Pharmacokinetics and pharmacodynamics of an oral formulation of decitabine and tetrahydrouridine.

BACKGROUND: Sickle cell disease (SCD) is caused by an inherited structural abnormality of adult hemoglobin causing polymerization. Fetal hemoglobin interferes with polymerization but is epigenetically silenced by DNA methyltransferase 1 (DNMT1) in adult erythropoiesis. Decitabine depletes DNMT1 and increases fetal and total hemoglobin in SCD patients, but is rapidly catabolized by cytidine deaminase (CDA) in vivo. Tetrahydrouridine (THU) inhibits CDA, safeguarding decitabine. METHODS: The pharmacokinetics and pharmacodynamics of three oral combination formulations of THU and decitabine, with different coatings producing different delays in decitabine release, were investigated in healthy participants. RESULTS: Tetrahydrouridine and decitabine were rapidly absorbed into the systemic circulation after a single combination oral dose, with relative bioavailability of decitabine ≥74% in fasted males compared with separate oral administration of THU followed by decitabine 1 h later. THU and decitabine Cmax and area under the plasma concentration versus time curve were higher in females versus males, and fasted versus fed states. Despite sex and food effect on pharmacokinetics, the pharmacodynamic effect of DNMT1 downregulation was comparable in males and females and fasted and fed states. Treatments were well tolerated. CONCLUSION: Combination oral formulations of THU with decitabine produced pharmacokinetics and pharmacodynamics suitable for oral DNMT1-targeted therapy.

Cs5Sn9(OH)·4NH3.

The title compound, penta-caesium nona-stannide hydroxide tetra-ammonia, crystallized from a solution of CsSnBi in liquid ammonia. The Sn9 (4-) unit forms a monocapped quadratic anti-prism. The hydroxide ion is surrounded by five caesium cations, which form a distorted quadratic pyramidal polyhedron. A three-dimensional network is formed by Cs-Sn [3.8881 (7) Što 4.5284 (7) Å] and Cs-NH3 [3.276 (7)-3.636 (7) Å] contacts.

(18-Crown-6)potassium(I) di-phenyl-stibate(-1).

Red crystals of the title salt, [K(C12H24O6)][Sb(C6H5)2], were obtained by the reaction of SbPh3, KSnBi and 18-crown-6 in liquid ammonia. The asymmetric unit contains one half of a [K(18-crown-6)](+) cation and one half of an SbPh2 (-) anion, with the central element lying on a twofold axis and a centre of inversion, respectively. In the crystal structure, the sequestered potassium cations show weak inter-actions with the π-electrons of the phenyl groups of the SbPh2 (-) anion [shortest K⋯C distances = 3.190 (2) and 3.441 (2) Å], leading to one-dimensional strands along the crystallographic c axis. These strands are aligned in a pseudo-hexa-gonal packing perpendicular to the ab plane.

Evidence of solubility of the acetylide ion C2(2-): syntheses and crystal structures of K2C2⋠2 NH3, Rb2C2⋠2 NH3, and Cs2C2⋠7 NH3.

Carbon anions in solution: C(2)(2-) dumbbells are well-known in solid-state compounds. The crystallization of the title compounds now shows that acetylide ions are existent in solution and therefore chemistry with small dissolved carbon anions may be within reach.

Pharmacokinetics, immunogenicity, safety, and preliminary efficacy of subcutaneous turoctocog alfa pegol in previously treated patients with severe hemophilia A (alleviate 1).

BACKGROUND: The current standard of care for patients with hemophilia A is regular prophylaxis with factor VIII (FVIII) administered intravenously. Interest in subcutaneous (s.c.) administration, to potentially increase convenience, reduce the treatment burden and improve compliance, is increasing. OBJECTIVES: Evaluate the pharmacokinetics (PK), immunogenicity, safety, and preliminary efficacy of s.c. administration of turoctocog alfa pegol (s.c. N8-GP) in adult or adolescent previously treated patients (PTPs) with severe hemophilia A (alleviate 1; NCT02994407). PATIENTS/METHODS: In part A, 24 PTPs received a single dose of s.c. N8-GP (12.5, 25, 50, or 100 IU/kg) with 6 patients per cohort. PK modelling of data from part A supported a suitable dose for part B. Part B comprised a multiple dose trial in 26 PTPs; patients <60 kg received 2000 IU and patients ≥60 kg received 4000 IU s.c. N8-GP daily for 3 months. RESULTS: Single-dose s.c. N8-GP supported dose linearity. Daily prophylaxis with s.c. N8-GP appeared well tolerated and efficacious, achieving a mean trough FVIII activity close to 10% at steady state. Five patients developed anti-N8-GP binding antibodies after 42 to 91 exposure days, one of whom developed an inhibitor to FVIII. Anti-N8-GP antibody appearance was associated with a decline in FVIII plasma activity in four of the five patients. Five patients reported a total of nine treatment-requiring bleeding episodes during prophylaxis. CONCLUSIONS: Subcutaneous administration of N8-GP is associated with a high incidence of antibodies in PTPs with severe hemophilia A. Further clinical development of s.c. N8-GP has been suspended.

A Step in Between: [Sn3Bi3](5-) and Its Structural Relationship to [Sn3Bi5](3-) and [Sn4Bi4](4.).

Extraction of "RbSnBi" in liquid ammonia yielded the cluster anion [Sn3Bi5](3-), which could be crystallized in the compound [Rb@[2.2.2]crypt]3[Sn3Bi5]⋅8.87 NH3. This anion is found to be derived from the formerly reported [Sn4Bi4](4-) by the formal substitution of one tin atom by bismuth. In contrast, the extraction of "RbSn2/Rb3Bi2" in liquid ammonia yielded the anion [Sn3Bi3](5-) in the compound Rb6[Sn3Bi3][Sn4]1/4⋅6.75 NH3. The structural correlation of the two novel clusters indicates that [Sn3Bi3](5-) might be an intermediate of the reaction pathway to [Sn3Bi5](3-) and [Sn4Bi4](4-). Each cluster is investigated by means of the electron localization function and further characterization was performed by using ESI-MS.

Synthesis of heteroatomic Zintl anions in liquid ammonia--the new highly charged [Sn4Bi4]4- and fully ordered [Sn2Bi2]2-.

The new Zintl anion [Sn(4)Bi(4)](4-), synthesized by dissolving CsSnBi in liquid ammonia, forms a monocapped nortricyclane-like cage. Its electron localization function (ELF) analysis shows evidence for 3-centre bonding. The analogous reaction with KSnBi results in a crystallographically fully ordered tetrahedral [Sn(2)Bi(2)](2-) ion.

Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs.

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.

Peripheral T-cell lymphoma in herpesvirus saimiri-infected tamarins: tumor cell lines reveal subgroup-specific differences.

Efficiency of lymphoma induction by herpesvirus saimiri (HVS) isolates correlates with the genetically defined viral subgroups A, B, and C. To compare subgroup-specific effects, highly susceptible tamarins were infected with HVS strain A-11, B-SMHI, or C-488. All animals developed T-cell lymphomas indistinguishable with respect to clinical, pathological, and virological parameters. Ex vivo T-cell lines were established readily from the HVS C-488 animal, less efficiently in the presence of HVS A-11, and from only a single HVS B-SMHI sample. These cultivated cells revealed strain-specific biochemical characteristics. HVS A-11 strongly induced the expression of tyrosine kinase Lyn. HVS C-488 led to the activation of STAT3, which is most likely linked to the association of virus-encoded Tip with tyrosine kinase Lck. The lack of these activities in HVS B-SMHI-transformed cells may correlate with the reduced oncogenic phenotype of this virus in species other than tamarins.

Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck.

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.

Anti-inflammatory effects of a cyclosporine receptor-binding compound, D-43787.

By virtue of its binding to cyclophilin, the cellular receptor for cyclosporine (CsA), we could identify a new compound D-43787 [N-[(1-tert-butyloxycarbonyl)-indolin-2-(S)-carbonyl]-indolin-2-(S)-carbonacid-[N-epsilon-benzyloxycarbonyl)-2-(S)-lysin methylester]-amide] exhibiting immunomodulating properties. It inhibited cell proliferation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA)/ionomycin and anti-CD3/CD28 with an IC(50) of 0.3 microM. The protein phosphatase calcineurin, which is the target of the CsA-cyclophilin complex, is not inhibited by D-43787. It inhibited T helper cell (Th) 2 cytokines interleukin (IL)-4, -5, and -13 more effectively than the Th1 cytokine interferon (IFN)-gamma in human primary T cells. The IC(50) for IL-5 and IL-13 in TPA/ionomycin-stimulated peripheral blood mononuclear cells (PBMC) is 0.7 +/- 0.1 and 0.5 +/- 0.1 microM, respectively, whereas the IC(50) for IFN-gamma is 2.0 +/- 0.4 microM. When PBMC were stimulated with anti-CD3/CD28, the IC(50) for IL-4, -5, and -13 were 1.5 +/- 0.2, 1.8 +/- 0.2, and 1.9 +/- 0.4 microM, respectively. IFN-gamma was only partially inhibited under these conditions. This effect was even more pronounced in pure CD4(+) T cells. Pretreatment of human monocytes with D-43787 inhibited lipopolysaccharide-induced proinflammatory cytokines IL-6 and TNFalpha with an IC(50) of 1.2 +/- 0.1 and 4.7 +/- 0.9 microM, respectively. In vivo, D-43787 potently inhibited late-phase eosinophilia in actively sensitized and challenged guinea pigs (10 mg/kg, i.p.: 51%) and Brown-Norway rats (1 mg/kg, intrapulmonary: 66% 30 mg/kg, i.p.: 50%). In adjuvant-induced arthritis, D-43787 (10-40 mg/kg, b.i.d., i.p.) dose dependently reduced edema development on both hind paws. The potency of D-43787 was comparable with that of indomethacin and dexamethasone. In conclusion, we characterized a novel Th2 selective immunosuppressive drug with possible anti-asthmatic/anti-inflammatory effects. Its mode of action is distinct from that of CsA.

Altered inactivation pathway of factor Va by activated protein C in the presence of heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'306) or after cleavage at Arg506 (k506) and subsequent cleavage at Arg306 (k306). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k506 by UFH was 12-fold, with the secondary cleavage at Arg306 (k306) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'306) two- to threefold. Low molecular weight heparin (Fragmin) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.