- Email: [email protected]
Ammonia-oxidizing bacteria accelerate wound closure in diabetic mice
Abstracts/Nitric Oxide 42 (2014) 99–153
inflammatory or anticancer agents. We have synthesized the first HNO-donating NSAIDs, which like structurally related NO donors show similar anti-inflammatory properties but significantly lower gastrointestinal ulceration than the NSAID alone. Both the HNO and NO donor prodrugs also exhibit higher toxicity to breast cancer cells and significantly reduce tumor growth of breast cancer xenografts in nude mice than the parent NSAID. Detailed analysis has indicated that the cytotoxic and antiproliferative effects of the NO and HNO donor adducts are induced by distinct pathways, which will be described. Overall, HNO-NSAIDs appear to be a unique and promising new class of chemotherapeutic agent. Keywords: Nitric oxide; Nitroxyl; NSAID; Anticancer.
Oral 1922-3. Nitric oxide synthase gene therapy: A potent therapeutic in metastatic breast cancer http://dx.doi.org/10.1016/j.niox.2014.09.039 Cian McCrudden, John McBride, Jonathan Coulter, Victoria Kett, Tracy Robson, Helen McCarthy Queen’s University, Belfast
Primary breast cancer, the most common malignancy in women, is a disease with many treatment options, and impressive response rates. However, treatment options are limited once tumors metastasize. Docetaxel is the front line therapy for metastatic breast cancer, but resistance eventually develops, limiting its potency. Nitric oxide (NO•) exerts anticancer effects by inducing DNA damage, modifying DNA repair proteins, and inhibiting anti-apoptotic factor NF-κB. We have previously reported the therapeutic benefit of inducible NO• synthase (iNOS) gene therapy in several xenograft models of human cancer. The purpose of the current study was to investigate the antimetastatic potential of NO• using a transcriptionally targeted iNOS gene construct and our novel delivery technology. We have developed a peptide-based gene therapy delivery vehicle that produces cationic nanoparticles (NPs) when incubated with plasmid iNOS (piNOS). The NPs overcome the biological barriers to gene therapy, efficiently delivering piNOS to the nucleus of breast cancer cells. piNOS-loaded NPs (constitutively active CMV or transcriptionally-regulated human osteocalcin [hOC] promoters) evoked iNOS gene expression in MDA-MB-231-Luc-D3H1 breast cancer cells in vitro, which manifested impressive nitrite accumulation in culture medium (indicative of NO• generation). C57/BL6 mice tolerated multiple administrations of pDNA-loaded NPs, and their sera lacked NP-neutralizing antibodies. These results indicated that iNOSloaded NPs have therapeutic potential in in vivo models of breast cancer metastasis. Metastatic breast cancer was established in female BALB/c SCID mice by inoculation with 2 × 10 5 MDA-MB-231-LucD3H1 via the left ventricle, and disease progression was assessed via bioluminescence imaging and body weight determination. Treated mice received 50 μg CMV-iNOS/hOC-iNOS in five intravenous biweekly doses. Serum nitrite levels were elevated in mice that received either iNOS gene therapy. Mice that received CMV-iNOS (median survival 40 days; p < 0.001) or hOCiNOS (38.5 days; p = 0.001) therapy had significantly improved survival compared with control mice (31.5 days). Moreover, combining docetaxel therapy with either CMV-iNOS (51 days) or hOC-iNOS (46 days) improved median survival of mice beyond that observed with docetaxel alone (44 days). Targeted NO• gene therapy has shown excellent therapeutic potential for metastatic breast cancer, and studies are ongoing to develop our NP formulation toward a potential clinical product.
111
Keywords: Gene therapy; Breast cancer; Metastasis; Nitric oxide. Oral 2011-1. The therapeutic effects of a nitric oxide generating lozenge that utilizes natural product chemistry http://dx.doi.org/10.1016/j.niox.2014.09.040 Nathan BryanPhD The University of Texas Health Science Center
The production and availability of nitric oxide (NO) is critical for cardiovascular health. The complex production of NO from L-arginine by nitric oxide synthase becomes compromised with age and certain elements of lifestyle. As this primary pathway for NO becomes dysfunctional, it leads to conditions of NO deficiency with subsequent clinical sequel including hypertension and onset and progression of cardiovascular disease. A complementary endogenous pathway for NO production emerged wherein inorganic nitrate and nitrite, produced from both the oxidation of NO and dietary nitrate-rich functional foods such as leafy green vegetables and beets, can be reduced back to NO through enterosalivary cycling. Utilizing intellectual property developed out of the University of Texas Health Science Center in Houston (US patents 8,298,589, 8,303,995 & 8,435,570), Neogenis Laboratories has licensed this technology and developed a GMP certified, over the counter, all natural formulation that provides a system for generating NO in an endothelium-dependent and independent manner. This technology has been clinically tested in placebo controlled designed studies. A single administration of an oral, active, naturebased NO supplement significantly lowers blood pressure, improves vascular compliance and restores endothelial function in subjects with hypertension. Thirty day administration also leads to normalization of blood pressure in pre-hypertensive patients. Pharmaceutical approaches for NO restoration present long-term safety issues; however, functional, nutraceutical approaches may offer select advantages for restoring NO availability. This treatment may be beneficial as routine supplementation for cardiovascular protection. Keywords: Nitrite; Nutraceutical; Diet. Oral 2011-2. Ammonia-oxidizing bacteria accelerate wound closure in diabetic mice http://dx.doi.org/10.1016/j.niox.2014.09.041 Ioannis Gryllos a, Shomir Ghosh a, Neeraja Vajrala b, David Whitlock a, Luis Sayavedra-Soto b, Spiros Jamas a a AOBIOME LLC, Cambridge, MA, USA b Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA
Diabetic foot ulcers (DFUs) are among the most common and serious complications of diabetes. They present in approximately 15% of diabetic patients, leading to ~80,000 amputations per year in the United States alone. Effective strategies for the treatment of DFUs are currently limited, thus highlighting an urgent unmet medical need. Topical delivery of nitric oxide (NO) at the wound site via NO-generating compounds, or by using acidified nitrite as a NO source, has been shown to promote healing in murine models of diabetes by reversing dysregulated expression of inflammatory cytokines, growth factors and matrix metalloproteinases among others. In the present study, we have evaluated the use of ammonia-oxidizing bacteria (AOB) of the genus Nitrosomonas as natural self-regulating biological delivery systems of NO/NOx in vivo. Nitrosomonas are ubiquitous Gram-negative obligate chemolithoautotrophic bacteria that exclusively oxidize ammonia as an energy source to generate nitrite and NOx species. As proof-of-concept, we proceeded to examine
112
Abstracts/Nitric Oxide 42 (2014) 99–153
the capacity of a novel Nitrosomonas eutropha strain to accelerate wound closure in diabetic db/db mice. This strain, designated D23, was initially isolated from organic soil, then extensively characterized for nitrite generation and propagated in continuous culture for increased biomass yields. For testing in the db/db model, three groups of eight mice each were treated daily for one week with D23 suspensions supplemented with ammonium chloride, or with vehicle control suspension only. Subsequently, full-size wounds generated on the back of each animal were treated topically once daily for 14 days with bacterial suspension or vehicle alone. Of the three AOB-treated groups, the group receiving the highest D23 doses showed significant improvement in wound closure from day 5 to day 15, with the most pronounced improvement of 83% observed on day 9 post-wounding. Based on these findings, we are exploring the potential therapeutic benefit(s) of AOB to DFUs, chronic wounds and other related indications. Keywords: Ammonia-oxidizing bacteria; Nitrosomonas; Nitrite; Nitric oxide; Wound healing; Diabetes.
Oral 2012-1. Interfacial residues promote an optimal alignment of the catalytic center in soluble guanylate cyclase http://dx.doi.org/10.1016/j.niox.2014.09.042 Elsa Garcin a, Franziska Seeger b, Royston Quintyn c, Akiko Tanimoto c, Gareth Williams d, John Tainer e, Vicki Wysocki c a University of Maryland Baltimore County b UMBC c Ohio State University d Lawrence Berkeley Laboratory e TSRI/LBL
Most of what we know about the guanylate cyclase catalytic mechanism derives from comparisons with related adenylate cyclases. As a structure of the active heterodimeric cyclase domain remains elusive, there is still much debate about structural requirements for cyclase activity and possible conformational changes needed to achieve full catalytic capacity. Several studies suggest that the heme-containing regulatory domain binds to the catalytic domain and maintains sGC in an auto-inhibited basal state. NO binding to heme, thought to be communicated to the cyclase domain via the regulatory and dimerization domains, releases the full catalytic potential of sGC. If this model is correct, isolated catalytic domains should display catalytic activity analogous to that of activated sGC. Using X-ray crystallography, activity measurements, and native mass spectrometry, we demonstrated that isolated catalytic domains are much less active than basal sGC, despite being able to form heterodimers. Our X-ray structure revealed key structural elements that modulate the dimer interface. We further propose a novel role for residues located in an interfacial flap and a hydrogen-bond network as key modulators of the orientation of the catalytic subunits. Our results unambiguously show that even in the absence of the regulatory domain, additional sGC domains are required to guide the appropriate conformation of the catalytic subunits associated with high activity. We propose a novel regulatory mechanism whereby sGC activity is tuned by distinct domain interactions that either promote or inhibit catalytic activity. These results further our understanding of sGC activation and regulation, and open additional drug discovery routes for targeting the NO–sGC–cGMP pathway via the design of small molecules that promote a productive conformation of the catalytic subunits or disrupt inhibitory domain interactions.
Keywords: Soluble guanylate cyclase; Enzyme mechanism; Regulation; X-ray crystallography; Conformational changes; Domain–domain interactions.
Oral 2012-2. Toward structural insights into activation and modulation of the soluble guanylyl cyclase http://dx.doi.org/10.1016/j.niox.2014.09.043 Vijay Kumar a, Jonathan Stamler b, Johannes-Peter Stasch b, Michael Hahn c, Martina Schaefer c, Athanassios Giannis c, Andreas Papapetropoulos c, Focco van den Akker d a CWRU b Bayer Pharma AG, Aprather Weg 18a, D-42096 Wuppertal, Germany c Department of Pharmacy, University of Patras, 26504 Patras, Greece d Case Western Reserve University
Objective/purpose: Soluble guanylyl cyclase (sGC), the principal receptor of nitric oxide (NO), converts GTP into cGMP that regulates several physiological functions. Under some abnormal cardiovascular processes, sGC activity is not optimal and thus spurs the need for further molecular studies investigating this as well as the development of novel stimulators and activators of sGC. Compounds BAY58-2667 (BAY58) and BAY602770 (BAY60) are NO-independent activators of sGC and had been already tested in clinical trials. Earlier our lab proposed the heme pivot–bend mechanism for sGC activation involving a shift of the H105-containing αF helix. In the current study we demonstrate how the study of existing and novel sGC activators may aid to a better understanding of sGC activation and ultimately to obtain better activators of sGC using X-ray crystallography. Results: Our lab solved the crystal structures of BAY58 and BAY60 bound to Nostoc (Ns) H-NOX domain. We noticed an interesting observation regarding the size of the substituents of the hydrophobic linker with respect to the size of heme pocket and the distortion of loop region following the important αF helix in H-NOX domain. Previous clinical trial studies for acute decompensated heart failure patients revealed that BAY58 leads to the dose-dependent hypotension development. On the basis of our structural information, an improved activator was developed with 5-fold higher cGMP-stimulatory properties compared to BAY58 and we probed the structural basis for its higher activation of sGC. Furthermore, we also solved the crystal structure of BAY60-bound Ns HNOX protein having the conserved C122 residue S-nitrosylated. S-nitrosylation conferred some subtle conformational changes nearby the heme binding cavity that could lead to the structural basis for the desensitization of sGC via S-nitrosylation. Conclusions: Using BAY58 and BAY60 bound Ns H-NOX structural comparisons; an improved activator with five-fold higher cGMP-stimulatory properties was developed. We could also utilize BAY60 for understanding the structural basis of desensitization of sGC via S-nitrosylation of C122. Keywords: Soluble guanylyl cyclase; Heme; Nitric oxide; Activation; BAY58; BAY60; S-nitrosylation.
Oral 2012-3. Nitric oxide and heat shock protein 90 activate soluble guanylate cyclase by driving rapid change in its subunit interactions and heme content http://dx.doi.org/10.1016/j.niox.2014.09.044 Arnab Ghosh a, Johannes-Peter Stasch b, Andreas Papapetropoulos c, Dennis Stuehr a a Department of Pathobiology, Cleveland Clinic, Cleveland, OH 44195, USA b Bayer Pharma AG, Aprather Weg 18a, D-42096 Wuppertal, Germany c Department of Pharmacy, University of Patras, 26504 Patras, Greece
inflammatory or anticancer agents. We have synthesized the first HNO-donating NSAIDs, which like structurally related NO donors show similar anti-inflammatory properties but significantly lower gastrointestinal ulceration than the NSAID alone. Both the HNO and NO donor prodrugs also exhibit higher toxicity to breast cancer cells and significantly reduce tumor growth of breast cancer xenografts in nude mice than the parent NSAID. Detailed analysis has indicated that the cytotoxic and antiproliferative effects of the NO and HNO donor adducts are induced by distinct pathways, which will be described. Overall, HNO-NSAIDs appear to be a unique and promising new class of chemotherapeutic agent. Keywords: Nitric oxide; Nitroxyl; NSAID; Anticancer.
Oral 1922-3. Nitric oxide synthase gene therapy: A potent therapeutic in metastatic breast cancer http://dx.doi.org/10.1016/j.niox.2014.09.039 Cian McCrudden, John McBride, Jonathan Coulter, Victoria Kett, Tracy Robson, Helen McCarthy Queen’s University, Belfast
Primary breast cancer, the most common malignancy in women, is a disease with many treatment options, and impressive response rates. However, treatment options are limited once tumors metastasize. Docetaxel is the front line therapy for metastatic breast cancer, but resistance eventually develops, limiting its potency. Nitric oxide (NO•) exerts anticancer effects by inducing DNA damage, modifying DNA repair proteins, and inhibiting anti-apoptotic factor NF-κB. We have previously reported the therapeutic benefit of inducible NO• synthase (iNOS) gene therapy in several xenograft models of human cancer. The purpose of the current study was to investigate the antimetastatic potential of NO• using a transcriptionally targeted iNOS gene construct and our novel delivery technology. We have developed a peptide-based gene therapy delivery vehicle that produces cationic nanoparticles (NPs) when incubated with plasmid iNOS (piNOS). The NPs overcome the biological barriers to gene therapy, efficiently delivering piNOS to the nucleus of breast cancer cells. piNOS-loaded NPs (constitutively active CMV or transcriptionally-regulated human osteocalcin [hOC] promoters) evoked iNOS gene expression in MDA-MB-231-Luc-D3H1 breast cancer cells in vitro, which manifested impressive nitrite accumulation in culture medium (indicative of NO• generation). C57/BL6 mice tolerated multiple administrations of pDNA-loaded NPs, and their sera lacked NP-neutralizing antibodies. These results indicated that iNOSloaded NPs have therapeutic potential in in vivo models of breast cancer metastasis. Metastatic breast cancer was established in female BALB/c SCID mice by inoculation with 2 × 10 5 MDA-MB-231-LucD3H1 via the left ventricle, and disease progression was assessed via bioluminescence imaging and body weight determination. Treated mice received 50 μg CMV-iNOS/hOC-iNOS in five intravenous biweekly doses. Serum nitrite levels were elevated in mice that received either iNOS gene therapy. Mice that received CMV-iNOS (median survival 40 days; p < 0.001) or hOCiNOS (38.5 days; p = 0.001) therapy had significantly improved survival compared with control mice (31.5 days). Moreover, combining docetaxel therapy with either CMV-iNOS (51 days) or hOC-iNOS (46 days) improved median survival of mice beyond that observed with docetaxel alone (44 days). Targeted NO• gene therapy has shown excellent therapeutic potential for metastatic breast cancer, and studies are ongoing to develop our NP formulation toward a potential clinical product.
111
Keywords: Gene therapy; Breast cancer; Metastasis; Nitric oxide. Oral 2011-1. The therapeutic effects of a nitric oxide generating lozenge that utilizes natural product chemistry http://dx.doi.org/10.1016/j.niox.2014.09.040 Nathan BryanPhD The University of Texas Health Science Center
The production and availability of nitric oxide (NO) is critical for cardiovascular health. The complex production of NO from L-arginine by nitric oxide synthase becomes compromised with age and certain elements of lifestyle. As this primary pathway for NO becomes dysfunctional, it leads to conditions of NO deficiency with subsequent clinical sequel including hypertension and onset and progression of cardiovascular disease. A complementary endogenous pathway for NO production emerged wherein inorganic nitrate and nitrite, produced from both the oxidation of NO and dietary nitrate-rich functional foods such as leafy green vegetables and beets, can be reduced back to NO through enterosalivary cycling. Utilizing intellectual property developed out of the University of Texas Health Science Center in Houston (US patents 8,298,589, 8,303,995 & 8,435,570), Neogenis Laboratories has licensed this technology and developed a GMP certified, over the counter, all natural formulation that provides a system for generating NO in an endothelium-dependent and independent manner. This technology has been clinically tested in placebo controlled designed studies. A single administration of an oral, active, naturebased NO supplement significantly lowers blood pressure, improves vascular compliance and restores endothelial function in subjects with hypertension. Thirty day administration also leads to normalization of blood pressure in pre-hypertensive patients. Pharmaceutical approaches for NO restoration present long-term safety issues; however, functional, nutraceutical approaches may offer select advantages for restoring NO availability. This treatment may be beneficial as routine supplementation for cardiovascular protection. Keywords: Nitrite; Nutraceutical; Diet. Oral 2011-2. Ammonia-oxidizing bacteria accelerate wound closure in diabetic mice http://dx.doi.org/10.1016/j.niox.2014.09.041 Ioannis Gryllos a, Shomir Ghosh a, Neeraja Vajrala b, David Whitlock a, Luis Sayavedra-Soto b, Spiros Jamas a a AOBIOME LLC, Cambridge, MA, USA b Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA
Diabetic foot ulcers (DFUs) are among the most common and serious complications of diabetes. They present in approximately 15% of diabetic patients, leading to ~80,000 amputations per year in the United States alone. Effective strategies for the treatment of DFUs are currently limited, thus highlighting an urgent unmet medical need. Topical delivery of nitric oxide (NO) at the wound site via NO-generating compounds, or by using acidified nitrite as a NO source, has been shown to promote healing in murine models of diabetes by reversing dysregulated expression of inflammatory cytokines, growth factors and matrix metalloproteinases among others. In the present study, we have evaluated the use of ammonia-oxidizing bacteria (AOB) of the genus Nitrosomonas as natural self-regulating biological delivery systems of NO/NOx in vivo. Nitrosomonas are ubiquitous Gram-negative obligate chemolithoautotrophic bacteria that exclusively oxidize ammonia as an energy source to generate nitrite and NOx species. As proof-of-concept, we proceeded to examine
112
Abstracts/Nitric Oxide 42 (2014) 99–153
the capacity of a novel Nitrosomonas eutropha strain to accelerate wound closure in diabetic db/db mice. This strain, designated D23, was initially isolated from organic soil, then extensively characterized for nitrite generation and propagated in continuous culture for increased biomass yields. For testing in the db/db model, three groups of eight mice each were treated daily for one week with D23 suspensions supplemented with ammonium chloride, or with vehicle control suspension only. Subsequently, full-size wounds generated on the back of each animal were treated topically once daily for 14 days with bacterial suspension or vehicle alone. Of the three AOB-treated groups, the group receiving the highest D23 doses showed significant improvement in wound closure from day 5 to day 15, with the most pronounced improvement of 83% observed on day 9 post-wounding. Based on these findings, we are exploring the potential therapeutic benefit(s) of AOB to DFUs, chronic wounds and other related indications. Keywords: Ammonia-oxidizing bacteria; Nitrosomonas; Nitrite; Nitric oxide; Wound healing; Diabetes.
Oral 2012-1. Interfacial residues promote an optimal alignment of the catalytic center in soluble guanylate cyclase http://dx.doi.org/10.1016/j.niox.2014.09.042 Elsa Garcin a, Franziska Seeger b, Royston Quintyn c, Akiko Tanimoto c, Gareth Williams d, John Tainer e, Vicki Wysocki c a University of Maryland Baltimore County b UMBC c Ohio State University d Lawrence Berkeley Laboratory e TSRI/LBL
Most of what we know about the guanylate cyclase catalytic mechanism derives from comparisons with related adenylate cyclases. As a structure of the active heterodimeric cyclase domain remains elusive, there is still much debate about structural requirements for cyclase activity and possible conformational changes needed to achieve full catalytic capacity. Several studies suggest that the heme-containing regulatory domain binds to the catalytic domain and maintains sGC in an auto-inhibited basal state. NO binding to heme, thought to be communicated to the cyclase domain via the regulatory and dimerization domains, releases the full catalytic potential of sGC. If this model is correct, isolated catalytic domains should display catalytic activity analogous to that of activated sGC. Using X-ray crystallography, activity measurements, and native mass spectrometry, we demonstrated that isolated catalytic domains are much less active than basal sGC, despite being able to form heterodimers. Our X-ray structure revealed key structural elements that modulate the dimer interface. We further propose a novel role for residues located in an interfacial flap and a hydrogen-bond network as key modulators of the orientation of the catalytic subunits. Our results unambiguously show that even in the absence of the regulatory domain, additional sGC domains are required to guide the appropriate conformation of the catalytic subunits associated with high activity. We propose a novel regulatory mechanism whereby sGC activity is tuned by distinct domain interactions that either promote or inhibit catalytic activity. These results further our understanding of sGC activation and regulation, and open additional drug discovery routes for targeting the NO–sGC–cGMP pathway via the design of small molecules that promote a productive conformation of the catalytic subunits or disrupt inhibitory domain interactions.
Keywords: Soluble guanylate cyclase; Enzyme mechanism; Regulation; X-ray crystallography; Conformational changes; Domain–domain interactions.
Oral 2012-2. Toward structural insights into activation and modulation of the soluble guanylyl cyclase http://dx.doi.org/10.1016/j.niox.2014.09.043 Vijay Kumar a, Jonathan Stamler b, Johannes-Peter Stasch b, Michael Hahn c, Martina Schaefer c, Athanassios Giannis c, Andreas Papapetropoulos c, Focco van den Akker d a CWRU b Bayer Pharma AG, Aprather Weg 18a, D-42096 Wuppertal, Germany c Department of Pharmacy, University of Patras, 26504 Patras, Greece d Case Western Reserve University
Objective/purpose: Soluble guanylyl cyclase (sGC), the principal receptor of nitric oxide (NO), converts GTP into cGMP that regulates several physiological functions. Under some abnormal cardiovascular processes, sGC activity is not optimal and thus spurs the need for further molecular studies investigating this as well as the development of novel stimulators and activators of sGC. Compounds BAY58-2667 (BAY58) and BAY602770 (BAY60) are NO-independent activators of sGC and had been already tested in clinical trials. Earlier our lab proposed the heme pivot–bend mechanism for sGC activation involving a shift of the H105-containing αF helix. In the current study we demonstrate how the study of existing and novel sGC activators may aid to a better understanding of sGC activation and ultimately to obtain better activators of sGC using X-ray crystallography. Results: Our lab solved the crystal structures of BAY58 and BAY60 bound to Nostoc (Ns) H-NOX domain. We noticed an interesting observation regarding the size of the substituents of the hydrophobic linker with respect to the size of heme pocket and the distortion of loop region following the important αF helix in H-NOX domain. Previous clinical trial studies for acute decompensated heart failure patients revealed that BAY58 leads to the dose-dependent hypotension development. On the basis of our structural information, an improved activator was developed with 5-fold higher cGMP-stimulatory properties compared to BAY58 and we probed the structural basis for its higher activation of sGC. Furthermore, we also solved the crystal structure of BAY60-bound Ns HNOX protein having the conserved C122 residue S-nitrosylated. S-nitrosylation conferred some subtle conformational changes nearby the heme binding cavity that could lead to the structural basis for the desensitization of sGC via S-nitrosylation. Conclusions: Using BAY58 and BAY60 bound Ns H-NOX structural comparisons; an improved activator with five-fold higher cGMP-stimulatory properties was developed. We could also utilize BAY60 for understanding the structural basis of desensitization of sGC via S-nitrosylation of C122. Keywords: Soluble guanylyl cyclase; Heme; Nitric oxide; Activation; BAY58; BAY60; S-nitrosylation.
Oral 2012-3. Nitric oxide and heat shock protein 90 activate soluble guanylate cyclase by driving rapid change in its subunit interactions and heme content http://dx.doi.org/10.1016/j.niox.2014.09.044 Arnab Ghosh a, Johannes-Peter Stasch b, Andreas Papapetropoulos c, Dennis Stuehr a a Department of Pathobiology, Cleveland Clinic, Cleveland, OH 44195, USA b Bayer Pharma AG, Aprather Weg 18a, D-42096 Wuppertal, Germany c Department of Pharmacy, University of Patras, 26504 Patras, Greece