Wolfram Steinke

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (α/ß) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. Through a high‐throughput screen we identified BAY 58‐2667, an amino dicarboxylic acid which potently activates sGC in an NO‐independent manner. In contrast to NO, YC‐1 and BAY 41‐2272, the sGC stimulators described recently, BAY 58‐2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. Binding studies with radiolabelled BAY 58‐2667 show a high affinity site on the enzyme. Using photoaffinity labelling studies we identified the amino acids 371 (α‐subunit) and 231 – 310 (ß‐subunit) as target regions for BAY 58‐2667. sGC activation by BAY 58‐2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. BAY 58‐2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58‐2667 and GTN are very similar on the arterial and venous system. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272

BackgroundThe most important receptor for nitic oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.ResultsWe developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the α1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236–290 of the α1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.ConclusionsOur data demonstrate that the region surrounding the cysteines 238 and 243 in the α1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

125I-endothelin-1 and 125I-big endothelin-1 in rat tissues: autoradiographic localization and receptor binding

SummaryThe potent vasoconstrictor peptide, endothelin-1 (ET-1), which exhibits a characteristically long-acting activity in vitro and in vivo, is thought to be generated in endothelial cells from a less active intermediate, big endothelin-1 (big ET-1). In addition to ET-1, big ET-1 is also present in the circulation. The autoradiographic localization of 125I-big ET-1 and 125I-ET-1 has been studied after intravenous administration in rat tissues. Highest enrichment of radioactivity was found in the kidney cortex for both peptides. Compared to blood levels, enrichment of radioactivity is also detected, in the vascular wall of the aorta. Comparing the radioactivity pattern of ET-1 and big ET-1, a nearly identical tissue distribution is observed, with the exception of the relative enrichment in the lung and the zona glomerulosa after administration of ET-1.Both radioligands show a specific and saturable binding to lung and kidney membranes. In the case of lung tissue, Ki values are 10−10M for endothelin-1 and 10−8M for big endothelin-1. This difference in affinities may account for the lack of binding of big endothelin-1 to lung tissue.

Pharmacologic activity and pharmacokinetics of metabolites of regorafenib in preclinical models

Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment‐refractory metastatic colorectal cancer. In clinical studies, steady‐state exposure to the M‐2 and M‐5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M‐2, and M‐5 in vitro and in murine xenograft models. M‐2 and M‐5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell‐based assays. In murine xenograft models, oral regorafenib, M‐2, and M‐5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M‐2 and M‐5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M‐2, and M‐5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M‐2, but not M‐5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M‐2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.

Abstract 3526: Subcutaneous administration of PSMA/CD3-bispecific BiTE antibody MT112/BAY 2010112 leads to complete remission of human prostate cancer xenografts in mice

Blinatumomab, a CD19/CD3-bispecific BiTE antibody has shown high response rates and durable remissions in patients with relapsed or refractory acute lymphocytic leukemia and non-Hodgkin9s lymphoma. For treatment of patients with prostate cancer (PCa), we have developed a novel BiTE antibody, MT112/BAY2010112, that is bispecific for prostate-specific membrane antigen (PSMA) and the CD3 epsilon subunit of the T cell receptor complex. MT112/BAY 2010112 binds PSMA and CD3 of human and macaque origin allowing for assessment of safety, pharmacodynamics and pharmacokinetics in a relevant animal species. PSMA expressing human PCa cell lines VCaP, 22Rv1, MDA PCa 2b, C4-2, PC3-PSMA and LNCaP were lysed by freshly isolated, unstimulated human T cells redirected by MT112/BAY2010112 at EC50 values ranging between 0.1 and 4 ng/ml (1.8-72 pM). Efficacy of lysis correlated with levels of surface expressed PSMA. No lysis was observed in PSMA-negative PCa cell lines PC3 and DU145, showing the target antigen-specific activity of MT112/BAY2010112. The relevance of cynomolgus monkeys for further nonclinical safety evaluation of MT112/BAY 2010112 was demonstrated by in vitro side-by-side comparison of the BiTE antibody9s pharmacological characteristics in human and cynomolgus monkey assay systems. For studying the activity of MT112/BAY 2010112 after subcutaneous (s.c.) administration, human 22Rv1 PCa xenografts were grown s.c. in mice to sizes of >200 mm3 in the absence of human T cells. Three days prior to treatment with BiTE, ex-vivo expanded human T cells were intraperitoneally injected. Animals were treated s.c. at 2.5 mg/kg/d or intravenously (i.v.) at 0.5 mg/kg/d for 28 consecutive days. S.c. administration of MT112/BAY 2010112 resulted in a similar drug exposure, rapid shrinkage and complete remission of established PCa xenografts as seen upon i.v. administration of the BiTE antibody. Treatment with human T cells or vehicle alone had no effect on tumor growth. Compared to i.v. administration, the relative bioavailability of the PSMA BiTE in serum after s.c. injection in mice was approximately 18%. Initial results will be presented showing that 14C-labeled MT112/BAY 2010112 accumulates in s.c. implanted LNCaP PCa tumors of mice after tail vein injection. Based on these preclinical in vivo pharmacology results, treatment of PCa patients with s.c. administered MT112/BAY2010112 appears to be justified. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3526. doi:1538-7445.AM2012-3526

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

SummaryWhole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.