Intraperitoneal administration of synthetic microRNA-214 elicits tumor suppression in an intraperitoneal dissemination mouse model of canine hemangiosarcoma.

Canine hemangiosarcoma (HSA) has an extremely poor prognosis, making it necessary to develop new systemic treatment methods. MicroRNA-214 (miR-214) is one of many microRNAs (miRNA) that can induce apoptosis in HSA cell lines. Synthetic miR-214 (miR-214/5AE), which showed higher cytotoxicity and greater nuclease resistance than mature miR-214, has been developed for clinical application. In this study, we evaluated the effects of miR-214/5AE on stage 2 HSA in a mouse model. Mice intraperitoneally administered with miR-214/5AE (5AE group) had significantly fewer intraperitoneal dissemination tumor foci (median number: 72.5 vs. 237.5; p < 0.05) and a lower median foci weight (0.26 g vs. 0.61 g; p < 0.05). Mice in the 5AE group had increased expression of p53 and cleaved caspase-3, and a significantly lower proportion of Ki-67-positive cells, than those in the non-specific miR group. Notably, no significant side effects were observed. These results indicate that intraperitoneal administration of miR-214/5AE exhibits antitumor effects in an intraperitoneal dissemination mouse model of HSA by inducing apoptosis and suppressing cell proliferation. These results provide a basis for future studies on the antitumor effect of miR-214/5AE for HSA.

Synthesis and evaluation of the anti-hepatitis B virus activity of 4'-Azido-thymidine analogs and 4'-Azido-2'-deoxy-5-methylcytidine analogs: structural insights for the development of a novel anti-HBV agent.

Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4'-Azido-thymidine/4'-Azido-2'-deoxy-5-methylcytidine derivatives (6, 10-15) were synthesized, and their anti-HBV activities evaluated. Compounds 10-15 were synthesized via an SNAr reaction of 18, in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11-15 showed no antiviral activity. However, 4'-Azido thymidine (6) and 4'-Azido-2'-deoxy-5-methylcytidine (10) displayed significant anti-HBV activity (EC50 = 0.63 and 5.99 µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100 µM.

CMCdG, a Novel Nucleoside Analog with Favorable Safety Features, Exerts Potent Activity against Wild-Type and Entecavir-Resistant Hepatitis B Virus.

We designed, synthesized, and characterized a novel nucleoside analog, (1S,3S,5S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4'-cyano-methylenecarbocyclic-2'-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG's in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBVWTCe) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBVETV-RL180M/S202G/M204V). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC50], ∼30 nM) and HBVWTCe plasmid-transfected Huh7 cells (IC50, 206 nM) and efficiently suppressed ETV-resistant HBVETV-RL180M/S202G/M204V (IC50, 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 µM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBVWTCe-infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of HBVETV-RL180M/S202G/M204V viremia by ∼1 log in HBVETV-RL180M/S202G/M204V-infected human liver-chimeric mice, while ETV (1 mg/kg/day q.d.) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body weights or serum human albumin levels. Structural analyses using homology modeling, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBVWTCe reverse transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBVETV-RL180M/S202G/M204V RT complex. However, CMCdG-TP retains good contacts with both the HBVWTCe RT and HBVETV-RL180M/S202G/M204V RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed light on the further development of more potent and safer anti-HBV agents.

Diastereoselective Synthesis of 6″-(Z)- and 6″-(E)-Fluoro Analogues of Anti-hepatitis B Virus Agent Entecavir and Its Evaluation of the Activity and Toxicity Profile of the Diastereomers.

A method for the diastereoselective synthesis of 6″-(Z)- and 6″-(E)-fluorinated analogues of the anti-HBV agent entecavir has been developed. Construction of the methylenecyclopentane skeleton of the target molecules has been accomplished by radical-mediated 5-exo-dig cyclization of the selenides 6 and 15 having the phenylsulfanylethynyl structure as a radical accepting moiety. In the radical reaction of the TBS-protected precursor 6, (Z)-anti-12 was formed as a major product. On the other hand, TIPS-protected 15 gave (E)-anti-12. The sulfur-extrusive stannylation of anti-12 furnished a mixture of geometric isomers of the respective vinylstannane, whereas benzoyl-protected 17 underwent the stannylation in the manner of retention of configuration. Following XeF2-mediated fluorination, introduction of the purine base and deoxygenation of the resulting carbocyclic guanosine gave the target (E)- and (Z)-3 after deprotection. Evaluation of the anti-HBV activity of 3 revealed that fluorine-substitution at the 6″-position of entecavir gave rise to a reduction in the cytotoxicity in HepG2 cells with retention of the antiviral activity.

Design, Synthesis, and Evaluation of Anti-HBV Activity of Hybrid Molecules of Entecavir and Adefovir: Exomethylene Acycloguanine Nucleosides and Their Monophosphate Derivatives.

Exomethylene acycloguanine nucleosides 4, 6 and its monophosphate derivatives 5, 7, and 8 have been synthesized. Mitsunobu-type coupling of 2-N-acetyl-6-O-diphenylcarbamoylguanine (11) with primary alcohols proceeded regioselectively to furnish the desired N(9)-substituted products in moderate yield. Evaluation of 4-8 for anti-HBV activity in HepG2 cells revealed that the phosphonate derivative 8 was found to exhibit moderated activity (EC50 value of 0.29 µM), but cytotoxicity (CC50 value of 39 µM) against the host cells was also observed.

2'-deoxy-4'-C-ethynyl-2-halo-adenosines active against drug-resistant human immunodeficiency virus type 1 variants.

One of the formidable challenges in therapy of infections by human immunodeficiency virus (HIV) is the emergence of drug-resistant variants that attenuate the efficacy of highly active antiretroviral therapy (HAART). We have recently introduced 4'-ethynyl-nucleoside analogs as nucleoside reverse transcriptase inhibitors (NRTIs) that could be developed as therapeutics for treatment of HIV infections. In this study, we present 2'-deoxy-4'-C-ethynyl-2-fluoroadenosine (EFdA), a second generation 4'-ethynyl inhibitor that exerted highly potent activity against wild-type HIV-1 (EC50 approximately 0.07 nM). EFdA retains potency toward many HIV-1 resistant strains, including the multi-drug resistant clone HIV-1A62V/V75I/F77L/F116Y/Q151M. The selectivity index of EFdA (cytotoxicity/inhibitory activity) is more favorable than all approved NRTIs used in HIV therapy. Furthermore, EFdA efficiently inhibited clinical isolates from patients heavily treated with multiple anti-HIV-1 drugs. EFdA appears to be primarily phosphorylated by the cellular 2'-deoxycytidine kinase (dCK) because: (a) the antiviral activity of EFdA was reduced by the addition of dC, which competes nucleosides phosphorylated by the dCK pathway, (b) the antiviral activity of EFdA was significantly reduced in dCK-deficient HT-1080/Ara-Cr cells, but restored after dCK transduction. Further, unlike other dA analogs, EFdA is completely resistant to degradation by adenosine deaminase. Moderate decrease in susceptibility to EFdA is conferred by a combination of three RT mutations (I142V, T165R, and M184V) that result in a significant decrease of viral fitness. Molecular modeling analysis suggests that the M184V/I substitutions may reduce anti-HIV activity of EFdA through steric hindrance between its 4'-ethynyl moiety and the V/I184 beta-branched side chains. The present data suggest that EFdA, is a promising candidate for developing as a therapeutic agent for the treatment of individuals harboring multi-drug resistant HIV variants.

2'-Deoxy-4'-C-ethynyl-2-fluoroadenosine: a nucleoside reverse transcriptase inhibitor with highly potent activity against all HIV-1 strains, favorable toxic profiles and stability in plasma.

A working hypothesis to solve the critical problems of existing HAART was proposed. The study based on the hypothesis proved the validity of the hypothesis and resulted in the development of 2'-deoxy-4'-C-ethynyl-2-fluoro-adenosine (4'Ed2FA), a nucleoside reverse transcriptase inhibitor (NRTI) with highly potent activity against all HIV-1 strains, very favourable toxic profiles, and stability in plasma.

Synthesis of 4'-C-ethynyl and 4'-C-cyano purine nucleosides from natural nucleosides and their anti-HIV activity.

Purine 2'-deoxynucleosides bearing an ethynyl or a cyano group at C-4' of the sugar moiety were synthesized from the corresponding 2'-deoxynucleosides. These compounds exhibited very potent anti-HIV activity, and remained active against drug resistant HIV strains.