Recombinant VWF fragments improve bioavailability of subcutaneous factor VIII in hemophilia A mice.

Conventional treatment of hemophilia A (HA) requires repetitive IV injection of coagulation factor VIII (FVIII). Subcutaneous administration of FVIII is inefficient because of binding to the extravascular matrix, in particular to phospholipids (PLs), and subsequent proteolysis. To overcome this, recombinant dimeric fragments of von Willebrand factor (VWF) containing the FVIII-stabilizing D3 domain were engineered. Two fragments, called VWF-12 and VWF-13, demonstrated high binding affinity to recombinant human FVIII (rhFVIII) and suppressed PL binding in a dose-dependent manner. High concentrations of VWF fragments did not interfere with the functional properties of full-length VWF in vitro. The HA mouse model was used to study the effects of VWF-12 or VWF-13 on the in vivo pharmacokinetics of rhFVIII, demonstrating (1) no significant impact on rhFVIII recovery or half-life after a single IV administration; (2) enhanced bioavailability (up to 18.5%) of rhFVIII after subcutaneous administration; and (3) slow absorption (peak concentration, 6 hours) and prolonged half-life (up to 2.5-fold) of rhFVIII after subcutaneous administration. Formation of anti-FVIII antibodies was not increased after administration of rhFVIII/VWF-12 subcutaneously compared with rhFVIII IV. A single subcutaneous dose of rhFVIII/VWF-12 provided protection in the HA tail-bleeding model for up to 24 hours. In summary, recombinant VWF fragments support FVIII delivery through the subcutaneous space into vascular circulation without interfering with VWF or FVIII function. Slow resorption and excretion of FVIII after subcutaneous administration highlight the potential application of VWF fragments for subcutaneous FVIII prophylaxis in HA.

1-Hydroxyanthraquinones Containing Aryl Substituents as Potent and Selective Anticancer Agents.

A series of 1,2-, 1,4-disubstituted or 1,2,4-trisubstituted anthraquinone-based compounds was designed, synthesized, characterized and biologically evaluated for anticancer efficacy. 2- or 4-arylated 1-hydroxy-9,10-antraquinones (anthracene-9,10-diones) were prepared by Suzuki-Miyaura cross-coupling reaction of 1-hydroxy-2-bromoanthraquinone, 1-hydroxy-4-iodoanthraquinone or 1-hydroxy-2,4-dibromoanthraquinone with arylboronic acids. The cross-coupling reaction of 2,4-dibromo-9,10-anthraquinone with arylboronic acids provide a convenient approach to 2,4-bis arylated 1-hydroxyanthraquinones with a variety of aryl substituent in the 2 and 4 position. The cytotoxicity of new anthraquinone derivatives was evaluated using the conventional MTT assays. The data revealed that six of the aryl substituted compounds among the entire series 3, 15, 16, 25, 27, 28 were comparable potent with the commercially available reference drug doxorubicin on the human glioblastoma cells SNB-19, prostate cancer DU-145 or breast cancer cells MDA-MB-231 and were relatively safe towards human telomerase (h-TERT)immortalized lung fibroblasts cells. The results suggested that the in vitro antitumor activity of synthesized 2-aryl, 4-aryl- and 2,4-diaryl substituted 1-hydroxyanthraquinones depends on the nature of the substituent within the cyclic backbone. Docking interaction of 2-, 4-substituted and 2,4-disubstituted 1-hydroxyanthraquinones indicates intercalative mode of binding of compounds with DNA topoisomerase. The interaction with the DNA of 4-aryl-13, 15, 16 and 4-(furan-3-yl)-23 1-hydroxyanthraquinones was experimentally confirmed through a change in electroforetic mobility. Further experiments with 1-hydroxy-4-phenyl-anthraquinone 13 demonstrated that the compound induced cell cycle arrest at sub-G1 phase in DU-145 cells in the concentration 1.1 µM, which is probably achieved by inducing apoptosis. 4-Arylsubstituted 1-hydroxyanthraquinones 13 and 16 induced the enhancement of DNA synthesis on SNB19 cell lines.

Dehydroabietylamine Ureas and Thioureas as Tyrosyl-DNA Phosphodiesterase 1 Inhibitors That Enhance the Antitumor Effect of Temozolomide on Glioblastoma Cells.

A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors was found among resin acid derivatives. Several novel ureas and thioureas derived from dehydroabietylamine were synthesized and tested for TDP1 inhibition. The synthesized compounds showed IC50 values in the range of 0.1 to 3.7 µM and demonstrated low cytotoxicity against the human tumor cell lines U-937, U-87MG, MDA-MB, SK-Mel8, A-549, MCF7, T98G, and SNB19. Several compounds showed enhancement of the cytotoxic activity of the alkylating agent temozolomide, which is used as a first line therapy against glioblastoma (GBM), in the GBM cell lines U-87MG and SNB19.

von Willebrand factor modulates immune complexes and the recall response against factor VIII in a murine hemophilia A model.

Achieving tolerance toward factor VIII (FVIII) remains an important goal of hemophilia treatment. Up to 40% of patients with severe hemophilia A (HA) develop neutralizing antibodies against FVIII, and the only proven treatment to achieve tolerance is infusion of FVIII over prolonged periods in the context of immune tolerance induction. Here, we addressed the role of von Willebrand factor (VWF) as a modulator of anti-FVIII antibody effector functions and the FVIII-specific recall response in an HA mouse model. Analytical ultracentrifugation was used to demonstrate formation of FVIII-containing immune complexes (FVIII-ICs). VWF did not fully prevent FVIII-IC formation but was rather incorporated into larger macromolecular complexes. VWF prevented binding of FVIII-ICs to complement C1q, most efficiently when it was preincubated with FVIII before the addition of antibodies. It also prevented binding to immobilized Fc-γ receptor and to bone marrow-derived dendritic cells. An in vitro model of the anti-FVIII recall response demonstrated that addition of VWF to FVIII abolished the proliferation of FVIII-specific antibody-secreting cells. After adoptive transfer of sensitized splenocytes into immunocompetent HA mice, the FVIII recall response was diminished by VWF. In summary, these data indicate that VWF modulates the formation and effector functions of FVIII-ICs and attenuates the secondary immune response to FVIII in HA mice.

Targets of autoantibodies in acquired hemophilia A are not restricted to factor VIII: data from the GTH-AH 01/2010 study.

The root cause of autoantibody formation against factor VIII (FVIII) in acquired hemophilia A (AHA) remains unclear. We aimed to assess whether AHA is exclusively associated with autoantibodies toward FVIII or whether patients also produce increased levels of autoantibodies against other targets. A case-control study was performed enrolling patients with AHA and age-matched controls. Human epithelial cell (HEp-2) immunofluorescence was applied to screen for antinuclear (ANA) and anticytoplasmic autoantibodies. Screening for autoantibodies against extractable nuclear antigens was performed by enzyme immunoassay detecting SS-A/Ro, SS-B/La, U1RNP, Scl-70, Jo-1, centromere B, Sm, double-stranded DNA, and α-fodrin (AF). Patients with AHA were more often positive for ANA than control patients (64% vs 30%; odds ratio [OR] 4.02, 1.98-8.18) and had higher ANA titers detected than controls. Cytoplasmic autoantibodies and anti-AF immunoglobulin A autoantibodies were also more frequent in patients with AHA compared with controls. Autoantibodies against any target other than FVIII were found in 78% of patients with AHA compared with 46% of controls (OR 4.16, 1.98-8.39). Results were similar preforming sensitivity analyses (excluding either subjects with autoimmune disorders, cancer, pregnancy, or immunosuppressive medication at baseline) and in multivariable binary logistic regression. To exclude that autoantibody staining was merely a result of cross-reactivity of anti-FVIII autoantibodies, we tested a mix of 7 well-characterized monoclonal anti-FVIII antibodies. These antibodies did not stain HEp-2 cells used for ANA detection. In conclusion, a diverse pattern of autoantibodies is associated with AHA, suggesting that a more general breakdown of immune tolerance might be involved in its pathology.

6-(4'-Aryl-1',2',3'-triazolyl)-spirostan-3,5-diols and 6-(4'-Aryl-1',2',3'-triazolyl)-7-hydroxyspirosta-1,4-dien-3-ones: Synthesis and analysis of their cytotoxicity.

In an endeavour to develop potent anti-tumor agents from diosgenin, a series of C-6 derived 1,2,3-triazolyl derivatives were designed and synthesized by employing Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition reaction of novel azides - (22R,25R)-6ß-azidospirostan-3ß,5α-diol and 6ß-azido-7α-hydroxyspirosta-1,4-dien-3-one with aryl(hetaryl)alkynes. All the derivatives were evaluated for cytotoxic activity by MTT assay against eight different human cancer cell lines: T-cellular leucosis (CEM-13), human monocytes (U-937), breast (MDA-MB-231, BT-474), prostate (DU-145) and glioblastoma (U-87MG, SNB-19, T98G). The results of this study suggested that 6-(4'-aryl-1',2',3'-triazolyl)spirostan-3,5-diols 2, 3, 4, 5 and 6 possessed a promising cytotoxic potential. The corresponding 6-substituted 7-hydroxy-1,4-spirostadien-3-ones shown less cytotoxity on the human cancer cells. Compounds 2, 3, 4, and 5 which demonstrated high grown inhibition against glioma cancer cells U-87 and T98G, and also on the human-derived N118669 primary glioblastoma cell line (with GI50 values in the range of 5-9 µM), were not affected the growth of SNB-19 cells. The data revealed that phenyl, 4-methoxyphenyl, 4-fluorophenyl, 3,4,5-trimethoxyphenyl or 2-pyridinyl substituent in the triazole moiety at the C-6 position significantly improved the anti-tumor activity. The mentioned position at the spirostan core may be favourable for the synthesis of potent anticancer leads from diosgenin.