An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies.

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Hepatoprotective effect of aged black garlic extract in rodents.

In this study, we investigated the hepatoprotective effects of aged black garlic (ABG) in rodent models of liver injury. ABG inhibited carbon tetrachloride-induced elevation of aspartate transaminase (AST) and alanine transaminase (ALT), which are markers of hepatocellular damage, in SD rats. D-galactosamineinduced hepatocellular damage was also suppressed by ABG treatment. However, ABG does not affect the elevation of alkaline phosphatase (ALP), a marker of hepatobilliary damage, in rats treated with carbon tetrachloride or D-galactosamine. We also examined the effect of ABG on high-fat diet (HFD)-induced fatty liver and subsequent liver damage. ABG had no significant effect on body weight increase and plasma lipid profile in HFD-fed mice. However, HFD-induced increase in AST and ALT, but not ALP, was significantly suppressed by ABG treatment. These results demonstrate that ABG has hepatoprotective effects and suggest that ABG supplementation might be a good adjuvant therapy for the management of liver injury.

EGR1-dependent PTEN upregulation by 2-benzoyloxycinnamaldehyde attenuates cell invasion and EMT in colon cancer.

There has been little evidence to support EGR1 and PTEN function on the EMT of cancer cells. We tried to evaluate how these genes affect cancer cell invasion and EMT through investigating the molecular mechanism(s) of 2'-benzoyloxycinnamaldehyde (BCA). Matrigel invasion and wound healing assay, and in vivo mice model were used to evaluate the effect of BCA on colon cancer cell migration. The molecular mechanism(s) of BCA were evaluated by knock-down or overexpression of EGR1 and PTEN. BCA at 50 nM increased E-cadherin and EGR1 expression without cytotoxicity. Cell migration was inhibited significantly by BCA both in vitro and in vivo. Moreover, BCA inhibits Snail and Vimentin expression, as well as ß-catenin nuclear accumulation. Suppression of EGR1 by siRNA attenuated the inhibition of matrigel invasion by BCA, indicating that EGR1 is responsible for BCA effect. PTEN was upregulated by BCA treatment or EGR1 overexpression. In addition, shPTEN transfection stimulated EMT and cell invasion in vitro. Our data suggest that BCA leads to a remarkable upregulation of EGR1 expression, and that EMT and invasion is decreased via EGR1-dependent PTEN activation. These data showed a critical role of EGR1-PTEN signaling pathway in the EMT of colon cancer, as well as metastasis.

Protective effect of silymarin against ethanol-induced gastritis in rats: role of sulfhydryls, nitric oxide and gastric sensory afferents.

Silymarin has been known to exert antioxidant, anti-carcinogenic and anti-inflammatory effects. In this study, we examined the effect of silymarin on gastritis in rats. Oral administration of silymarin dose-dependently decreased gastric lesions in ethanol-induced gastritis model. Silymarin also significantly suppressed the development of gastric lesions in aspirin- or water immersion-restraint stress-induced gastritis models. Further study demonstrated that the gastroprotective effect of silymarin was blocked by nitric oxide (NO) synthase inhibitor l-NAME, SH blocker N-ethylmaleimide or TRPV1 antagonist capsazepine in ethanol-induced gastritis model. In addition, ex vivo analysis revealed that ethanol-induced decrease in gastric mucus and non-protein sulfhydryl (NPSH) groups was significantly reversed by silymarin treatment and lipid peroxidation was also suppressed by silymarin in ethanol-induced gastritis model. Taken together, these results suggest that silymarin exerts gastroprotective effects and the gastroprotective effects of silymarin might be related to the protection of gastric mucosal NO and NP-SH and the modulation of capsaicin-sensitive gastric sensory afferents.

Facile synthesis of 2-O-iodoacetyl protected glycosyl iodides: useful precursors of 1-->2-linked 1,2-trans-glycosides.

The preparation and utilization of novel iodide glycosyl donors, 2-O-iodoacetyl-glycopyranosyl iodides, is described. The mechanism for the reaction of iodine with carbohydrate cyclic ketene acetal was investigated through low-temperature NMR experiments. 2-O-Iodoacetyl-glycopyranosyl iodides can serve as effective glycosyl donors giving 2-O-iodoacetyl 1,2-trans-glycosides in high yields and excellent stereoselectivities. The 2-O-iodoacetyl group was removed selectively with thiourea to afford 2-hydroxy 1,2-trans-glycosides in high yield without affecting other protecting groups and anomeric configurations.

Synthesis and in vitro photodynamic activities of water-soluble fluorinated tetrapyridylporphyrins as tumor photosensitizers.

A series of water-soluble fluorinated cationic porphyrins were designed, synthesized, and characterized. In vitro photocytotoxicity of these compounds was evaluated by MTT assay on HeLa cells. Their photocytotoxicity was dependent on the positions of the cations and the fluorines in the pyridine ring, and 5,10,15,20-tetrakis-(N-methyl-2-fluoro-pyridin-3-yl)-porphyrin (8) showed the most potent photo-induced cytotoxicity without photobleaching. PDT-induced ROS inside HeLa cells was measured with flow cytometry using ROS-sensitive fluorometric probe, 2,7-dichlorofluororescin (DCF), which revealed high correlations of ROS with cellular cytotoxicity. FACS analysis shows that PDT with porphyrin 8 induced apoptosis in HeLa cells. In summary, efficient generation of ROS, biological effectiveness, and good photostability of porphyrin 8 indicate its potential application in photodynamic therapy (PDT) in the near future.

Bicyclo[3.2.1]octa-3,6-dien-2-yl cation: a bishomoantiaromate.

Antiaromatic compounds with a closed loop of 4n p-electrons are relatively unstable and often difficult to study. We report in this article the synthesis of alcohols 2-(4'-fluorophenyl)bicyclo[3.2.1]octan-2-ol 11, 2-(4'-fluorophenyl)bicyclo[3.2.1]oct-3-en-2-ol 12, and 2-(4'-fluorophenyl)bicyclo[3.2.1]octa-3,6-dien-2-ol 13 and their transformations into corresponding carbocations 14-16, respectively, in a superacidic medium (FSO3H/SO2ClF) at -120 degrees C. Cations 14-16 are characterized by NMR analysis (1H, 13C, 19F), and 15 and 16 are further characterized by quenching in NaOCH3/H3COH at -120 degrees C. The relative stabilities of 14-16 are determined experimentally by 19F NMR spectroscopy. Cation 16 is found to be experimentally less stable than cation 15 by 3.7 kcal/mol. DFT calculations (structure and energy: B3LYP/6-31G(d); NMR: B3LYP/6-311+G(2d,p)) are performed for alcohols 11-13 and bicyclo[3.2.1]octyl cations 6, 7, 9, 14-16, 26, 28, and 30. In the case of 11-16, data from DFT calculations is in good agreement with experimental data. Because 6,7-dimethylenebicyclo[3.2.1]oct-3-en-2-yl cation 26 is more stable than cation 7 by 1.69 kcal/mol, the inductive effect of sp(2)-hybridized carbon atoms C6 and C7 in carbocations 6 and 16 cannot be the reason for the destabilization of 6 relative to 7 and 16 relative to 15. Destabilization of 6 relative to 7 and 16 relative to 15 and the calculated NICS of 6 (+4.17 ppm) and 16(+3.3 ppm) document that 6 and 16 are bishomoantiaromates.

Design, synthesis, and anti-Helicobacter pylori activity of erythromycin A (E)-9-oxime ether derivatives.

The synthesis and anti-Helicobacter pylori (H. pylori) activity evaluation of a new series of erythromycin A (E)-9-oxime ether derivatives are described. These compounds exhibited comparable in vitro anti-H. pylori activity and improved acid stability compared to the reference compound clarithromycin.

Intramolecular ionic Diels-Alder reactions of alpha-acetylenic acetals.

The intramolecular ionic Diels-Alder reaction of alpha-acetylenic acetals as a precursor of the propargyl cation has been investigated in the presence of Lewis acids and in protic acids. The reaction of diene-tethered alpha-acetylenic acetals (1-2) with formic acid yielded the regioselective intramolecular ionic Diels-Alder reaction products, bicyclodienal (9) and bicyclodienone (11) derivatives, in good yields.