Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection.

Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore, there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1 domain of the viral L protein to the Na+/taurocholate cotransporting polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/hepatitis D virus (HDV) infection and has been approved as Hepcludex in Europe for the treatment of patients with chronic HDV infection. We established a monoclonal antibody (MAb), N6HB426-20, that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo, administration of the N6HB426-20 MAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84 to 87 in ECL1 and aa 157 to 165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope mapping and the three-dimensional (3D) model of the NTCP structure suggested that the N6HB426-20 MAb may recognize aa 276/277 at the tip of ECL4 and interfere with binding of HBV to the region from aa 84 to 87. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleoside/nucleotide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic MAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these MAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations, and the presence of subviral particles. Although N6HB426-20 requires a higher dose than myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time postadministration in proportion to the half-life of an IgG MAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.

Current state of therapeutic development for rare cancers in Japan, and proposals for improvement.

This article discusses current obstacles to the rapid development of safe and effective treatments for rare cancers, and considers measures required to overcome these challenges. In order to develop novel clinical options for rare cancers, which tend to remain left out of novel therapeutic development because of their paucity, efficient recruitment of eligible patients, who tend to be widely dispersed across the country and treated at different centers, is necessary. For this purpose, it is important to establish rare cancer registries that are linked with clinical studies, to organize a central pathological diagnosis system and biobanks for rare cancers, and to consolidate patients with rare cancers to facilities that can conduct clinical studies meeting international standards. Establishing an all-Japan cooperative network is essential. Clinical studies of rare cancers have considerable limitations in study design and sample size as a result of paucity of eligible patients and, as a result, the level of confirmation of the efficacy and safety shown by the studies is relatively low. Therefore, measures to alleviate these weaknesses inherent to external conditions need to be explored. It is also important to reform the current research environment in order to develop world-leading treatment for rare cancers, including promotion of basic research, collaboration between industry and academia, and improvement of the infrastructure for clinical studies. Collaboration among a wide range of stakeholders is required to promote the clinical development of treatment for rare cancers under a nationwide consensus.

A pyrrolo-pyrimidine derivative targets human primary AML stem cells in vivo.

Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

FK419, a nonpeptide platelet glycoprotein IIb/IIIa antagonist, ameliorates brain infarction associated with thrombotic focal cerebral ischemia in monkeys: comparison with tissue plasminogen activator.

The binding of platelet glycoprotein (GP) IIb/IIIa to fibrinogen is the final common pathway in platelet aggregation, a process known to play a key role in the pathogenesis of ischemic brain damage. We compared the effects of FK419, a novel nonpeptide GPIIb/IIIa antagonist, with recombinant tissue plasminogen activator (rt-PA) on middle cerebral artery (MCA) patency and ischemic brain damage in a thrombotic stroke model in squirrel monkeys. FK419 not only inhibited in vitro platelet aggregation (IC50: 88 nmol/L), but also showed disaggregatory activity to aggregated platelet (EC50: 286 nmol/L). FK419 dose-dependently reduced the time to first reperfusion and total occlusion time of MCA blood flow when administered immediately after the termination of photoirradiation. FK419 reduced cerebral infarction and ameliorated neurologic deficits with similar dose-dependency. Although rt-PA reduced the time to first reperfusion, total occlusion time, and cerebral infarction, it did not significantly ameliorate neurologic deficits and induced petechial intracerebral hemorrhages. These results indicate: (1) FK419 restored cerebral blood flow after thrombotic occlusion of MCA, (2) FK419 reduced ischemic brain injury by its thrombolytic actions in a non-human primate stroke model, and (3) FK419 has superior antithrombotic efficacy and is safer than rt-PA.

Ultrafast all optical switching using pulse trapping in birefringent fibers.

An ultrafast all-optical switching using pulse trapping by orthogonally polarized soliton pulse in birefringent fiber is investigated both experimentally and numerically. The signal pulse, which is polarized along the fast axis, is trapped by the control soliton pulse along the slow axis; they subsequently copropagate along the fiber. Their wavelengths are red-shifted by the soliton self-frequency shift. The trapped pulse is shaped as a sech2 ultrashort pulse. It is also amplified by the Raman gain of the control pulse. Temporal response of the pulse trapping is estimated as 250 fs for 150 fs signal pulses. Ultrafast all-optical switching is demonstrated for the 0.84 THz pulse train.

Neuroprotective effect of tacrolimus (FK506) on ischemic brain damage following permanent focal cerebral ischemia in the rat.

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemic cell death with respect to cytochrome c translocation and DNA fragmentation, which are pivotal events in the necrotic and apoptotic signaling pathway, using permanent focal cerebral ischemia in rats. Immunohistochemically, cytochrome c was observed in the cytoplasm as early as 1 h after middle cerebral artery (MCA) occlusion in the infarcted hemisphere. Cytosolic release of cytochrome c after MCA occlusion was also confirmed by Western blot analysis and enzyme immunoassay. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) showed DNA fragmentation evolving in the ipsilateral cortex and the caudate putamen after 3 and 6 h, respectively, following MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, significantly attenuated the release of cytochrome c in the ischemic region, the number of TUNEL-positive cells in the ischemic penumbra zone, and the size of cortical ischemic lesions. This study demonstrated that tacrolimus ameliorated the accumulation of cytochrome c in the cytosol and the increase of TUNEL-positive cells induced by cerebral ischemia, indicating that the neuroprotective action of tacrolimus on ischemic brain injury caused by permanent focal cerebral ischemia could partially be attributed to the attenuation of the activation of the apoptotic execution machinery.

Flatly broadened, wideband and low noise supercontinuum generation in highly nonlinear hybrid fiber.

We present wideband of 1180-2100 nm, flatly broadened supercontinuum (SC) generation using highly nonlinear hybrid fibers and femtosecond fiber laser. Stable and smooth spectra without fine structure are demonstrated. The hybrid fibers are constructed by fusion splicing fibers with different properties. The SC spectra can be properly controlled by the optimal design of the hybrid fiber based on the numerical analysis. The generated SC pulse shows the low relative intensity noise (RIN).

Differential effects of FK506 and methotrexate on inflammatory cytokine levels in rat adjuvant-induced arthritis.

OBJECTIVE: To investigate the effects of prophylactic and therapeutic treatments with FK506 (tacrolimus), an immunosuppressive drug that specifically inhibits T cell activation, and methotrexate (MTX) on inflammatory cytokines, tumor necrosis factor (TNF)-a, interleukin (IL)-1beta, and IL-6 levels in rat adjuvant-induced arthritis (AIA). METHODS: AIA was induced in female Lewis rats. Arthritis was assessed by hindpaw swelling. TNF-a, IL-1beta, and IL-6 levels in paw extracts were determined by ELISA. To assess the effects on cytokine levels, rats were treated prophylactically with FK506 (3 mg/kg) or MTX (0.1 mg/kg) from day 1 to day 17, and therapeutically with FK506 (5 mg/kg) or MTX (1 mg/kg) from day 15 to day 17 (3-day treatment) or day 15 to 20 (6-day treatment) by oral administration. RESULTS: TNF-a, IL-1beta, and IL-6 levels in paw tissue were found to significantly increase between day 15 and day 21 after adjuvant injection, when the arthritis was in a developed stage. Prophylactic treatment with FK506 and MTX suppressed arthritis and reduced the levels of those inflammatory cytokines. FK506 caused a marked reduction of TNF-a and IL-1beta levels in paw tissue even in short-term (3-day) therapeutic treatment. It reduced all levels of TNF-a, IL-1beta, and IL-6 in paws in 6-day therapeutic treatment. In contrast, therapeutic treatment with MTX affected neither TNF-a or IL-6 levels in paws. MTX reduced IL-1beta levels only in the 6-day treatment. CONCLUSION: FK506 is more effective than MTX in reducing elevated levels of inflammatory cytokines TNF-a, IL-1beta, and IL-6 in established stages of AIA. Our findings suggest that inhibition of T cell activation results in a rapid reduction of inflammatory cytokine levels even after the arthritis is established in AIA.

Calcineurin inhibitors exert rapid reduction of inflammatory pain in rat adjuvant-induced arthritis.

1. FK506 and cyclosporin A (CsA) are immunosuppressive drugs, that specifically inhibit T-cell activation via calcineurin inhibition. This study was undertaken to investigate whether calcineurin inhibitors exert analgesic actions in rat adjuvant-induced arthritis (AIA), an animal model of rheumatoid arthritis (RA). 2. AIA was induced in female Lewis rats. Single doses of FK506 and CsA were orally administered to arthritic rats 17 days after arthritis induction. Intensity of hyperalgesia was assessed by measuring the pain threshold of hind paws. Tumor necrosis factor (TNF)-alpha, IL-1beta and PGE(2) levels in paw extracts were determined by ELISA. TNF activity was measured by L929 cell cytotoxicity assay. IL-1beta and cyclooxygenase (COX) mRNA expression in arthritic paws were measured by RT-PCR. 3. Single doses of FK506 and CsA markedly reduced joint hyperalgesia 24 h after drug administration, without affecting inflammation in an advanced stage of AIA. 4. The calcineurin inhibitors partially reduced the elevated level of TNF-alpha in arthritic paws, however, the analgesic effects of these drugs were not associated with the reduction in TNF-alpha level. 5. Moreover, treatment with anti-rat TNF-alpha antibody did not affect the hyperalgesia, when TNF-alpha activity was suppressed in arthritic paws by that treatment. 6. Both calcineurin inhibitors reduced the elevated level of IL-1beta in arthritic paws to a normal level, 24 h after drug administration. 7. FK506 reduced IL-1beta and COX-2 mRNA expression and PGE(2) level in arthritic paws. 8 In conclusion, calcineurin inhibitors rapidly reduce joint hyperalgesia probably by downregulating IL-1beta, but not TNF-alpha, in AIA. Our findings may provide a new strategy for the treatment of pain in RA.

Neuroprotective action of tacrolimus (FK506) in focal and global cerebral ischemia in rodents: dose dependency, therapeutic time window and long-term efficacy.

Tacrolimus (FK506), a potent immunosuppressive drug, is effective in attenuating brain infarction after cerebral ischemia. However, there has been no report characterizing the neuroprotective action and therapeutic time window of tacrolimus systematically using different types of stroke models and extended observation periods. Therefore, we evaluated the neuroprotective effect of tacrolimus in three different animal models of cerebral ischemia: transient and permanent focal ischemia in rats and transient global ischemia in gerbils. Tacrolimus at doses higher than 0.1 mg/kg (i.v.) produced a statistically significant reduction in ischemic brain damage following permanent and transient focal ischemia in rats when administered immediately after the onset of ischemia. Tacrolimus (1 mg/kg, i.v.) demonstrated similar neuroprotective activity even after delayed administration (2 h after permanent or 1 h after transient focal ischemia). The neuroprotective effect of tacrolimus was still present 2 weeks after transient focal ischemia and 1 week after permanent focal ischemia. After transient global ischemia in gerbils, tacrolimus (1 mg/kg, i.v.) given immediately after reperfusion also produced long-lasting neuroprotective effects with a protective time-window of 1-2 h. Taken together, the results clearly indicate that tacrolimus exerts potent, long-term neuroprotective effects with a favorable therapeutic time-window, regardless of the model of cerebral ischemia. These results strengthen the notion that tacrolimus might be of clinical value for the treatment of acute stroke.

Serum insulinlike growth factor-I in biliary atresia.

BACKGROUND/PURPOSE: Low level of Insulinlike growth factor-I (IGF-I) has been reported in children with chronic liver disease like biliary atresia (BA) awaiting liver transplantation. However, there has been no report on IGF-I in BA managed without liver transplantation. METHODS: The authors measured IGF-I and growth hormone (GH) in 21 postoperative BA, and 17 choledochal cysts (CC) as a control with normal liver function. To avoid an influence of aging, IGF-I was analyzed after converting them into a newly defined index "IGF%." IGF% is proportional to the lower limit of the value of IGF-I in gender- and age-matched normal control previously reported in literature. RESULTS: IGF% in BA was significantly lower than that in CC. IGF% tended to be lower in Kasai's type III (atresia at the porta hepatis) and higher in the jaundice-free group. IGF% in patients with esophageal varices was significantly lower. The correlation between choline esterase and IGF% was positive and that for TTT and IGF% was negative. CONCLUSIONS: Low level of IGF-I is a characteristic finding in BA, especially in patients without need of liver transplantation. And it may reflect the severity of pathologic changes (ie, hepatic fibrosis and reduced volume of normally functioning liver) in BA liver.

Ultrafast all optical switching by use of pulse trapping across zero-dispersion wavelength.

Ultrafast all optical switching by use of pulse trapping across zero dispersion wavelength in optical fiber is demonstrated both experimentally and numerically for the first time. Only an arbitrary single pulse among four pulses with temporal separation of about 1.5 ps is successfully picked off with almost perfect extinction ratio. The spectrogram of the optical switching is directly observed using the X-FROG technique. The characteristics of all optical switching are analyzed numerically by the use of strict coupled nonlinear Schrödinger equations and the numerical results are in agreement with the experimental ones. It is interesting to note that although the other pulses are also overlapped with the soliton pulse, they are not trapped.

FK960, a potential anti-dementia drug, increases synaptic density in the hippocampal CA3 region of aged rats.

There is accumulating evidence suggesting that synapse formation in the adult brain is dynamically regulated, and that this regulation plays a role in cognitive function. A decrease in synaptic density is reportedly related to memory deficits in aged animals as well as in Alzheimer's patients. FK960 [N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate], a novel anti-dementia drug, has been shown to ameliorate experimental amnesia in rats and monkeys through activation of the somatostatinergic nervous system in the hippocampus. Furthermore, FK960 has been shown to be considerably more effective in a model of spontaneous amnesia in aged rats than cholinesterase inhibitors. In the present electron microscopy study, we demonstrated that the density of axodendritic and axosomatic synapses in the hippocampal CA3 region of aged rats was reduced compared to young rats, and that repeated treatment with FK960 for either 3 or 21 days dose-dependently reversed these deficits in aged rats. This FK960-induced increase in synaptic density was transient and density returned to basal levels at 8 days after the final dose. In contrast, FK960 did not alter synaptic density in the cingulate cortex or hippocampal CA1 region in aged rats, nor the CA3 region of young rats. Collectively, these results suggest that FK960 can selectively and reversibly increase synaptic density in the hippocampal CA3 region of aged rats, and that this activity may play a role in its cognitive-enhancing action.

Effects of FK317, a novel anti-cancer agent, on survival of mice bearing B16BL6 melanoma and Lewis lung carcinoma.

The effects of FK317 (11-acetyl-8-carbamoyloxymethyl-4-formyl-6-methoxy-14- oxa-1,11-diazatraacylo[7.4.1.0(2.7).0(10.2)]-tetradeca-2,4,6-trien-9-yl acetate), a novel anti-cancer agent, and mitomycin C (MMC) on survival time of mice bearing B16BL6 melanoma and Lewis lung carcinoma (LLC), induced by intravenous inoculation of the tumor, were investigated. Treatment with FK317 resulted in a significant prolongation of survival time in both tumor models. Four of ten mice bearing B16BL6 were disease-free following FK317 treatment. In contrast, MMC was not effective in prolonging survival time. Overall, this study demonstrated that FK317 shows more potent survival extension in mice bearing B16BL6 and LLC than MMC, suggesting that FK317 may have therapeutic utility for cancer chemotherapy.

A combined treatment with tacrolimus (FK506) and recombinant tissue plasminogen activator for thrombotic focal cerebral ischemia in rats: increased neuroprotective efficacy and extended therapeutic time window.

The authors evaluated the therapeutic efficacy of tacrolimus (FK506), administered alone or in combination with recombinant tissue plasminogen activator (t-PA), on brain infarction following thrombotic middle cerebral artery (MCA) occlusion. Thrombotic occlusion of the MCA was induced by a photochemical reaction between rose bengal and green light in Sprague-Dawley rats, and the volume of ischemic brain damage was determined 24 hours later. Intravenous administration of tacrolimus or t-PA dose-dependently reduced the volume of ischemic brain infarction, whether administered immediately or 1 hour after MCA occlusion. When tacrolimus or t-PA was administered 2 hours after MCA occlusion, each drug showed a tendency to reduce ischemic brain damage. However, combined treatment with both drugs resulted in a significant reduction in ischemic brain damage. On administration 3 hours after MCA occlusion, tacrolimus alone showed no effect, and t-PA tended to worsen ischemic brain damage. However, the combined treatment with both drugs not only ameliorated the worsening trend seen with t-PA alone, but also tended to reduce ischemic brain damage. In conclusion, tacrolimus, used in combination with t-PA, augmented therapeutic efficacy on brain damage associated with focal ischemia and extended the therapeutic time window compared to single-drug treatments.

The structure of human recombinant aldose reductase complexed with the potent inhibitor zenarestat.

The crystal structure of the complex of human recombinant aldose reductase (AR) with zenarestat, one of its potent inhibitors, has been solved at 2.5 A resolution. Zenarestat fits neatly in the hydrophobic active site and induces unique and dramatic conformational changes. For example, the benzene ring of zenarestat occupies a gap in the side chains of Leu300 and Trp111 that interact directly and forms a CH-pi interaction in the native holoenzyme. As a result, the benzene ring of the inhibitor and these side chains form a CH-pi-pi interaction. Such structural information is key to understanding the mode of action of this class of inhibitors and for rational design of better therapeutics.

FK506 induces chondrogenic differentiation of clonal mouse embryonic carcinoma cells, ATDC5.

FK506 (Tacrolimus) and cyclosporin A exert their immunosuppressive effects via a common mechanism, calcineurin inhibition, after binding to intracellular proteins termed immunophilins: FK506-binding protein (FKBP) and cyclophilin. In this study, FK506 was found to induce chondrogenic differentiation of ATDC5 cells (clonal mouse embryonal carcinoma cells) in a concentration-dependent manner (0.1-1000 ng/ml). Immunohistochemical staining showed that ATDC5 cells induced to differentiate by FK506 produced proteoglycan and type II collagen, main components of the extracellular matrix of cartilage. Rapamycin, an immunosuppressant that binds to FKBP, antagonized the effect of FK506. Cyclosporin A did not induce chondrogenesis at concentrations up to 1000 ng/ml. Taken together, these results suggest that FK506 induces chondrogenic differentiation of ATDC5 cells via a calcineurin-independent mechanism, after binding to FKBP.

Differential effect of FR122047, a selective cyclo-oxygenase-1 inhibitor, in rat chronic models of arthritis.

We investigated the effects of FR122047 (1-[(4,5-bis(4-methoxyphenyl)-2-thiazoyl)carbonyl]-4-methylpiperazine hydrochloride), a selective cyclo-oxygenase (COX)-1 inhibitor, in rat type II collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AIA). Using an ex vivo rat whole blood assay, FR122047 (0.032 - 3.2 mg kg(-1)) inhibited COX-1-derived thromboxane (TX) B(2) production with ED(50) value of 0.059 mg kg(-1), indicating that it was orally active, but did not inhibit lipopolysaccharide-induced prostaglandin (PG) E(2) production derived by COX-2. Oral administration of FR122047 showed a dose-dependent anti-inflammatory effect in rat CIA with ED(50) value of 0.56 mg kg(-1). This drug also dose dependently suppressed the levels of PGE(2) and TXB(2) in CIA rat paws with ED(50) values of 0.24 and 0.13 mg kg(-1), respectively. FR122047 had no effect in rat AIA model. In contrast, indomethacin, a non-selective COX inhibitor, was anti-inflammatory and reduced the formation of PGs in AIA rat paws. Unlike indomethacin, chronic treatment of FR122047 did not damage the stomach mucosa in CIA rats. These results demonstrate that COX-1 contributes to the oedema and the formation of PGE(2) and TXB(2) in rat CIA model, but not in rat AIA model. We conclude that FR122047 has an orally active and anti-inflammatory effect mediated by inhibition of PGE(2) and TXB(2) produced by COX-1 at a site of inflammation induced by type II collagen and it may be a useful tool for studying the involvement of COX-1 in various in vivo models of inflammation.

Trapped pulse generation by femtosecond soliton pulse in birefringent optical fibers.

A novel phenomenon of trapped pulse generation by orthogonally polarized femtosecond soliton pulse is discovered in low birefringent optical fiber. As pulse propagation, wavelengths of soliton pulse and trapped pulse are shifted toward longer wavelength side due to effects of soliton selffrequency shift and pulse trapping. The energy of trapped pulse is increased exponentially through Raman gain of soliton pulse and orthogonally polarized and temporally overlapped two colored femtosecond twin pulses are generated. The spectrogram of output pulses is observed using crosscorrelation frequency resolved optical gating technique. The characteristics of this phenomenon are also analyzed numerically and numerical results are almost in agreement with experimental ones.

Characteristics of pulse trapping by ultrashort soliton pulse in optical fibers across zerodispersion wavelength.

Characteristics of pulse trapping by ultrashort soliton pulse in optical fibers across zero-dispersion wavelength are analyzed both experimentally and numerically. The spectrogram of pulse trapping is observed using the technique of cross correlation frequency resolved optical gating and the phenomenon of pulse trapping is confirmed directly. The pulse trapping is numerically analyzed using the coupled strict nonlinear Schrödinger equations and the numerical results are well in agreement with the experimental ones. It is clarified that the pulse trapping occurs due to the sequential cross phase modulation by the Raman shifted soliton pulse.