significant effects on food consumption, body weight, or behavior were observed. In addition, there were no test article-related changes in hematology, clinical biochemistry and histopathology. The no observed adverse effect level (NOAEL) was 200 mg/kg. The toxicokinetic study demonstrated a dose-dependent increase in the systemic exposure to QF-036 after 4 weeks of oral administration. There were no marked sex differences or drug accumulation observed for repeated doses of QF-036.

Preclinical pharmacokinetics of a recombinant humanized rabbit anti-VEGF monoclonal antibody in rabbits and monkeys.

TMAB001 is a humanized rabbit monoclonal antibody (mAb) designed to bind and neutralize human vascular endothelial growth factor (VEGF)-165. The purpose of the study was to investigate the pharmacokinetics (PK) and ocular tissue distribution after a single intravitreal (IVT) dose in rabbits and monkeys. Rabbits (2.5 mg/eye; n = 40) and monkeys (2.5 mg/eye; n = 12) received TMAB001 as a bilateral IVT dose. TMAB001 concentrations were measured in ocular tissues in all rabbits and monkeys by enzyme-linked immunosorbent assay (ELISA). TMAB001 and VEGF concentrations were measured in serum of monkeys by ELISA. Following a single bilateral IVT injection of TMAB001 2.5 mg/eye, the highest concentration was in vitreous humor, followed by retina and choroid, and the lowest concentration was in lens. In rabbits, TMAB001 was still detectable in ocular tissues at day 21 after single IVT dose, with the highest level in the vitreous humor and then retina, with longest t1/2 in aqueous humor and shortest t1/2 in choroid. In monkeys, tmax in serum was 43 h and t1/2 was approximately 5.5 days. Cmax in serum was much lower than that in vitreous, nearly 1/200. After IVT injection of TMAB001, total VEGF concentrations in serum and ocular tissues increased over time. VEGF concentration in retina and choroid increased over time, up to 336 h after administration. This study demonstrated that TMAB001 could reach the drug target sites-retina and choroid after a single bilateral IVT administration in rabbits and monkeys, with a long t1/2 in vitreous humor. TMAB001 also showed strong capability to neutralize VEGF. The study further confirmed that full-length antibodies can also efficiently diffuse and distribute in ocular tissues.

Recombinant humanized anti-vascular endothelial growth factor monoclonal antibody efficiently suppresses laser-induced choroidal neovascularization in rhesus monkeys.

Neovascular age-related macular degeneration, characterized by abnormal choroidal neovascularization (CNV), is a major cause of blindness worldwide. Anti-vascular endothelial growth factor (VEGF) antibodies have demonstrated significant efficacy in improving visual acuity. TMAB001 is a new recombinant humanized rabbit anti-VEGF monoclonal antibody. It presents high activities in vitro studies. In the binding affinity assay, TMAB001 exhibited a high binding capability to VEGF with an affinity constant of 10-11M. In the receptor antagonist activity assay, IC50 of TMAB001 was 0.15µg/ml. In a cell-based assay, TMAB001 inhibited VEGF165-induced HUVEC cells proliferation in a dose-dependent manner. Furthermore, in the rhesus monkey model of laser-induced CNV, results showed the growth and leakage of experimental CNV were significantly decreased with a single bilateral intravitreal injection of TMAB001, and the grade 4 lesions were complete absence in TMAB001 groups. The efficacy of TMAB001 was maintained for at least 28days. In a mice model of oxygen-induced retinopathy, the retina fluorescence leakage was reduced and the vascular morphology in retina was normalized by TMAB001 intraperitoneal administration. In conclusion, those results indicate that TMAB001 might be a potential drug candidate for wet AMD.

Effects of GW002, a novel recombinant human glucagon-like peptide-1 (GLP-1) analog fusion protein, on CHO recombinant cells and BKS-db mice.

AIMS: GLP-1-based strategies have many advantages in treatment of type 2 diabetes mellitus (T2DM), but native GLP-1 has a short half-life in the circulation, which limits its clinical application. The purpose of this study was to evaluate the effects of GW002, a novel recombinant GLP-1 analog fusion protein produced by linking the human GLP-1 analog C-terminus to the N-terminus of human serum albumin via a linker, in vitro and in BKS-db mice. METHODS: To determine whether GW002 can activate the GLP-1 receptor in cells, the level of luciferase expression was evaluated in vitro. In vivo, body weight, food intake, non-fasting and fasting blood glucose, oral glucose tolerance test, blood glucose and insulin levels, liver histology, liver function parameters and antibody levels in BKS-db mice were investigated to evaluate the effects of GW002. Albiglutide was chosen as a positive comparator. RESULTS: Cyclic adenosine monophosphate levels were increased in a dose-dependent manner in cells. In vivo studies demonstrated that GW002 lowers non-fasting and fasting blood glucose levels and improves glucose tolerance and insulin secretion in BKS-db mice. The degree of hepatic steatosis and hepatic biochemical indexes was also decreased. In this study, the mice body weight was not reduced significantly. CONCLUSIONS: The above results showed that the efficacy of GW002 in BKS-db mice displayed a significant hypoglycemic effect, which indicated that GW002 might be a potential candidate for the treatment of T2DM.

Arctigenin alleviates ER stress via activating AMPK.

AIM: To investigate the protective effects of arctigenin (ATG), a phenylpropanoid dibenzylbutyrolactone lignan from Arctium lappa L (Compositae), against ER stress in vitro and the underlying mechanisms. METHODS: A cell-based screening assay for ER stress regulators was established. Cell viability was measured using MTT assay. PCR and Western blotting were used to analyze gene and protein expression. Silencing of the CaMKKß, LKB1, and AMPKα1 genes was achieved by RNA interference (RNAi). An ATP bioluminescent assay kit was employed to measure the intracellular ATP levels. RESULTS: ATG (2.5, 5 and 10 µmol/L) inhibited cell death and unfolded protein response (UPR) in a concentration-dependent manner in cells treated with the ER stress inducer brefeldin A (100 nmol/L). ATG (1, 5 and 10 µmol/L) significantly attenuated protein synthesis in cells through inhibiting mTOR-p70S6K signaling and eEF2 activity, which were partially reversed by silencing AMPKα1 with RNAi. ATG (1-50 µmol/L) reduced intracellular ATP level and activated AMPK through inhibiting complex I-mediated respiration. Pretreatment of cells with the AMPK inhibitor compound C (25 µmol/L) rescued the inhibitory effects of ATG on ER stress. Furthermore, ATG (2.5 and 5 µmol/L) efficiently activated AMPK and reduced the ER stress and cell death induced by palmitate (2 mmol/L) in INS-1 ß cells. CONCLUSION: ATG is an effective ER stress alleviator, which protects cells against ER stress through activating AMPK, thus attenuating protein translation and reducing ER load.

17-Acetoxyjolkinolide B irreversibly inhibits IkappaB kinase and induces apoptosis of tumor cells.

Nuclear factor-kappaB (NF-kappaB) is critically important for tumor cell survival, growth, angiogenesis, and metastasis. One of the key events in the NF-kappaB signaling is the activation of inhibitor of NF-kappaB kinase (IKK) in response to stimuli of various cytokines. We have identified 17-acetoxyjolkinolide B (17-AJB) from a traditional Chinese medicinal herb Euphorbia fischeriana Steud as a novel small-molecule inhibitor of IKK. 17-AJB effectively inhibited tumor necrosis factor-alpha-induced NF-kappaB activation and induced apoptosis of tumor cells. 17-AJB had no effect on binding of tumor necrosis factor-alpha to its receptor or on binding of NF-kappaB to DNA. It inhibited NF-kappaB nuclear translocation. Detailed analysis revealed that the direct target of 17-AJB was IKK. 17-AJB kept IKK in its phosphorylated form irreversibly. This irreversible modification of IKK inactivated its kinase activity, leading to its failure to activate NF-kappaB. The effect of 17-AJB on IKK was specific. It had no effect on other kinases such as p38, p44/42, and JNK. In addition, 17-AJB induced apoptosis in tumor cells. The effects of 17-AJB on apoptosis correlated with inhibition of expression of the NF-kappaB-regulated genes. Taken together, our data suggest that 17-AJB is a novel type NF-kappaB pathway inhibitor. Its unique interaction mechanism with IKK may render it a strong apoptosis inducer of tumor cells and a novel type anticancer drug candidate.

Production of native protein by using Synechocystis sp. PCC6803 DnaB mini-intein in Escherichia coli.

To directly express native recombinant proteins in Escherichia coli, a new expression vector pSB was constructed using Ssp DnaB mini-intein. Using the vector, native proteins could be produced with the help of C-terminal self-cleavage of the intein. In this study, we cloned hIFNalpha-4 gene into pSB and used E. coli strain Origami B (DE3) as the host. Expression experiments were carried out both in Shake flasks and a 5 L bioreactor. The results indicated hIFNalpha-4 could be expressed in the form of soluble protein with correct folding in E. coli. The maximal hIFNalpha-4 content was 21.7% of total protein, and the antiviral activity of the protein was 1.2x10(8 )IU mg(-1). Overall, good effects were achieved with this system. This intein-mediated protein expression system opens up a useful method for production of native recombinant protein in E. coli.