mRNA-HPV vaccine encoding E6 and E7 improves therapeutic potential for HPV-mediated cancers via subcutaneous immunization.

The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA-HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell-mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor-implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV-treated mice. Furthermore, tumor expansion was significantly reduced upon TC-1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV-related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.

Herb-Drug Interaction of Red Ginseng Extract and Ginsenoside Rc with Valsartan in Rats.

The purpose of this study was to investigate the herb-drug interactions involving red ginseng extract (RGE) or ginsenoside Rc with valsartan, a substrate for organic anion transporting polypeptide (OATP/Oatp) transporters. In HEK293 cells overexpressing drug transporters, the protopanaxadiol (PPD)-type ginsenosides- Rb1, Rb2, Rc, Rd, Rg3, compound K, and Rh2-inhibited human OATP1B1 and OATP1B3 transporters (IC50 values of 7.99-68.2 µM for OATP1B1; 1.36-30.8 µM for OATP1B3), suggesting the herb-drug interaction of PPD-type ginsenosides involving OATPs. Protopanaxatriol (PPT)-type ginsenosides-Re, Rg1, and Rh1-did not inhibit OATP1B1 and OATP1B3 and all ginsenosides tested didn't inhibit OCT and OAT transporters. However, in rats, neither RGE nor Rc, a potent OATP inhibitor among PPD-type ginsenoside, changed in vivo pharmacokinetics of valsartan following repeated oral administration of RGE (1.5 g/kg/day for 7 days) or repeated intravenous injection of Rc (3 mg/kg for 5 days). The lack of in vivo herb-drug interaction between orally administered RGE and valsartan could be attributed to the low plasma concentration of PPD-type ginsenosides (5.3-48.4 nM). Even high plasma concentration of Rc did not effectively alter the pharmacokinetics of valsartan because of high protein binding and the limited liver distribution of Rc. The results, in conclusion, would provide useful information for herb-drug interaction between RGE or PPD-type ginsenosides and Oatp substrate drugs.

In Vitro Inhibitory Effects of APINACA on Human Major Cytochrome P450, UDP-Glucuronosyltransferase Enzymes, and Drug Transporters.

APINACA (known as AKB48, N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide), an indazole carboxamide synthetic cannabinoid, has been used worldwide as a new psychoactive substance. Drug abusers take various drugs concomitantly, and therefore, it is necessary to characterize the potential of APINACA-induced drug-drug interactions due to the modulation of drug-metabolizing enzymes and transporters. In this study, the inhibitory effects of APINACA on eight major human cytochrome P450s (CYPs) and six uridine 5'-diphospho-glucuronosyltransferases (UGTs) in human liver microsomes, as well as on the transport activities of six solute carrier transporters and two efflux transporters in transporter-overexpressed cells, were investigated. APINACA exhibited time-dependent inhibition of CYP3A4-mediated midazolam 1'-hydroxylation (Ki, 4.5 µM; kinact, 0.04686 min-1) and noncompetitive inhibition of UGT1A9-mediated mycophenolic acid glucuronidation (Ki, 5.9 µM). APINACA did not significantly inhibit the CYPs 1A2, 2A6, 2B6, 2C8/9/19, or 2D6 or the UGTs 1A1, 1A3, 1A4, 1A6, or 2B7 at concentrations up to 100 µM. APINACA did not significantly inhibit the transport activities of organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)1, OCT2, P-glycoprotein, or breast cancer resistance protein at concentrations up to 250 µM. These data suggest that APINACA can cause drug interactions in the clinic via the inhibition of CYP3A4 or UGT1A9 activities.

Heterologous expression and enzymatic characterization of γ-glutamyltranspeptidase from Bacillus amyloliquefaciens.

γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of γ-glutamyl compounds and the transfer of γ-glutamyl moiety to water or to amino acid/peptide acceptors. GGT can be utilized for the generation of γ-glutamyl peptides or glutamic acid, which are used as food taste enhancers. In the present study, Bacillus amyloliquefaciens SMB469 with high GGT activity was isolated from Doenjang, a traditional fermented soy food of Korea. The gene encoding GGT from B. amyloliquefaciens SMB469 (BaGGT469) was cloned from the isolate, and heterologously expressed in E. coli and B. subtilis. For comparison, three additional GGT genes were cloned from B. subtilis 168, B. licheniformis DSM 13, and B. amyloliquefaciens FZB42. The BaGGT469 protein was composed of 591 amino acids. The final protein comprises two separate polypeptide chains of 45.7 and 19.7 kDa, generated via autocatalytic cleavage. The specific activity of BaGGT469 was determined to be 17.8 U/mg with γ-L-glutamyl-p-nitroanilide as the substrate and diglycine as the acceptor. GGTs from B. amyloliquefaciens showed 1.4- and 1.7-fold higher transpeptidase activities than those from B. subtilis and B. licheniformis, respectively. Especially, recombinant B. subtilis expressing BaGGT469 demonstrated 11- and 23-fold higher GGT activity than recombinant E. coli and the native B. amyloliquefaciens, respectively, did. These results suggest that BaGGT469 can be utilized for the enzymatic production of various γ-glutamyl compounds.