Alessandro Bressan

Bradykinin receptor antagonists – a review of the patent literature 2005 – 2008

Background: For > 20 years, pharmaceutical companies and academic centers have been developing bradykinin antagonists. The patent literature on these molecules (up to and including 2004) has been analyzed previously in this journal in two review articles. Objective: The aim of this review is to provide an update (from 2005 to early 2009) on the patenting activity in the field of bradykinin antagonists (including patents on their formulation). Where possible, the information from the patents has been supplemented with that from the primary literature, clinical trial databases and company websites in an attempt to give a more complete picture. Conclusions: In the past 4 years, nearly 50 new patents have been filed on bradykinin antagonists – in the case of several filings, only the original source has been considered in this analysis – the vast majority of these (> 93%) on B1 antagonists. However, despite this large amount of work, only one compound, icatibant – a hydrophilic decapeptide selective for the B2 receptor – has reached the market, although it needs to be administered parenterally.

SAHA/Vorinostat induces the expression of the CD137 receptor/ligand system and enhances apoptosis mediated by soluble CD137 receptor in a human breast cancer cell line.

HDAC inhibitors (HDACis) represent a class of anticancer agents including suberoylanilide hydroxamic acid (SAHA, Vorinostat), which has shown a strong antitumor effect, both in vitro and in vivo. Induction of apoptotic genes is an important pathway of SAHA cytotoxic mechanism of action and it has been largely described that SAHA induces sensitization of cell death receptor-resistant breast cancer cells to apoptosis. In this study, we investigated the activation of some apoptotic genes which could be responsible for the in vivo antitumor potency of SAHA in a model of human breast cancer. We found that the apoptotic gene pattern induced by SAHA in the MDA-MB-231 cell line involves the upregulation of some molecules belonging to the TNF superfamily. In particular, we demonstrated that the upregulation of the CD137 receptor/ligand system correlates with a synergistic cytotoxic effect when MDA-MB-231 cells are treated with the combination of SAHA and soluble CD137 receptor. To our knowledge, this is the first study to indicate that this member of the TNF superfamily, CD137, is modulated by SAHA treatment in breast cancer cells, suggesting that the combination of SAHA with this TNF-related receptor could be a new therapeutic approach for the treatment of tumors.

OC125, M11 and OV197 epitopes are not uniformly distributed in the tandem-repeat region of CA125 and require the entire SEA domain.

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments in E. coli and Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

Antitumor activity of delimotecan against human metastatic melanoma: Pharmacokinetics and molecular determinants

Delimotecan (MEN 4901/T‐0128) is a new cytotoxic prodrug constituted by a camptothecin analog (T‐2513) bound to carboxymethyl dextran through a triglycine linker. A significant antitumor activity of delimotecan against human metastatic melanoma xenograft model Me15392 is reported. Dacarbazine, the drug approved for the treatment of metastatic melanoma, was ineffective in this melanoma model. Pharmacokinetic studies, together with the expression analysis of mRNA for enzymes involved in delimotecan metabolism, showed that T‐2513 and other cytotoxic metabolites of delimotecan (SN 38 and T‐0055) are generated in greater quantities in the tumor tissue than in toxicity target tissues, such as liver, thus accounting for the antitumoral activity. Moreover, we demonstrated that human metastatic melanoma cells are able to phagocytose delimotecan and cleave it to release the cytotoxic moieties T‐2513 in the tumoral environment. Further flow cytometric analysis showed a higher recruitment of macrophages in xenografted human metastatic melanoma, when compared with other human tumors. Thus, the antitumoral activity of delimotecan exerted on metastatic melanoma is due to several factors: (i) the ability of melanoma cells to phagocytose and metabolise delimotecan; (ii) the accumulation of delimotecan in tumoral mass; (iii) the recruitment of macrophage cells to the melanoma nodule and (iv) the expression in melanoma cells of a pattern of enzymes that converts delimotecan into cytotoxic metabolites. Based on these results, delimotecan might be exploited as a new anticancer agent for the therapy of metastatic melanoma because of its high efficacy and good selectivity, and therefore clinical trials for this indication are warranted.

Expression and characterization of biologically active human fas ligand produced in CHO cells

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh-FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 µg/L × 106 cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. Synthesis and structure-relationships of α,α-tetrahydropyranylglycine

A series of α,α-cycloalkylglycine sulfonamide compounds of general formula 1 has previously been identified by our group as selective human B(2)(hB(2)) receptor antagonists. Here we report the in vitro and in vivo BK antagonist activity of a further evolution of the series, consisting in compounds of the general formula 2, containing either an alkyl piperazine or a 4-alkyl piperidine ring bearing various positively charged groups (R). These studies unexpectedly revealed quite a flat nanomolar/subnanomolar SAR for the binding affinity, while differences were seen in the in vitro functional activities. We propose that variations in the residence time may explain these results.

Abstract 3646: Characterization of the novel antibody drug conjugate MEN1309 and its target antigen Ly75

Ly75 (CD205, DEC-205) is a type I transmembrane glycoprotein and a C-type lectin receptor involved in antigen uptake and processing, mainly expressed by antigen presenting cells (APC). The short cytoplasmic tail contains motifs for amino acid-based endocytosis, making this receptor an ideal target antigen for an antibody drug conjugate (ADC)-based antitumoral therapy. MEN1309 is a novel fully humanized ADC which binds to Ly75 with high affinity as shown by ELISA and FACS analysis. The antibody is conjugated to a maytansinoid DM4, a potent tubulin inhibitor, through a cleavable linker.The ability of Ly75 to internalize the antibody after binding was determined using an immunoflourescence assay that showed a rapid, efficient, and near complete internalization over a one hour time course.The expression of Ly75 mRNA and protein was investigated in human cancer cell lines derived from different histotypes and revealed high expression in pancreas, bladder, triple negative breast cancer (TNBC) cells and in diffuse large B-cell lymphoma (DLBCL). Indeed, MEN1309 shows a powerful (pM range) in vitro cytotoxic activity on different cancer cell lines expressing Ly75, whereas it exerts a weaker effect on antigen-negative cells. Besides the mechanism of action (MoA) of MEN1309 as an ADC, the putative efficacy of the antibody to drive an ADCC response was investigated through in vitro binding and functional assays. In spite of a high binding affinity of MEN1309 to FcγRIIIa, no ADCC response was observed, suggesting that the high internalization rate of the antigen could hamper the triggering of NK responses.Moreover, in order to characterize the functional role of Ly75 in cancer cell lines, its expression was downregulated by siRNA demonstrating an inhibition of the proliferation rate in cells from different histotypes.Finally, we investigated if some cancer cell lines could show a higher expression of two intergenically spliced forms derived from Ly75 and DCL-1 genes recently reported in literature. We found that the intergenically spliced forms were expressed on average 30 fold less than CD205 mRNA in all the cancer cell lines analyzed, suggesting that these variants derive just from an intergenic readthrough without a specific transcriptional regulation. Citation Format: Alessandro Bressan, Alessio Fiascarelli, Giuseppe Merlino, Corrado Carrisi, Daniela Bellarosa, Rachel Dusek, Rahel Awdew, Sudha Swaminathan, Arnima Bisht, To Uyen T. Do, San Lin Lou, Dee Aud, Jonathan Terrett, Keith Wilson, Christian Rohlff, Monica Binaschi. Characterization of the novel antibody drug conjugate MEN1309 and its target antigen Ly75 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3646. doi:10.1158/1538-7445.AM2017-3646