Inhibitory effect of fidaxomicin on biofilm formation in Clostridioides difficile.

Clostridioides difficile infection results from a disturbance of the normal microbial flora of the colon, allowing proliferation of C. difficile and toxin production by toxigenic strains. Fidaxomicin, a macrocyclic antibiotic that prevents RNA synthesis in C. difficile and inhibits spore formation, toxin production, and cell proliferation, is clinically effective in treating C. difficile infection. As recent studies have suggested that biofilm formation influences C. difficile colonization and infection in the colon, we undertook the present study to determine the effects of fidaxomicin on C. difficile biofilm formation. Sub-minimum inhibitory concentrations (MICs) of fidaxomicin inhibited biofilm formation by C. difficile UK027 and delayed planktonic growth. Sub-MICs of vancomycin did not inhibit biofilm formation or affect planktonic growth. In C. difficile UK027 exposed to sub-MICs of fidaxomicin, mRNA expression of biofilm-related flagellin gene fliC was slightly increased compared with that of other biofilm-related genes (pilA1, cwp84, luxS, dccA, and spo0A). In conclusion, this study indicates that sub-MICs of fidaxomicin inhibit C. difficile UK027 biofilm formation by influencing cell growth and fliC transcription.

The gut microbiome diversity of Clostridioides difficile-inoculated mice treated with vancomycin and fidaxomicin.

OBJECTIVE: To investigate the effect of vancomycin and fidaxomicin on the diversity of intestinal microbiota in a mouse model of Clostridioides difficile infection. METHODS: Mice were divided into 11 models (4 mice per model): 6 uninoculated models and 5 models inoculated with C. difficile BI/NAP1/027. Inoculated models were prepared using intraperitoneal clindamycin followed by inoculation with C. difficile BI/NAP1/027. Uninoculated and C. difficile-inoculated mice received 2 or 7 days' vancomycin or fidaxomicin. Clostridium butyricum MIYAIRI 588 probiotic and lactoferrin prebiotic were administered for 10 days to uninoculated mice. Intestinal microbiome composition was investigated by sequence analyses of bacterial 16S rRNA genes from faeces, and microbiota diversity estimated. RESULTS: In uninoculated, untreated ('normal') mice, Clostridia (57.8%) and Bacteroidia (32.4%) accounted for the largest proportions of gut microbiota. The proportion of Clostridia was numerically reduced in C. difficile-inoculated versus normal mice. Administration of vancomycin to C. difficile-inoculated mice reduced the proportions of Bacteroidia and Clostridia, and increased that of Proteobacteria. Administration of fidaxomicin to C. difficile-inoculated mice reduced the proportion of Clostridia to a lesser extent, but increased that of Bacteroidia. Microbiota diversity was lower in C. difficile-inoculated versus normal mice (164.5 versus 349.1 operational taxonomic units (OTUs), respectively); treatment of C. difficile-inoculated mice with 7 days' vancomycin reduced diversity to a greater extent than did 7 days' fidaxomicin treatment (26.2 versus 134.2 OTUs, respectively). CONCLUSIONS: Both C. difficile inoculation and treatment with vancomycin or fidaxomicin reduced microbiota diversity; however, dysbiosis associated with fidaxomicin was milder than with vancomycin.

Antiviral efficacy of the helicase-primase inhibitor amenamevir in murine models of severe herpesvirus infection.

Existing treatments have limited efficacy against severe infection associated with herpes simplex virus (HSV) and herpes zoster virus (VZV), particularly in immunocompromized patients and those with multidermatomal infection. This issue, along with issues regarding drug resistance, support the need for improved therapeutic options. To investigate the antiviral effect of amenamevir, a VZV and HSV helicase-primase inhibitor, in severe infection conditions, mouse models of severe HSV-1 infection were developed by immunosuppression or multidermatomal infection. Mice with cyclosporin-induced immunosuppression and HSV-1 infection via inoculation of a dorsolateral area of skin were orally treated with amenamevir (10-100 mg/kg/day) for different durations (2-5 days). Immunosuppressed mice maintained high skin HSV-1 titers in the absence of treatment. Amenamevir successfully reduced HSV-1 titers at all tested doses in immunosuppressed mice, but required a longer treatment period to avoid a rebound in viral titers due to immunosuppression. To compare the efficacy of amenamevir and valacyclovir, a murine model of multidermatomal HSV-1 infection was generated by scarifying the dorsolateral area of skin in a line and inoculating broadly with HSV-1. The mice were treated with amenamevir or valacyclovir starting on Day 3, 4, or 5 post-infection for 5 days. Although both drugs similarly reduced disease scores when treatment was started on Day 3, amenamevir also reduced disease severity when treatment was initiated on Day 4, whereas valacyclovir did not. Amenamevir was not affected by the host's immune status in terms of effective oral doses and was more efficacious in treating severe cutaneous infection even when treatment initiation was delayed.

Integrative pharmacokinetic-pharmacodynamic modeling and simulation of amenamevir (ASP2151) for treatment of recurrent genital herpes.

Amenamevir is a novel drug that targets the viral helicase-primase complex. While dose-dependent efficacy had been observed in non-clinical studies, no clear dose dependence has been observed in humans. We therefore developed a pharmacokinetic/pharmacodynamic (PK/PD) model to explain this inconsistency between species and to clarify the immune-related healing of amenamevir in humans. The model consisted of a non-linear kinetic model for a virtual number of virus plaques as a built-in biomarker. Lesion score was defined as an endpoint of antiviral efficacy, and logit model analysis was applied to the ordered-categorical lesion score. The modeling results suggested the time course profiles of lesion score could be explained with the efficacy terms in the logit model, using change in number of virus plaques as an indicator of the effects of amenamevir and time elapsed as an indicator of the healing of the immune response. In humans, the PD effect was almost dose-independent, and immune-related healing may have been the driving force behind the reduction in lesion scores. Drug efficacy is occasionally masked in diseases healed by the immune response, such as genital herpes. The PK/PD model proposed in the present study must be useful for explanation the PK/PD relationship of such drugs.

Pharmacokinetics and pharmacodynamics of ASP2151, a helicase-primase inhibitor, in a murine model of herpes simplex virus infection.

ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus 1 (HSV-1), HSV-2, and varicella zoster virus. Here, to determine and analyze the correlation between the pharmacodynamic (PD) and pharmacokinetic (PK) parameters of ASP2151, we examined the PD profile of ASP2151 using in vitro plaque reduction assay and a murine model of HSV-1 infection. ASP2151 inhibited the in vitro replication of HSV-1 with a mean 50% effective concentration (EC(50)) of 14 ng/ml. In the cutaneously HSV-1-infected mouse model, ASP2151 dose dependently suppressed intradermal HSV-1 growth, with the effect reaching a plateau at a dose of 30 mg/kg of body weight/day. The dose fractionation study showed that intradermal HSV-1 titers were below the detection limit in mice treated with ASP2151 at 100 mg/kg/day divided into two daily doses and at 30 or 100 mg/kg/day divided into three daily doses. The intradermal HSV-1 titer correlated with the maximum concentration of drug in serum (C(max)), the area under the concentration-time curve over 24 h (AUC(24h)), and the time during which the concentration of ASP2151 in plasma was above 100 ng/ml (T(>100)). The continuous infusion of ASP2151 effectively decreased intradermal HSV-1 titers below the limit of detection in mice in which the ASP2151 concentration in plasma reached 79 to 145 ng/ml. Our findings suggest that the antiviral efficacy of ASP2151 is most closely associated with the PK parameter T(>100) in HSV-1-infected mice. Based on these results, we propose that a plasma ASP2151 concentration exceeding 100 ng/ml for 21 to 24 h per day provides the maximum efficacy in HSV-1-infected mice.

Synergistic activity of amenamevir (ASP2151) with nucleoside analogs against herpes simplex virus types 1 and 2 and varicella-zoster virus.

ASP2151 (amenamevir) is a helicase-primase complex inhibitor with antiviral activity against herpes simplex virus HSV-1, HSV-2, and varicella-zoster virus (VZV). To assess combination therapy of ASP2151 with existing antiherpes agents against HSV-1, HSV-2, and VZV, we conducted in vitro and in vivo studies of two-drug combinations. The combination activity effect of ASP2151 with nucleoside analogs acyclovir (ACV), penciclovir (PCV), or vidarabine (VDB) was tested via plaque-reduction assay and MTS assay, and the data were analyzed using isobolograms and response surface modeling. In vivo combination therapy of ASP2151 with valaciclovir (VACV) was studied in an HSV-1-infected zosteriform spread mouse model. The antiviral activity of ASP2151 combined with ACV and PCV against ACV-susceptible HSV-1, HSV-2, and VZV showed a statistically significant synergistic effect (P<0.05). ASP2151 with VDB was observed to have additive effects against ACV-susceptible HSV-2 and synergistic effects against VZV. In the mouse model of zosteriform spread, the inhibition of disease progression via combination therapy was more potent than that of either drugs as monotherapy (P<0.05). These results indicate that the combination therapies of ASP2151 with ACV and PCV have synergistic antiherpes effects against HSV and VZV infections and may be feasible in case of severe disease, such as herpes encephalitis or in patients with immunosuppression.

Characterization of virus strains resistant to the herpes virus helicase-primase inhibitor ASP2151 (Amenamevir).

ASP2151 is an antiherpes agent targeting the helicase-primase complex of herpes simplex virus (HSV)-1, HSV-2, and varicella-zoster virus (VZV). We characterized the ASP2151-resistant HSV-1 and HSV-2 variants or mutants based on findings from sequencing analysis, growth, pathogenicity, and susceptibility testing, identifying several single base-pair substitutions resulting in amino acid changes in the helicase and primase subunit of ASP2151-resistant mutants. Amino acid alterations in the helicase subunit were clustered near helicase motif IV in the UL5 helicase gene of both HSV-1 and HSV-2, while the primase subunit substitution associated with reduced susceptibility, R367H, was found in ASP2151-resistant HSV-1 mutants. However, while susceptibility in the ASP2151-resistant HSV mutants to existing antiherpes agents was equivalent to that in wild-type HSV strains, ASP2151-resistant HSV mutants showed attenuated in vitro growth capability and in vivo pathogenicity compared with the parent strains. Taken together, our present findings demonstrated that important amino acid substitutions associated with reduced susceptibilities of HSV-1 and HSV-2 to ASP2151 exist in both the helicase and primase subunits of the helicase-primase complex, and that mutations in this complex against ASP2151 might confer defects in viral replication and pathogenicity.

Susceptibility of herpes simplex virus isolated from genital herpes lesions to ASP2151, a novel helicase-primase inhibitor.

ASP2151 (amenamevir) is a helicase-primase inhibitor against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus. To evaluate the anti-HSV activity of ASP2151, susceptibility testing was performed on viruses isolated from patients participating in a placebo- and valacyclovir-controlled proof-of-concept phase II study for recurrent genital herpes. A total of 156 HSV strains were isolated prior to the dosing of patients, and no preexisting variants with less susceptibility to ASP2151 or acyclovir (ACV) were detected. ASP2151 inhibited HSV-1 and HSV-2 replication with mean 50% effective concentrations (EC(50)s) of 0.043 and 0.069 µM, whereas ACV exhibited mean EC(50)s of 2.1 and 3.2 µM, respectively. Notably, the susceptibilities of HSV isolates to ASP2151 and ACV were not altered after dosing with the antiviral agents. Taken together, these results demonstrate that ASP2151 inhibits the replication of HSV clinical isolates more potently than ACV, and HSV resistant to this novel helicase-primase inhibitor as well as ACV may not easily emerge in short-term treatment for recurrent genital herpes patients.

Efficacy of ASP2151, a helicase-primase inhibitor, against thymidine kinase-deficient herpes simplex virus type 2 infection in vitro and in vivo.

ASP2151 was developed as a novel inhibitor of herpes simplex virus (HSV) and varicella-zoster virus helicase-primase. The anti-HSV activity of ASP2151 toward a clinical HSV isolate with acyclovir (ACV)-resistant/thymidine kinase (TK)-deficiency was characterized in vitro and in vivo using a plaque reduction assay and the ear pinna infection in mice. The IC(50) ranged from 0.018 to 0.024 µg/ml, indicating the susceptibility of TK-deficient HSV-2 was similar to that of wild-type HSV-2 strains. Anti-HSV activity of ASP2151 in vivo was evaluated in mice infected with wild-type HSV-2 and TK-deficient HSV-2. ASP2151 significantly reduced the copy numbers of wild-type HSV-2 and TK-deficient HSV-2 at the inoculation ear pinna, while valacyclovir significantly reduced the copy number of wild type HSV-2 but not that of TK-deficient HSV-2 in the inoculated ear pinna. Thus, ASP 2151 showed therapeutic efficacy in mice infected with both wild-type and TK-deficient HSV-2. In conclusion, ASP2151 is a promising novel herpes helicase-primase inhibitor that indicates the feasibility of ASP2151 for clinical application for the treatment of HSV infections, including ACV-resistant/TK-deficient HSV infection.

Effect of ASP2151, a herpesvirus helicase-primase inhibitor, in a guinea pig model of genital herpes.

ASP2151 is a herpesvirus helicase-primase inhibitor with antiviral activity against varicella zoster virus and herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Here, we examined the potency and efficacy of ASP2151 against HSV in vitro and in vivo. We found that ASP2151 was more potent in inhibiting the replication of HSV-1 and HSV-2 in Vero cells in the plaque reduction assay and had greater anti-HSV activity in a guinea pig model of genital herpes than did acyclovir and valacyclovir (VACV), respectively. Oral ASP2151 given from the day of infection reduced peak and overall disease scores in a dose-dependent manner, resulting in complete prevention of symptoms at the dose of 30 mg/kg. The 50% effective dose (ED(50)) values for ASP2151 and VACV were 0.37 and 68 mg/kg, respectively, indicating that ASP2151 was 184-fold more potent than VACV. When ASP2151 was administered after the onset of symptoms, the disease course of genital herpes was suppressed more effectively than by VACV, with a significant reduction in disease score observed one day after starting ASP2151 at 30 mg/kg, whereas the therapeutic effect of VACV was only evident three days after treatment at the highest dose tested (300 mg/kg). This indicated that ASP2151 possesses a faster onset of action and wider therapeutic time window than VACV. Further, virus shedding from the genital mucosa was significantly reduced with ASP2151 at 10 and 30 mg/kg but not with VACV, even at 300 mg/kg. Taken together, our present findings demonstrated the superior potency and efficacy of ASP2151 against HSV.

ASP2151, a novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes simplex virus types 1 and 2.

OBJECTIVES: To evaluate and describe the anti-herpesvirus effect of ASP2151, amenamevir, a novel non-nucleoside oxadiazolylphenyl-containing herpesvirus helicase-primase complex inhibitor. METHODS: The inhibitory effect of ASP2151 on enzymatic activities associated with a recombinant HSV-1 helicase-primase complex was assessed. To investigate the effect on viral DNA replication, we analysed viral DNA in cells infected with herpesviruses [herpes simplex virus (HSV), varicella-zoster virus (VZV) and human cytomegalovirus]. Sequencing analyses were conducted on an ASP2151-resistant VZV mutant. In vitro and in vivo antiviral activities were evaluated using a plaque reduction assay and an HSV-1-infected zosteriform-spread model in mice. RESULTS: ASP2151 inhibited the single-stranded DNA-dependent ATPase, helicase and primase activities associated with the HSV-1 helicase-primase complex. Antiviral assays revealed that ASP2151, unlike other known HSV helicase-primase inhibitors, exerts equipotent activity against VZV, HSV-1 and HSV-2 through prevention of viral DNA replication. Further, the anti-VZV activity of ASP2151 (EC(50), 0.038-0.10 microM) was more potent against all strains tested than that of aciclovir (EC(50), 1.3-27 microM). ASP2151 was also active against aciclovir-resistant VZV. Amino acid substitutions were found in helicase and primase subunits of ASP2151-resistant VZV. In a mouse zosteriform-spread model, ASP2151 was orally active and inhibited disease progression more potently than valaciclovir. CONCLUSIONS: ASP2151 is a novel herpes helicase-primase inhibitor that warrants further investigation for the potential treatment of both VZV and HSV infections.

Mucosal acid causes gastric mucosal microcirculatory disturbance in nonsteroidal anti-inflammatory drug-treated rats.

The mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) suppress gastric mucosal blood flow is not fully understood, although the depletion of mucosal prostaglandin E2 has been proposed as one possible explanation. We investigated the role of gastric acid on gastric mucosal blood flow in NSAID-treated rats. A rat stomach was mounted in an ex vivo chamber, and gastric mucosal blood flow was measured sequentially in a 5-mm2 area of the gastric corpus using a scanning laser Doppler perfusion image system. Results showed that diclofenac (5 mg/kg s.c.) and indomethacin (10 mg/kg s.c.) did not affect gastric mucosal blood flow, although both strongly decreased mucosal prostaglandin E2 when saline was instilled into the gastric chamber. On replacement of the saline in the chamber with 100 mM hydrochloric acid, these drugs caused a decrease in gastric mucosal blood flow levels within 30 min. The specific cyclooxygenase (COX)-2 inhibitors celecoxib (50 mg/kg s.c.) and rofecoxib (25 mg/kg s.c.) did not affect mucosal prostaglandin E2 level, nor did they decrease gastric mucosal blood flow, even when hydrochloric acid was added to the chamber. Furthermore, measurement of vasoconstrictive factors present in the mucosa showed that endothelin-1 levels increased after administration of diclofenac s.c. in the presence of intragastric hydrochloric acid. This indicates that the presence of mucosal hydrochloric acid plays an important role in the NSAID-induced decrease in gastric mucosal blood flow, while the COX-1-derived basal prostaglandin E2, which is unlikely to control gastric mucosal blood flow itself, protects microcirculatory systems from mucosal hydrochloric acid.

A cell model study of calcium influx mechanism regulated by calcium-dependent potassium channels in Purkinje cell dendrites.

The present study was designed to elucidate the roles of dendritic voltage-gated K+ channels in Ca2+ influx mechanism of a rat Purkinje cell using a computer simulation program. First, we improved the channel descriptions and the maximum conductance in the Purkinje cell model to mimic both the kinetics of ion channels and the Ca2+ spikes, which had failed in previous studies. Our cell model is, therefore, much more authentic than those in previous studies. Second, synaptic inputs that mimic stimulation of parallel fibers and induce sub-threshold excitability were simultaneously applied to the spiny dendrites. As a result, transient Ca2+ responses were observed in the stimulation points and they decreased with the faster decay rate in the cell model including high-threshold Ca2+-dependent K+ channels than in those excluding these channels. Third, when a single synaptic input was applied into a spiny dendrite, Ca2+-dependent K+ channels suppressed Ca2+ increases at stimulation and recording points. Finally, Ca2+-dependent K+ channels were also found to suppress the time to peak Ca2+ values in the recording points. These results suggest that the opening of Ca2+-dependent K+ channels by Ca2+ influx through voltage-gated Ca2+ channels hyperpolarizes the membrane potentials and deactivates these Ca2+ channels in a negative feedback manner, resulting in local, weak Ca2+ responses in spiny dendrites of Purkinje cells.

Non-ocular dermal photoreception in the pond snail Lymnaea stagnalis.

Based on the results of previous behavioral experiments, researchers believe that sensitivity to light stimuli is not restricted to the eyes in the pond snail Lymnaea stagnalis. To determine the presence of a non-ocular dermal photoreception system and to examine the synaptic connections between this peripheral system and the central nervous system, we electrophysiologically examined the activities of the pedal nerves in L. stagnalis by light stimulation. The results demonstrated that light stimulation evokes non-ocular dermal photosensitive responses in the foot, that these responses exert inhibitory, afferent influences through the inferior pedal nerves to the pedal ganglia, and that these responses were independent of the ocular photoreception system in L. stagnalis.