ALBERT

Description: The polymerization of partially methylated β-cyclodextrin (CRYSMEB) with epichlorohydrin was carried out in the presence of a known amount of toluene as imprinting agent. Three different preparations (D1, D2 and D3) of imprinted polymers were obtained and characterized by solid-state 13 C NMR spectroscopy under cross-polarization magic angle spinning (CP-MAS) conditions. The polymers were prepared by using the same synthetic conditions but with different molar ratios of imprinting agent/monomer, leading to morphologically equivalent materials but with different absorption properties. The main purpose of the work was to find a suitable spectroscopic descriptor accounting for the different imprinting process in three homogeneous polymeric networks. The polymers were characterized by studying the kinetics of the cross-polarization process. This approach is based on variable contact time CP-MAS spectra, referred to as VCP-MAS. The analysis of the VCP-MAS spectra provided two relaxation parameters: T CH (the CP time constant) and T 1ρ (the proton spin-lattice relaxation time in the rotating frame). The results and the analysis presented in the paper pointed out that T CH is sensitive to the imprinting process, showing variations related to the toluene/cyclodextrin molar ratio used for the preparation of the materials. Conversely, the observed values of T 1ρ did not show dramatic variations with the imprinting protocol, but rather confirmed that the three polymers are morphologically similar. Thus the combined use of T CH and T 1ρ can be helpful for the characterization and fine tuning of imprinted polymeric matrices. Beilstein J. Org. Chem. 2015, 11, 2785–2794. doi:10.3762/bjoc.11.299

Description: Two different formulations of cyclodextrin nanosponges (CDNS), obtained by polycondensation of β-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn), were treated with aqueous solutions of ibuprofen sodium salt (IbuNa) affording hydrogels that, after lyophilisation, gave two solid CDNS-drug formulations. 1 H fast MAS NMR and 13 C CP-MAS NMR spectra showed that IbuNa was converted in situ into its acidic and dimeric form (IbuH) after freeze-drying. 13 C CP-MAS NMR spectra also indicated that the structure of the nanosponge did not undergo changes upon drug loading compared to the unloaded system. However, the 13 C NMR spectra collected under variable contact time cross-polarization (VCT-CP) conditions showed that the polymeric scaffold CDNS changed significantly its dynamic regime on passing from the empty CDNS to the drug-loaded CDNS, thus showing that the drug encapsulation can be seen as the formation of a real supramolecular aggregate rather than a conglomerate of two solid components. Finally, the structural features obtained from the different solid-state NMR approaches reported matched the information from powder X-ray diffraction profiles. Beilstein J. Org. Chem. 2017, 13, 182–194. doi:10.3762/bjoc.13.21

Description: Tricyclic fused-ring cyclobenzaprine ( 1 ) and amitriptyline ( 2 ) form 1:1 inclusion complexes with β-cyclodextrin (β-CD) in the solid state and in water solution. Rotating frame NOE experiments (ROESY) showed the same geometry of inclusion for both 1 /β-CD and 2 /β-CD complexes, with the aromatic ring system entering the cavity from the large rim of the cyclodextrin and the alkylammonium chain protruding out of the cavity and facing the secondary OH rim. These features matched those found in the molecular dynamics (MD) simulations in solution and in the solid state from single-crystal X-ray diffraction of 1 /β-CD and 2 /β-CD complexes. The latter complex was found in a single conformation in the solid state, whilst the MD simulations in explicit water reproduced the conformational transitions observed experimentally for the free molecule. Beilstein J. Org. Chem. 2017, 13, 714–719. doi:10.3762/bjoc.13.70

Description: This work aims to investigate and characterize the photo-ignition phenomenon of MWCNT/ferrocene mixtures by using a continuous wave (CW) xenon (Xe) light source, in order to find the power ignition threshold by employing a different type of light source as was used in previous research (i.e., pulsed Xe lamp). The experimental photo-ignition tests were carried out by varying the weight ratio of the used mixtures, luminous power, and wavelength range of the incident Xe light by using selective optical filters. For a better explanation of the photo-induced ignition process, the absorption spectra of MWCNT/ferrocene mixtures and ferrocene only were obtained. The experimental results show that the luminous power (related to the entire spectrum of the Xe lamp) needed to trigger the ignition of MWCNT/ferrocene mixtures decreases with increasing metal nanoparticles content according to previously published results when using a different type of light source (i.e., pulsed vs CW Xe light source). Furthermore, less light power is required to trigger photo-ignition when moving towards the ultraviolet (UV) region. This is in agreement with the measured absorption spectra, which present higher absorption values in the UV–vis region for both MWCNT/ferrocene mixtures and ferrocene only diluted in toluene. Finally, a chemo-physical interpretation of the ignition phenomenon is proposed whereby ferrocene photo-excitation, due to photon absorption, produces ferrocene itself in its excited form and is thus capable of promoting electron transfer to MWCNTs. In this way, the resulting radical species, FeCp2 +∙ and MWCNT−, easily react with oxygen giving rise to the ignition of MWCNT/ferrocene samples.

Description: Tricyclic fused-ring cyclobenzaprine (1) and amitriptyline (2) form 1:1 inclusion complexes with β-cyclodextrin (β-CD) in the solid state and in water solution. Rotating frame NOE experiments (ROESY) showed the same geometry of inclusion for both 1/β-CD and 2/β-CD complexes, with the aromatic ring system entering the cavity from the large rim of the cyclodextrin and the alkylammonium chain protruding out of the cavity and facing the secondary OH rim. These features matched those found in the molecular dynamics (MD) simulations in solution and in the solid state from single-crystal X-ray diffraction of 1/β-CD and 2/β-CD complexes. The latter complex was found in a single conformation in the solid state, whilst the MD simulations in explicit water reproduced the conformational transitions observed experimentally for the free molecule.

Description: Two different formulations of cyclodextrin nanosponges (CDNS), obtained by polycondensation of β-cyclodextrin with ethylenediaminetetraacetic acid dianhydride (EDTAn), were treated with aqueous solutions of ibuprofen sodium salt (IbuNa) affording hydrogels that, after lyophilisation, gave two solid CDNS-drug formulations. 1H fast MAS NMR and 13C CP-MAS NMR spectra showed that IbuNa was converted in situ into its acidic and dimeric form (IbuH) after freeze-drying. 13C CP-MAS NMR spectra also indicated that the structure of the nanosponge did not undergo changes upon drug loading compared to the unloaded system. However, the 13C NMR spectra collected under variable contact time cross-polarization (VCT-CP) conditions showed that the polymeric scaffold CDNS changed significantly its dynamic regime on passing from the empty CDNS to the drug-loaded CDNS, thus showing that the drug encapsulation can be seen as the formation of a real supramolecular aggregate rather than a conglomerate of two solid components. Finally, the structural features obtained from the different solid-state NMR approaches reported matched the information from powder X-ray diffraction profiles.

Description: The polymerization of partially methylated β-cyclodextrin (CRYSMEB) with epichlorohydrin was carried out in the presence of a known amount of toluene as imprinting agent. Three different preparations (D1, D2 and D3) of imprinted polymers were obtained and characterized by solid-state 13C NMR spectroscopy under cross-polarization magic angle spinning (CP-MAS) conditions. The polymers were prepared by using the same synthetic conditions but with different molar ratios of imprinting agent/monomer, leading to morphologically equivalent materials but with different absorption properties. The main purpose of the work was to find a suitable spectroscopic descriptor accounting for the different imprinting process in three homogeneous polymeric networks. The polymers were characterized by studying the kinetics of the cross-polarization process. This approach is based on variable contact time CP-MAS spectra, referred to as VCP-MAS. The analysis of the VCP-MAS spectra provided two relaxation parameters: T CH (the CP time constant) and T 1ρ (the proton spin-lattice relaxation time in the rotating frame). The results and the analysis presented in the paper pointed out that T CH is sensitive to the imprinting process, showing variations related to the toluene/cyclodextrin molar ratio used for the preparation of the materials. Conversely, the observed values of T 1ρ did not show dramatic variations with the imprinting protocol, but rather confirmed that the three polymers are morphologically similar. Thus the combined use of T CH and T 1ρ can be helpful for the characterization and fine tuning of imprinted polymeric matrices.

Description: Background: The highly unfavorable outcome of patients with recurrent HL, who progress after stem cell transplantation or are ineligible for such procedure make the development of new active agents an impellent medical need in this clinical setting. EDO-S101 is fusion molecule combining the DNA damaging effects of bendamustine (BDM) with the pan-histone deacetylase (HDAC) inhibitor, vorinostat. Given that BDM and HDAC inhibitors are active agents in recurrent HL we investigated the preclinical activity of EDO-S101 in this malignancy. Methods: We assessed the patterns of EDO-S101 cytotoxicity (0.39 to 50 µmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its regulatory effects on genes involved in DNA-damage/repair response, apoptosis and cell cycle checkpoints. As a further model we exploited an L1236 cell clone (R100) selected for resistance (R) to BDM through continuous exposure to increasing concentrations of the agent. R100 cells display a growth pattern indistinguishable from parental L1236 cells when cultured in the presence of BDM (100 µmol/L). Clonal identity of R100 cells with parental L1236 was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions. Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all HL cell lines. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 1.88 µmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 2.06, 2.53, 2.26 and 16.2 µmol/L, respectively. These values were about 10-fold lower than the IC50 of BDM in the same cell lines. While exposure of L1236 cells to EDO-S101 caused cell accumulation in S-phase, qRT-PCR disclosed that cell death was mainly dependent on triggering of apoptosis, as shown by the early (24 hrs) and sustained (48 hrs) upregulation of NOXA, p21 and p27 genes. Data were confirmed by the significant increase (〉150%) of Annexin V-expressing L1236 cells. In contrast, expression levels of PLK1, AKA and cyclin B1 genes remained unchanged or were increased. This excluded induction of the mitotic catastrophe (MC) as a major determinant of cytotoxic activity for EDO-S101 in L1236 cells. Exposure to EDO-S101 induced a strong DNA stress/repair response as shown by the activation of pATR/pATM and increase of the downstream DNA damage checkpoint proteins pCHK1-/-2 and CCNB1, along with the upregulation of the EXO1 gene. Most intriguingly, BDM-resistant L1236 cells (R100) were highly sensitive to EDO-S101, with an IC50 of only 4.56 µmol/L, but less responsive to vorinostat (IC50: 6.17 µmol/L) than parental L1236 cells (IC50: 0.58 µmol/L). Differently from native cells, EDO-S101 induced a late downregulation of transcripts for PLK1 and AKA genes and of cyclin B1 gene and protein in R100 cells, along with the early induction of NOXA and p21, but not p27 genes. In both L1236 and R100 cells, expression of MC-genes was unaffected by exposure to vorinostat. This suggests a more complex mechanism for EDO-S101 in BDM-resistant HL cells involving activation of the both apoptotic and MC pathways. Notably, we documented that EDO-S101 corrected the constitutive ATM/ATR unbalance of R100 cells by triggering the early (24 hrs) upregulation of ATR and a late (48 hrs) downregulation of ATM transcripts and proteins, along with increased levels of EXO1 and MGMT at 24 hrs. Vorinostat induced a similar effect. Finally, while baseline expression levels of HDAC isoforms were comparable among HL cell lines, EDO-S101 caused a significant (〉40%) late downregulation of transcripts for all HDAC isoforms (HDAC-1 to -8) in R100 cells but only of HDAC-6 in native L1236. This pattern diverged from results obtained in both L1236, i.e. increase of all HDAC isoform transcripts except HDAC-6, and R100 cells, i.e. upregulation of all isoforms and reduction of HDAC-6, with vorinostat and BDM as single agents. Conclusions: We have described for the first time that EDO-S101 is effective in preclinical models of HL including cells resistant to BDM. The combined functions of in one molecule of a bifunctional alkylator and panHDAC inhibitor confer this agent unique antitumor property different from both of its single drug components. Following a strong DNA damage response, triggering of apoptosis and/or MC may take place in HL cells according to their sensitivity status to BDM. A phase 1/2 study in recurrent HL, including patients pretreated with BDM, is next to be launched. Disclosures Mehrling: 4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Pinto:Takeda, Celgene, Roche, TEVA: Honoraria; Takeda: Research Funding.

Description: Chronic Lymphocytic Leukemia (CLL) represents the most frequent adult leukemia, and remains incurable with current standard therapies. Natural Killer (NK) cell count is predictive of CLL disease progression and their dysfunction in mediating cytokine release and direct or antibody dependent cellular cytotoxicity (ADCC) against CLL B-cells is well documented. Detailed mechanistic insight into the etiology of NK-cell dysfunction in CLL patients is currently lacking. CLL B-cells overexpress HLA-E, the natural ligand for heterodimer CD94/NKG2A receptor complex that is expressed on the surface of NK cells, and this interaction suppresses NK cell activation. While NKG2A/CD94/HLA-E interaction is known to assist NK cells in recognizing "self", tumor cells utilize this mechanism to evade effector cell killing. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mab) we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in patients with CLL. We hypothesized that limiting the interaction of HLA-E/NKG2A will reverse NK cell anergy and result in increased direct cytotoxicity of CLL cells. Our results confirm the over expression of HLA-E on CLL B-cells and demonstrate NKG2A expression on CD16+ NK cells from CLL patients. Next, we examined the effect of anti-NKG2A mab on NK cell direct cytotoxicity. Treatment of NK cells, from both healthy donor and CLL patients, with anti-NKG2A mab increased direct cytotoxicity over isotype control on targets at various effector to target ratios of 25:1 (54% vs. 46%, p〈 0.05, n= 12), 12:1 (43% vs. 35%, p

Description: Ligation of the B-cell receptor (BCR) results in activation of intracellular signaling as well as internalization and processing of ligand/receptor complexes. BCR responsiveness has been shown to vary markedly between patients with chronic lymphocytic leukaemia (CLL) and is linked to prognosis. Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, relatively little is known about the capacity of CLL B-cells to internalize ligands that bind to the BCR. In the present study we investigated whether, like normal B-cells, CLL cells can internalize their BCR following stimulation. First, we assessed to what extent this varies between various prognostic subgroups. Second, given that BCR signaling is thought to be more pronounced within lymphoid tissue, we investigated whether internalization varies between different anatomic sites of the same individual. Finally, we examined the effect of agents that inhibit BCR function by comparing BCR expression and internalization in a cohort of patient before and during therapy with Bruton's tyrosine kinase inhibitors (BTKi). BCR internalization was assessed in two ways. First, we used a pH sensitive dye linked to agonistic anti-IgM (pHrodo-αIgM) to detect the uptake and retention of ligand/receptor complexes in acidified endosomes. Second, BCR internalization was assessed directly by measuring the rate of disappearance of surface IgM following ligation by agonistic anti-IgM. An increase in the percentage of cells showing pHrodo fluorescence above control was detected in all CLL cases studied (mean percentage pHrodo-αIgM uptake = 30.2±2.5%, p