Synthesis of novel entecavir analogues having 4'-cyano-6''-fluoromethylenecyclopentene skeletons as an aglycone moiety as highly potent and long-acting anti-hepatitis B virus agent.

Encouraged by our recent findings that 4'-cyano-deoxyguanosine (2), entecavir analogues 4 and 5 are highly potent anti-hepatitis B virus (HBV) agents, we designed and synthesized 6 having a hybridized structure of 4 and 5. The chiral quaternary carbon portion at the 4'-position, which is substituted by cyano- and 5'-hydroxymethyl groups, was stereospecifically constructed by radical-mediated 5-exo-dig mode cyclization of 10. The introduction of the fluorine atom into the 6''-position was achieved by radical-mediated stannylation of sulfide (E)-11 and subsequent electrophilic fluorination of (E)-12. The desired (E)-6 was obtained after the introduction of the guanine base into (E)-18 under Mitsunobu conditions and following global deprotection. The stereoisomer (Z)-6 was also prepared by the same procedure using (Z)-12. Compound (E)-6 showed highly potent anti-HBV activity (EC50 = 1.2 nM) with favorable cytotoxicity (CC50 = 93 µM).

Hybrid and Conventional Endoscopic Submucosal Dissection for Early Gastric Neoplasms: A Multi-Center Randomized Controlled Trial.

BACKGROUND & AIMS: Hybrid endoscopic submucosal dissection (H-ESD), which incorporates endoscopic submucosal dissection (ESD) with endoscopic mucosal resection, has been developed to make ESD technically easier. This study aimed to determine if H-ESD is superior to conventional ESD (C-ESD) for small early gastric neoplasms (EGNs). METHODS: We conducted a multi-center, prospective, open-label, randomized controlled trial to compare the treatment outcomes of H-ESD and C-ESD (Hybrid-G Trial). Patients with differentiated type intramucosal EGN ≤20 mm in diameter and without ulceration were randomly assigned (1:1) to groups that underwent H-ESD or C-ESD. A single multi-functional snare, SOUTEN (ST1850-20, Kaneka, Medix, Tokyo, Japan), was used for H-ESD. The primary outcome was procedure time. Secondary outcomes included mucosal incision time, time and speed of submucosal dissection, curability, and endoscopic procedural adverse events. RESULTS: A total of 39 and 40 patients underwent H-ESD and C-ESD, respectively. The procedure time of H-ESD was significantly shorter than that of C-ESD (33.16 min vs 62.46 min; H-ESD/C-ESD ratio: 0.53; 95% confidence interval, 0.41-0.69; P < .0001). There was no significant difference in mucosal incision time between the 2 groups; the time and speed of submucosal dissection of H-ESD were significantly shorter than those of C-ESD. No difference was observed between the 2 groups in other outcomes. CONCLUSIONS: H-ESD has significantly shorter procedure time than C-ESD, with high and comparable curability and safety for both H-ESD and C-ESD. H-ESD can be a good option for the endoscopic treatment of small EGNs. (UMIN Clinical Trials Registry, Numbers: UMIN000041244).

Negligible procedure-related dissemination risk of mucosal incision-assisted biopsy for gastrointestinal stromal tumors versus endoscopic ultrasound-guided fine-needle aspiration/biopsy.

BACKGROUND: Mucosal incision-assisted biopsy (MIAB) is a valuable alternative to endoscopic ultrasound-guided fine-needle aspiration/biopsy (EUS-FNAB) for sampling gastric subepithelial lesions (SELs). This study aimed to evaluate the potential risk of dissemination and impact on postoperative prognosis associated with MIAB, which has not yet been investigated. METHODS: Study 1: A prospective observational study was conducted to examine the presence or absence and growth rate of tumor cells in gastric juice before and after the procedure in patients with SELs who underwent MIAB (n = 25) or EUS-FNAB (n = 22) between September 2018 and August 2021. Study 2: A retrospective study was conducted to examine the impact of MIAB on postoperative prognosis in 107 patients with gastrointestinal stromal tumors diagnosed using MIAB (n = 39) or EUS-FNAB (n = 68) who underwent surgery between January 2001 and July 2020. RESULTS: In study 1, although no tumor cells were observed in gastric juice in MIAB before the procedure, they were observed in 64% of patients after obtaining samples (P < 0.001). In contrast, no tumor cells were observed in the gastric juice in EUS-FNAB before and after the procedure. In study 2, there was no significant difference in 5-year disease-free survival between MIAB (100%) and EUS-FNAB (97.1%) (P = 0.27). CONCLUSION: MIAB is safe, with little impact on postoperative prognosis, although the procedure releases some tumor cells after damaging the SEL's pseudocapsule.

Comparison of the procedure time differences between hybrid endoscopic submucosal dissection and conventional endoscopic submucosal dissection in patients with early gastric neoplasms: a study protocol for a multi-center randomized controlled trial (Hybrid-G trial).

BACKGROUND: Endoscopic submucosal dissection (ESD) is widely accepted as a local treatment for gastrointestinal tract tumors. As a simplified endoscopic procedure, hybrid ESD (H-ESD) has been performed for colorectal neoplasms in recent times. However, whether H-ESD is superior to conventional ESD (C-ESD) for patients with early gastric neoplasms (EGN) remains unclear. In this trial, we will compare the treatment outcomes of H-ESD and C-ESD. We hypothesize that the procedure time for H-ESD is shorter than that for C-ESD. METHODS: This is an investigator-initiated, multi-center, prospective, randomized, open-label, parallel-group trial to be conducted beginning in August 2020 at nine institutions in Japan. We will determine if H-ESD is superior to C-ESD in terms of procedure time in patients with EGN diagnosed as macroscopically intramucosal (T1a) differentiated carcinoma ≤ 20 mm in diameter without ulcerative findings according to current Japanese gastric cancer treatment guidelines. A total of 82 patients will be recruited and randomly assigned to either the C-ESD or the H-ESD group. The primary outcome is ESD procedure time. Secondary outcomes include mucosal incision, time and speed of submucosal dissection, en bloc resection, complete resection, curability, adverse events related to the ESD procedure, extent of dissection before snaring, volume of injection solution, number and time of hemostasis, thickness of the submucosal layer in the resected specimen, and handover to another operator. The stated sample size was determined based on the primary outcome. According to a previous report comparing the procedure times of C-ESD and H-ESD, we hypothesized that H-ESD would provide a 0.2 reduction in logarithmically concerted procedure time (-37%). We estimated that a total of 82 participants were needed to reach a power of 80% for a t-test with a significance level of 0.05 and considering a 10% dropout. DISCUSSION: This trial will provide high-quality data on the benefits and risks of H-ESD for EGN patients. The results of this study could lead to improved outcomes in patients with EGN undergoing ESD. The results will be presented at national and international meetings and published in peer-reviewed journals. TRIAL REGISTRATION: UMIN-CTR UMIN000041244 . Registered on July 29, 2020.

4-Thiofuranoid Glycal: Versatile Glycosyl Donor for the Selective Synthesis of ß-anomer of 4'-thionucleoside and its Biological Activities.

The first highly diastereoselective synthesis of ß-anomers of 4'-thionucleosides has been carried out by means of electrophilic glycosidation utilizing 3,5-O-(di-tertbutylsilylene) (DTBS)-4-thiofuranoid glycal as a glycosyl donor. The resulting glycosides were transformed into ribo-, 2'-deoxy-, and arabinofuranosyl nucleosides through a chemical transformation of the 2'-substituent. The additive Pummerer reaction of the glycal Soxide gave 1,2-di-O-acetyl-3,5-O-DTBS-4-thioribofuranose. The utility of the DTBSprotected 4-thioribofuranose has been demonstrated by the preparation of 4'-thio analogues of pyrimidine- and purine-4'-thioribonucleosides based on the Vorbrüggen glycosidation. Synthesis of 4'-thio-counterpart of C-nucleoside antibiotic tiazofurin has also been carried out. α-Face selective hydroboration of 1-C-aryl- or 1-C-heteroaryl-glycals obtained by cross-coupling of 1-tributylstannylglycal has furnished the respective ß- anomer of 4'-thio-C-ribonucleosides, including 4'-thio analogue of nucleoside antibiotic pseudouridine and 9-deazaadenosine. On the basis of lithiation chemistry, 1-C- and 2-Ccarbon- carbon-substituted 3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3- diyl) (TIPDS)- 4- thiofuranoid glycal were synthesized. These glycals enabled us to prepare 1'-C- and 2'-ß- C-carbon-substituted 2'-deoxy-4'-thionucleosides, including thio-counterpart of antitumor nucleoside antibiotic angustmycin C. Furthermore, 1'-C-methyl-4'-thiothymidine emerged as a potent inhibitor of angiogenesis. In addition, 1'-C-methyl-4'-thiothymidine exhibited more potent inhibitory activity against thymidine kinase-deficient mutant of herpes virus than that of ganciclovir. Among the 4'-substituted 4'-thiothymidines, the 4'- C-cyano- and 4'-C-ethynyl derivatives inhibited replication of HIV variant resistant to 3TC (HIVM184V) as potently as HIV-1IIIB. In terms of the value of selectivity index (SI), 4'-C-cyano-4'-thiothymidine showed a 3-fold selective index (SI) than that of the corresponding thymidine derivative. Furthermore, 4'-C-ethynyl-2'-deoxy-4'-thioguanosine has a 20-fold better value (>18,200) than that of 2'-deoxyguanosine counterpart (933). Furthermore, 4'-azido-4'-thiothymidine emerged as a selective and potent anti-EBV agent. In terms of antineoplastic activity, 4'-azido- and 4'-C-fluoromethyl-2'-deoxy-4'-thiocytidine inhibited proliferation of human B-cell (CCRF-SB) and T-cell leukemia (Molt-4) cell lines, although the parent compound 2'-deoxy-4'-thiocytidine did not exhibit any cytotoxicity up to 100 µM. These facts concerning the biological activities suggested that replacement of the furanose oxygen with a sulfur atom is a promising approach for the development of less toxic antiviral and antineoplastic nucleoside antimetabolites. 4'- Thionucleoside also acts as a monomer for oligonucleotides (ONs) therapeutics, exhibiting superior biological properties. Therefore, this review provides a wide range of potential monomers for antisense ON and siRNA.

Efficacy of hybrid endoscopic submucosal dissection with SOUTEN in gastric lesions: An ex vivo porcine model basic study.

BACKGROUND: Hybrid endoscopic submucosal dissection (ESD) that comprises mucosal incision and partial submucosal dissection followed by snaring in a planned manner, has been developed for endoscopic resection of gastrointestinal neoplasms to overcome the technical barrier of ESD. Although the superiority of hybrid ESD with SOUTEN, a single multifunctional device, over conventional ESD has been indicated, the actual effect of snaring itself remains unclear since SOUTEN could be applied to hybrid ESD group, but not to the conventional ESD group, due to ethical issue in clinical practice. AIM: To determine whether and how hybrid ESD was superior to conventional ESD in the endoscopic treatment of gastric lesions in an ex vivo porcine model basic study. METHODS: Sixteen endoscopists participated in this basic study in August 2020 at Kyushu University, performing 32 procedures each for hybrid ESD and conventional ESD. Mock lesions (10-15 mm, diameter) were created in the porcine stomach. The primary outcome was total procedure time and secondary outcomes were en bloc or complete resection, perforation, procedure time/speed for both, mucosal incision, and submucosal dissection. Factors associated with difficulty in ESD including longer procedure time, incomplete resection, and perforation, were also investigated. Categorical and continuous data were analyzed using the chi-square test or Fisher's exact test and the Mann-Whitney U test, respectively. RESULTS: The median total procedure time of hybrid ESD was significantly shorter than that of conventional ESD (median: 8.3 min vs 16.2 min, P < 0.001). Time, speed, and the amount of hyaluronic acid during submucosal dissection were more favorable in hybrid ESD than conventional ESD (time, 5.2 min vs 10.4 min, P < 0.001; speed, 43.7 mm2/min vs 23.8 mm2/min, P < 0.00; injection volume, 1.5 mL vs 3.0 mL, P < 0.001), although no significant differences in those factors were observed between both groups during mucosal incision. There was also no significant difference between both groups in the en bloc/complete resection rate and perforation rate (complete resection, 93.8% vs 87.5%, P = 0.67; perforation, 0% vs 3.1%, P = 1). Selection of conventional ESD as the treatment method was significantly associated with difficulties during ESD (odds ratio = 10.2; highest among factors). CONCLUSION: Hybrid ESD with SOUTEN improves the treatment outcomes of gastric lesions. It also has the potential to reduce medical costs since SOUTEN is a single multifunctional device that is inexpensive.

Endoscopic mucosal resection vs endoscopic submucosal dissection for superficial non-ampullary duodenal tumors.

BACKGROUND: The selection of endoscopic treatments for superficial non-ampullary duodenal epithelial tumors (SNADETs) is controversial. AIM: To compare the efficacy and safety of endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) for SNADETs. METHODS: We retrospectively analyzed the data of patients with SNADETs from a database of endoscopic treatment for SNADETs, which included eight hospitals in Fukuoka, Japan, between April 2001 and October 2017. A total of 142 patients with SNADETs treated with EMR or ESD were analyzed. Propensity score matching was performed to adjust for the differences in the patient characteristics between the two groups. We analyzed the treatment outcomes, including the rates of en bloc/complete resection, procedure time, adverse event rate, hospital stay, and local or metastatic recurrence. RESULTS: Twenty-eight pairs of patients were created. The characteristics of patients between the two groups were similar after matching. The EMR group had a significantly shorter procedure time and hospital stay than those of the ESD group [median procedure time (interquartile range): 6 (3-10.75) min vs 87.5 (68.5-136.5) min, P < 0.001, hospital stay: 8 (6-10.75) d vs 11 (8.25-14.75) d, P = 0.006]. Other outcomes were not significantly different between the two groups (en bloc resection rate: 82.1% vs 92.9%, P = 0.42; complete resection rate: 71.4% vs 89.3%, P = 0.18; and adverse event rate: 3.6% vs 17.9%, P = 0.19, local recurrence rate: 3.6% vs 0%, P = 1; metastatic recurrence rate: 0% in both). Only one patient in the ESD group underwent emergency surgery owing to intraoperative perforation. CONCLUSION: EMR has significantly shorter procedure time and hospital stay than ESD, and provides acceptable curability and safety compared to ESD. Accordingly, EMR for SNADETs is associated with lower medical costs.

Superiority of mucosal incision-assisted biopsy over ultrasound-guided fine needle aspiration biopsy in diagnosing small gastric subepithelial lesions: a propensity score matching analysis.

BACKGROUND: Gastric subepithelial lesions, including gastrointestinal stromal tumors, are often found during routine gastroscopy. While endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) has been the gold standard for diagnosing gastric subepithelial lesions, alternative open biopsy procedures, such as mucosal incision-assisted biopsy (MIAB) has been reported useful. The aim of this study is to evaluate the efficacy of MIAB for the diagnosis of gastric SELs compared with EUS-FNAB. METHODS: We retrospectively analyzed medical records of 177 consecutive patients with gastric SELs who underwent either MIAB or EUS-FNAB at five hospitals in Japan between January 2010 and January 2018. Diagnostic yield, procedural time, and adverse event rates for the two procedures were evaluated before and after propensity-score matching. RESULTS: No major procedure-related adverse events were observed in either group. Both procedures yielded highly-accurate diagnoses once large enough samples were obtained; however, such successful sampling was more often accomplished by MIAB than by EUS-FNAB, especially for small SELs. As a result, MIAB provided better diagnostic yields for SELs smaller than 20-mm diameter. The diagnostic yields of both procedures were comparable for SELs larger than 20-mm diameter; however, MIAB required significantly longer procedural time (approximately 13 min) compared with EUS-FNAB. CONCLUSIONS: Although MIAB required longer procedural time, it outperformed EUS-FNAB when diagnosing gastric SELs smaller than 20-mm diameter.

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.

An alternative method for the synthesis of 2'-halogeno-1',2'-unsaturated uridine derivatives through syn-elimination of pivalic acid of 2'-halogeno- 2'-deoxy-1'-pivaloyloxyuracil nucleoside: preparation of its 2'-C-branched nucleosides.

An alternative method for the preparation of 2'-bromo- (5b) and 2'-iodo- (5c) 1',2'-unsaturated uracil nucleosides has been developed. The protocol was on the basis of the syn-elimination of pivalic acid from 2'-bromo-(7a,b) and 2'-iodo-(9a,b) 1'-pivaloyloxy-2'-deoxyuridine derivatives, which were derived from the halo-pivaloyloxylation of 3',5'-bis-O-TBDMS-1',2'-unsaturated uridine 1. Compounds 5b and 5c were shown to serve as versatile synthons for the respective 2'-C-branched 1',2'-unsaturated uracil nucleosides, through palladium-catalyzed cross-coupling or halogen-lithium exchange reactions.

Objective validity of the Japan Narrow-Band Imaging Expert Team classification system for the differential diagnosis of colorectal polyps.

BACKGROUND AND AIM: The Japan Narrow-Band Imaging (NBI) Expert Team (JNET) classification is a recently proposed NBI magnifying endoscopy-based classification system for colorectal tumors. Although the usefulness of this system has been reported by JNET experts, its objective validity remains unclear. We tested its validity and usefulness for the diagnosis of colorectal polyps by including colonoscopy experts and non-experts as test participants. METHODS: Forty NBI images of polyps of various JNET types were shown to 22 doctors (11 experts and 11 non-gastrointestinal [GI] trainees) who had not examined the patients. The doctors diagnosed the polyps based solely on the surface and vessel patterns in the magnified images and the JNET classification system. Concordance rates of their diagnoses with the pathological findings of the polyps were determined, and the results for experts and non-GI trainees were compared. RESULTS: Both for colonoscopy experts and non-GI trainees, the JNET classification system was particularly useful for classifying polyps as benign or malignant. Although the accuracy rates for classifying polyps into each JNET type varied among colonoscopy experts, those who were familiar with the JNET classification system were able to diagnose polyps with approximately 90% accuracy. Common mistakes were attributable to misunderstandings of the wording in the JNET classification chart and lack of proper training. CONCLUSION: The JNET classification system is a practical approach for the diagnosis of colorectal polyps. Training is required even for experienced colonoscopists to adopt the system properly. Common pitfalls must be shared among colonoscopists to improve the accuracy of the diagnosis.

Mucosal Profiles of Immune Molecules Related to T Helper and Regulatory T Cells Predict Future Relapse in Patients With Quiescent Ulcerative Colitis.

BACKGROUND: T helper (Th)- and regulatory T (Treg) cell-related immune molecules are implicated in ulcerative colitis (UC). However, the association between their mucosal expression during remission and the subsequent clinical course of UC is unknown. METHODS: The expression of cytokines and transcription factors related to Th1, Th2, Th17, and Treg in endoscopic mucosal biopsy specimens from 40 UC patients in clinical remission and 9 controls was measured by quantitative polymerase chain reaction. The relationship between their expression patterns, as stratified by Mayo Endoscopic Subscore (MES), and any future relapse was evaluated by univariate and multivariate analyses. RESULTS: Six of 40 patients (baseline MES 0/1/2, 22/14/4) experienced a relapse during the study period (median, 37 months). At baseline, even in the MES0 patients, the interleukin (IL)-17A of the patients was significantly upregulated in comparison with controls (P = 0.0351). Future relapse was associated with a higher baseline expression of IL-17A, IL-17F, and IL-21 in MES0/1, and the upregulation of IL-17F and IL-21 remained statistically significant when limited to MES0 patients. Kaplan-Meier analysis revealed that as a single marker, a higher IL-21 level best grouped patients with an increased risk of relapse (P = 0.0042). Furthermore, a multivariate model that consisted of IL-21 and T-bet showed an even greater value (P = 0.0001). CONCLUSIONS: The profiles of Th/Treg-related gene expression in the colonic mucosa are altered, even during clinical and endoscopic remission of UC, with a detectable Th17-predominant profile predicting future relapse. This association might represent latent immune dysregulation during disease quiescence and has the potential to be utilized to improve patient care.

Synthesis, Anti-HBV, and Anti-HIV Activities of 3'-Halogenated Bis(hydroxymethyl)-cyclopentenyladenines.

Synthesis of 3'-halogeno analogues (5a-d) of 9-[c-4,t-5-bis(hydroxymethyl)-cyclopent-2-en-r-1-yl]-9H-adenine (BCA, 3) was accomplished by means of dual utilization of the vinyl sulfone functional moieties in both 10 and 16 utilizing a SN2' conjugate-addition reaction and a sulfur-extrusive stannylation, respectively. Evaluation of the antiviral activities of 5a-d revealed that introduction of a halogeno-substituent into the 3'-position of (-)-BCA diminished its anti-HIV-1 activity but increased the inhibitory activity for the reverse transcriptase of HBV in that the 3'-fluorinated BCA 5d exhibited the highest activity without significant cytotoxicity.

A novel entecavir analogue constructing with a spiro[2.4]heptane core structure in the aglycon moiety: Its synthesis and evaluation for anti-hepatitis B virus activity.

Synthesis of a novel 2'-deoxy-guanine carbocyclic nucleoside 4 constructed with spiro[2.4]heptane core structure in the aglycon moiety was carried out. Radical-mediated 5-exo-dig mode cyclization and following cyclopropanation proceeded efficiently to furnish the spiro alcohol 10. Subsequent Mitsunobu-type glycosylation between 13 and 14, deoxygenation of the 2'-hydroxyl group of 16 and deprotection of 17 gave the title compound 4. Compound 4 demonstrated moderate anti-HBV activity (EC50 value of 0.12 ± 0.02 µM) and no cytotoxicity against HepG2 cells was observed up to 100 µM.

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.

4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus.

UNLABELLED: Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4'-C-cyano-2-amino-2'-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50 ] = 0.4 nM) and HIV-1 (IC50 = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50 = 0.7 nM), but is much less active against HIV-1 (IC50 = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA; IC50 = 0.3 nM) is less active against HBV (IC50 = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50 = 7.2 nM) and ETV-resistant HBV (IC50 = 69.6 nM for HBVETV-RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV-RL180M/S202G/M204V DNA even at 1 µM. Once-daily peroral administration of CAdA reduced HBVETV-RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV-RL180M/S202G/M204V-infected mice, whereas ETV completely failed to reduce HBVETV-RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4'-cyano of CAdA-triphosphate (TP), but not the longer 4'-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4'-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. CONCLUSION: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients.

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.

Hepcidin/ferroportin expression levels involve efficacy of pegylated-interferon plus ribavirin in hepatitis C virus-infected liver.

AIM: To investigate the relationship between the iron-metabolism-related gene expression profiles and efficacy of antiviral therapy in chronic hepatitis C patients. METHODS: The hepatic expression profile of iron-metabolism-related genes was analyzed and its association with virological response to pegylated-interferon plus ribavirin combination therapy was evaluated. A hundred patients with chronic hepatitis C (genotype1b, n = 50; genotype 2, n = 50) were enrolled and retrospectively analyzed. Liver biopsy samples were subjected to quantitative polymerase chain reaction for iron-metabolism-related genes and protein expression (Western blotting analysis) for ferroportin. As a control, normal liver tissue was obtained from 18 living donors of liver transplantation. Serum hepcidin level was measured by sensitive liquid chromatography/electrospray ionization tandem mass spectrometry. RESULTS: Iron overload is associated with liver damage by increasing oxidative stress and hepatitis C virus (HCV) is reported to induce iron accumulation in hepatocytes in vivo. Conversely, iron administration suppresses HCV replication in vitro. Therefore, the association between HCV infection and iron metabolism remains unclear. Compared with controls, patients had significantly higher gene expression for transferrin, iron-regulatory proteins 1 and 2, divalent metal transporter 1, and ferroportin, but similar for transferrin receptors 1 and 2, and hepcidin. When the expression profiles were compared between sustained virological response (SVR) and non-SVR patients, the former showed significantly lower transcription and protein expression of hepcidin and ferroportin. Expression of hepcidin-regulating genes, BMPR1, BMPR2, and hemojuvelin, was significantly increased, whereas BMP2 was decreased in HCV-infected liver. BMPR2 and hemojuvelin expression was significantly lower in the SVR than non-SVR group. HCV infection affects the expression of iron-metabolism-related genes, leading to iron accumulation in hepatocytes. CONCLUSION: Decreased expression of hepcidin and ferroportin in SVR patients indicates the importance of hepatocytic iron retention for viral response during pegylated-interferon plus ribavirin treatment.