Synthesis and evaluation of novel dolastatin 10 derivatives for versatile conjugations.

Dolastatin 10 (1) is a highly potent cytotoxic microtubule inhibitor (cytotoxicity IC50 < 5.0 nM) and several of its analogs have recently been used as payloads in antibody drug conjugates. Herein, we describe the design and synthesis of a series of novel dolastatin 10 analogs useful as payloads for conjugated drugs. We explored analogs containing functional groups at the thiazole moiety at the C-terminal of dolastatin 10. The functional groups included amines, alcohols, and thiols, which are representative structures used in known conjugated drugs. These novel analogs showed excellent potency in a tumor cell proliferation assay, and thus this series of dolastatin 10 analogs is suitable as versatile payloads in conjugated drugs. Insights into the structure-activity relationships of the analogs are also discussed.

Novel pegylated interferon-ß as strong suppressor of the malignant ascites in a peritoneal metastasis model of human cancer.

Malignant ascites manifests as an end-stage event during the progression of a number of cancers and lacks a generally accepted standard therapy. Interferon-ß (IFN-ß) has been used to treat several cancer indications; however, little is known about the efficacy of IFN-ß on malignant ascites. In the present study, we report on the development of a novel, engineered form of human and murine IFN-ß, each conjugated with a polyethylene glycol molecule (PEG-hIFN-ß and PEG-mIFN-ß, respectively). We provide evidence that these IFN-ß molecules retain anti-viral potency comparable to unmodified IFN-ß in vitro and manifested improved pharmacokinetics in vivo. Interestingly, PEG-mIFN-ß significantly inhibited the accumulation of ascites fluid and vascular permeability of the peritoneal membrane in models of ovarian cancer and gastric cancer cell xenograft mice. We further show that PEG-hIFN-ß directly suppresses VEGF165 -induced hyperpermeability in a monolayer of human vascular endothelial cells and that PEG-mIFN-ß enhanced gene expression for a number of cell adhesion related molecules in mouse vascular endothelial cells. Taken together, these findings unveil a hitherto unrecognized potential of IFN-ß in maintaining vascular integrity, and provide proof-of-mechanism for a novel and long-acting pegylated hIFN-ß for the therapeutic treatment of malignant ascites.

Interferon-beta reduces the mouse liver fibrosis induced by repeated administration of concanavalin A via the direct and indirect effects.

Type I interferons (IFNs), IFN-alpha and IFN-beta, are widely used for treating chronic hepatitis C. Although retrospective studies have suggested that type I IFNs have direct antifibrotic effects, little is known about these mechanisms. The present study was designed to clarify the preventive mechanisms of type I IFNs in the progression of fibrosis for the establishment of a more effective therapy. A murine fibrosis model comprising immunological reactions was induced by the administration of concanavalin A (0.3 mg/body) into mice once a week for 4 weeks. Liver injury and the degree of fibrosis were determined by measuring the serum alanine aminotransferase activities and liver hydroxyproline contents with or without IFN-beta pretreatment. IFN-beta suppressed the hepatocellular injury and increased the hydroxyproline content induced by repeated concanavalin A injections, but had no effect on established fibrosis. Furthermore, IFN-beta reduced the expressions of transforming growth factor-beta, basic fibroblast growth factor, collagen type I A2 and tissue inhibitor of metalloproteinase 1 messenger RNAs, which are related to the progression of liver fibrosis. The IFN-beta reduced the liver injury and fibrosis induced by immunological reactions. These data suggest that type I IFNs suppress the progression of cirrhosis through inhibition of repeated hepatocellular injury and/or factors that promote the liver fibrosis induced by hepatitis virus infection.

The combination of type I interferon and ribavirin has an inhibitory effect on mouse hepatitis virus infection.

AIM: To determine the differences in efficacy between therapy using IFN-beta and ribavirin, and using IFN-alpha and ribavirin. METHODS: We studied the effect of combination therapy consisting of IFN-beta and ribavirin on mouse hepatitis virus (MHV) infection in mice. RESULTS: Combination treatment of ribavirin and IFN-alpha was more effective than IFN-alpha mono-treatment in the MHV-mouse system, and combination treatment with ribavirin and IFN-beta was more effective than IFN-beta mono-treatment in the MHV-mouse system. Furthermore, administering IFN-beta once or twice one day before combination treatment using ribavirin and IFN-alpha was more effective than administering IFN-alpha once or twice one day before the combination treatment using ribavirin and IFN-alpha. CONCLUSION: These data indicate that this MHV-infection system is a good animal model to assess anti-HCV activity for therapy using IFN and ribavirin, and suggest that administering IFN-beta before the start of combination therapy with ribavirin and IFN-alpha promotes the therapeutic effects of combination treatment with ribavirin and IFN-alpha ?in chronic hepatitis C patients.

Interferon-ß Mediates Signaling Pathways Uniquely Regulated in Hepatic Stellate Cells and Attenuates the Progression of Hepatic Fibrosis in a Dietary Mouse Model.

The results of clinical and experimental studies suggest that type I interferons (IFNs) may have direct antifibrotic activity in addition to their antiviral properties. However, the mechanisms are still unclear; in particular, little is known about the antifibrotic activity of IFN-ß and how its activity is distinct from that of IFN-α. Using DNA microarrays, we demonstrated that gene expression in TWNT-4 cells, an activated human hepatic stellate cell line, was remarkably altered by IFN-ß more than by IFN-α. Integrated pathway enrichment analyses revealed that a variety of IFN-ß-mediated signaling pathways are uniquely regulated in TWNT-4 cells, including those related to cell cycle and Toll-like receptor 4 (TLR4) signaling. To investigate the antifibrotic activity of IFN-ß and the involvement of TLR4 signaling in vivo, we used mice fed a choline-deficient l-amino acid-defined diet as a model of nonalcoholic steatohepatitis-related hepatic fibrosis. In this model, the administration of IFN-ß significantly attenuated augmentation of the area of liver fibrosis, with accompanying transcriptional downregulation of the TLR4 adaptor molecule MyD88. Our results provide important clues for understanding the mechanisms of the preferential antifibrotic activity of IFN-ß and suggest that IFN-ß itself, as well as being a modulator of its unique signaling pathway, may be a potential treatment for patients with hepatic fibrosis in a pathogenesis-independent manner.

Relevance of c-erbB2, PLU-1 and survivin mRNA expression to diagnostic assessment of breast cancer.

The positivity rates of mRNA expression in breast cancer of the tumor-related genes for c-erbB2, PLU-1 and survivin are unclear. We quantitatively analyzed tissue samples from 39 breast cancers and non-cancerous parts of the same specimens for the above three mRNAs using a TaqMan reverse transcription-polymerase chain reaction (RT-PCR). Using the mean + 2SD of non-cancerous sample as a cut-off value, the positivity rates of the tumors for c-erbB2, PLU-1 and survivin were 20.5%, 7.7% and 69.2%, respectively. Combining consideration of survivin with c-erbB2 and or PLU-1 increased the positivity ratio (survivin plus either of others, 76.9%; survivin plus both, 79.5%). Analysis by histological type indicated that survivin showed the highest positivity in ductal carcinoma and that survivin and PLU-1 showed the same positivity rate (40.0%) in the five carcinomas classified histologically as either solid-tubular or mucinous. Further, all cases that were positive for PLU-1 were negative for survivin. Survivin mRNA expression appeared more useful as a marker for diagnosis of breast cancer than c-erbB2 or PLU-1. However, PLU-1 appeared to vary independently of survivin, enhancing the usefulness of assays considering both in combination.

Survivin mRNA expression in patients with breast cancer.

We measured survivin mRNA expression in breast cancers using a fluorogenically detected reverse transcription-polymerase chain reaction, seeking relationships between expression and clinical and histological variables. The mean relative expression of survivin mRNA was 0.347+/-0.466 in breast cancers and 0.017+/-0.023 in noncancerous breast tissue resected with the tumors. A cut-off value for positivity was set at the mean + SD for expression in the noncancerous tissues; positivity for survivin mRNA expression was 90.2% (37 out of 41 cases) in breast cancers and 23% (3 out of 13) in adjacent noncancerous breast tissues. Expression in tumors varied independently of clinicopathological factors except for lymphatic invasion. As measured by our sensitive detection system, survivin mRNA is a useful diagnostic marker for breast cancer.

RECOT: a tool for the coordinate transformation of next-generation sequencing reads for comparative genomics and transcriptomics.

BACKGROUND: The whole-genome sequences of many non-model organisms have recently been determined. Using these genome sequences, next-generation sequencing based experiments such as RNA-seq and ChIP-seq have been performed and comparisons of the experiments between related species have provided new knowledge about evolution and biological processes. Although these comparisons require transformation of the genome coordinates of the reads between the species, current software tools are not suitable to convert the massive numbers of reads to the corresponding coordinates of other species' genomes. RESULTS: Here, we introduce a set of programs, called REad COordinate Transformer (RECOT), created to transform the coordinates of short reads obtained from the genome of a query species being studied to that of a comparison target species after aligning the query and target gene/genome sequences. RECOT generates output in SAM format that can be viewed using recent genome browsers capable of displaying next-generation sequencing data. CONCLUSIONS: We demonstrate the usefulness of RECOT in comparing ChIP-seq results between two closely-related fruit flies. The results indicate position changes of a transcription factor binding site caused sequence polymorphisms at the binding site.

Interferon-{beta} inhibits bleomycin-induced lung fibrosis by decreasing transforming growth factor-{beta} and thrombospondin.

Pulmonary fibrosis is the result of abnormal processes of repair that occur after lung injury. Transforming growth factor (TGF)-beta is a key molecule in the progression of pulmonary fibrosis. Although clinical use of interferon (IFN)-beta did not improve survival in patients with idiopathic pulmonary fibrosis, because some preclinical studies have suggested that IFN-beta is a potent inhibitor of fibrogenesis, beneficial effects of IFN-beta have been expected. We therefore attempted to determine effects of IFN-beta and investigated the mechanism of action of IFN-beta in bleomycin-induced pulmonary fibrosis. Bleomycin at Day 0 and IFN-beta for 4 wk were administered intravenously to ICR mice. At 28 d after bleomycin injection, histologic and chemical analysis was performed for evaluation of effects of IFN-beta. Tissue distribution and amounts of TGF-beta1 and thrombospondin (TSP)-1/2 were analyzed. IFN-beta attenuated prolylhydroxylase activity, resulting in inhibition of pulmonary fibrosis. Bleomycin-induced increase in TGF-beta1 in epithelial cells and extracellular matrix was attenuated by IFN-beta. TSP-1/2 was limited in platelets of control mice, but was present in foamy cells in fibrotic regions induced by bleomycin. These findings suggest that the antifibrotic effect of IFN-beta is inhibition of TGF-beta and its activation via decrease in TSP-1/2 in lung tissue and change in location of TSP-1/2 from platelets to foamy cells.