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Table S2 is a summary of AO-176 tumor cell killing in solid and hematologic tumor cell lines

Table S1 shows the affinity and kinetics of AO-176 and AO-104 to human and cynomolgus monkey CD47 determined by surface plasmon resonance

Figure S2 shows that AO-176 does not bind to mouse CD47

Figure S5 demonstrates that AO-176 does not mediate antibody dependent cell cytotoxicity of tumor cells

Table S3 is a summary of AO-176 mediated phagocytosis of solid and hematologic tumor cells

Figure S3 shows the specificity of AO-176 for human CD47

Figure S1 depicts the sequence and analytical characteristics of AO-176

Figure S4 demonstrates the minimal reduction in RBC and hemoglobin levels in cynomolgus monkeys treated with AO-176

Toward the goal of using more livers for transplantation, transplant centers are looking to increase the use of organs from "marginal" donors. Livers from these donors, however, have been shown to be more susceptible to preservation and reperfusion injury.Using a porcine model of donation after circulatory death, we studied the use of antibody-mediated CD47 blockade to further improve liver graft function undergoing normothermic machine perfusion. Livers from 20 pigs (5 per group) were brought under either 30 or 60 min of warm ischemia time followed by the administration of CD47 monoclonal antibody (CD47mAb) treatment or immunoglobulin G control antibodies and 6 h of normothermic extracorporeal liver perfusion.After 6 h of normothermic extracorporeal liver perfusion, CD47mAb-treated livers with 30 or 60 min warm ischemia time had significantly lower alanine transaminase levels and higher bile production compared with their respective control groups. Blockade of the CD47 signaling pathway resulted in significantly lower thrombospondin-1 protein levels, lower expression of caspase-3, and higher expression of phosphorylated extracellular signal-regulated kinase.These findings suggested that CD47mAb treatment decreases ischemia/reperfusion injury through CD47/thrombospondin-1 signaling downregulation and the presence of necrosis/apoptosis after reperfusion and could increase liver regeneration during normothermic perfusion of the liver.

Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and overexpressed on many tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggers a “don't eat me” signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next-generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPα interaction to induce tumor cell phagocytosis, but also induces tumor cytotoxicity in hematologic and solid human tumor cell lines, but not normal noncancerous cells, by a cell autonomous mechanism (not ADCC). AO-176 also binds preferentially to tumor versus many normal cell types. In particular, AO-176 binds negligibly to RBCs in contrast to tumor cells, even at high concentrations up to 200 μg/mL and does not agglutinate RBCs up to 1 mg/mL in vitro. These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. When tested in cynomolgus monkeys, AO-176 was well tolerated with no adverse effects. Finally, we show that AO-176 demonstrates dose-dependent antitumor activity in tumor xenograft models. Taken together, the unique properties and antitumor activity of our next-generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPα axis targeting agents in clinical development.

CD47 is a cell surface transmembrane protein that binds to signal regulatory protein alpha (SIRPα) on macrophages and results in a “don't eat me” signal that inhibits phagocytosis. Breast cancer cells, both primary and metastatic, frequently overexpress CD47 and exploit this pathway to evade macrophage-mediated destruction. Anti-CD47 monoclonal antibodies (mAbs) block the CD47/SIRPα interaction and promote tumor cell destruction via phagocytosis. Anti-CD47 mAbs also contribute to an anti-tumor T-cell response in immune-competent mice. Therefore, anti-CD47 antibodies represent a new class of immune checkpoint inhibitors that modulate both the innate and adaptive immune systems. CD47 is expressed on multiple cell types, including tumor cells and normal cells. Many anti-CD47 mAbs block the CD47/SIRPα interaction and cause phagocytosis of tumor cells, but do not directly induce the death of human tumor cells. Tioma has created new humanized anti-CD47 mAbs with novel and differentiated functional profiles to enhance functional heterogeneity. Ti-104, Ti-176 and Ti-108 block the binding of SIRPα to CD47 and increase phagocytosis of human tumor cells. They also induce cell death of human hematological and solid tumor cell lines (including breast cancer lines) in a cell autonomous manner. Cell death was determined by an increase in phosphatidylserine-positive/7AAD-positive tumor cells assessed by flow cytometry following incubation in media containing anti-CD47 mAb or a negative control immunoglobulin. In vitro, these mAbs bind to human tumor cell lines with apparent binding affinities ranging from low pM to low nM, depending on the cell line and method of analysis (solid-phase or cell-based ELISA, flow cytometry or surface plasmon resonance). In vitro, Ti-104 and Ti-108 bind to human RBCs, whereas Ti-176 has markedly reduced binding to human and cynomolgus monkey RBCs. In a four-week (once-weekly dosing) exploratory safety study in cynomolgus monkeys with Ti-176 and Ti-108, no dose-limiting toxicity or gross pathological or microscopic findings were identified after an initial dose of 5 mg/kg (Week 1) followed by doses of 50 mg/kg (Weeks 2, 3, and 4). Ti-176 treatment resulted in minimal decrease in red cell mass, hemoglobin and hematocrit, which corresponded in vitro to markedly reduced binding to cynomolgus monkey RBCs. Ti-108 caused transient reduction of RBC parameters comparable to some previously reported anti-CD47 mAbs. Ti-104, Ti-176 and Ti-108 showed potent, dose-dependent efficacy in multiple mouse tumor models, including in the MDA-MB-231 triple-negative breast cancer orthotopic model. These data provide the preclinical rationale for further evaluation of Ti-104, Ti-176 and Ti-108 as breast cancer treatments. Citation Format: Karr RW, Liu K, Hiebsch R, Capoccia B, Donio M, Crowley K, Puro R, Chanda S, Manning P. Development of humanized anti-CD47 monoclonal antibodies with differentiated functional profiles [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-15.

It remains controversial whether renal allografts from donation after circulatory death (DCD) have a higher risk of acute rejection (AR). In the porcine large animal kidney transplant model, we investigated the AR and function of DCD renal allografts compared to the non-DCD renal allografts and the effects of increased immunosuppression. We found that the AR was significantly increased along with elevated MHC-I expression in the DCD transplants receiving low-dose immunosuppression; however, AR and renal function were significantly improved when given high-dose immunosuppressive therapy postoperatively. Also, high-dose immunosuppression remarkably decreased the mRNA levels of ifn-g, il-6, tgf-b, il-4, and tnf-a in the allograft at day 5 and decreased serum cytokines levels of IFN-g and IL-17 at day 4 and day 5 after operation. Furthermore, Western blot analysis showed that higher immunosuppression decreased phosphorylation of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells-p65, increased phosphorylation of extracellular-signal-regulated kinase, and reduced the expression of Bcl-2-associated X protein and caspase-3 in the renal allografts. These results suggest that the DCD renal allograft seems to be more vulnerable to AR; enhanced immunosuppression reduces DCD-associated AR and improves early allograft function in a preclinical large animal model.

Background The susceptibility of extended criteria livers to ischemia reperfusion injury is a major obstacle in organ cold preservation. Normothermic extracorporeal liver perfusion (NELP) has been investigated to reduce ischemic damage, restore physiologic function, and assess viability of the liver prior to transplant. The goal of this study is to compare physiological parameters of livers maintained continuously on NELP to those preserved in cold solution. Methods Livers from 9 female landrace pigs were subjected to either 20 minutes (W20-NELP), 40 minutes (W40-NELP), or 60 minutes (W60-NELP) of warm ischemia followed by 6 hours of NELP followed by a 2-hour NELP evaluation phase. This was compared with 3 livers subjected to 40 minutes of warm ischemia time followed by 6 hours of cold storage (W40-Cold) and a 2-hour NELP evaluation phase. Groups were compared with the 2-way analysis of variance test. Results NELP stabilized transaminases accompanied by significant improvement in bile production and decline in lactate and INR values in all W-NELP groups. Histologic analysis demonstrated significant improvement from 0 hour (mild-to-moderate sinusoidal dilation and zone 3 necrosis) to the end of the NELP run (minimal necrosis and mild IRI). Comparison of W40-NELP and W40-Cold revealed greater bile production and oxygen extraction ratio in W40-NELP. In contrast, markers of cellular and functional damage were increased in the W40-Cold group. Conclusion NELP improves metabolic and functional parameters of livers with either short or extended warm ischemia times compared with livers subjected to comparable cold ischemia times.

Orthotopic liver transplantation (OLT) remains the standard treatment option for nonresponsive liver failure. Because ischemia/reperfusion injury (IRI) is an important impediment to the success of OLT, new therapeutic strategies are needed to reduce IRI. We investigated whether blocking the CD47/thrombospondin-1 inhibitory action on nitric oxide signaling with a monoclonal antibody specific to CD47 (CD47mAb400) would reduce IRI in liver grafts. Syngeneic OLT was performed with Lewis rats. Control immunoglobulin G or CD47mAb400 was administered to the donor organ at procurement or to both the organ and the recipient at the time of transplant. Serum transaminases, histological changes of the liver, and animal survival were assessed. Oxidative stress, inflammatory responses, and hepatocellular damage were also quantified. A significant survival benefit was not achieved when CD47mAb400 was administered to the donor alone. However, CD47mAb400 administration to both the donor and the recipient increased animal survival afterward. The CD47mAb400-treated group showed lower serum transaminases, bilirubin, oxidative stress, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining, caspase-3 activity, and proinflammatory cytokine expression of tumor necrosis factor α, interleukin-1β, and interleukin-6. Thus, CD47 blockade with CD47mAb400 administered both to the donor and the recipient reduced liver graft IRI in a rat liver transplantation model. This may translate to decreased liver dysfunction and increased survival of liver transplant recipients.

Modulation of nitric oxide (NO) activity through blockade of CD47 signaling has been shown to reduce ischemia-reperfusion injury (IRI) in various models of tissue ischemia. Here, we evaluate the potential effect of an antibody-mediated CD47 blockade in a syngeneic and an allogeneic DCD rat kidney transplant model. The donor organ was subjected to 1 hr of warm ischemia time after circulatory cessation, then flushed with a CD47 monoclonal antibody (CD47mAb) in the treatment group, or an isotype-matched immunoglobulin in the control group. We found that CD47mAb treatment improved survival rates in both models. Serum markers of renal injury were significantly decreased in the CD47mAb treated group compared with the control group. Histologically the CD47mAb treated group had significantly reduced scores of acute tubular injury and acute tubular necrosis. The expression of biomarkers related to mitochondrial stress and apoptosis also were significantly lower in the CD47mAb treated groups. Overall, the protective effects of CD47 blockade were greater in the syngeneic model. Our data show that CD47mAb blockade decreased the IRI of DCD kidneys in rat transplant models. This therapy has the potential to improve DCD kidney transplant outcomes in the human setting. This article is protected by copyright. All rights reserved.

Ischemia-reperfusion injury (IRI) significantly contributes to delayed graft function and inflammation, leading to graft loss. Ischemia-reperfusion injury is exacerbated by the thrombospondin-1-CD47 system through inhibition of nitric oxide signaling. We postulate that CD47 blockade and prevention of nitric oxide inhibition reduce IRI in organ transplantation.We used a syngeneic rat renal transplantation model of IRI with bilaterally nephrectomized recipients to evaluate the effect of a CD47 monoclonal antibody (CD47mAb) on IRI. Donor kidneys were flushed with CD47mAb OX101 or an isotype-matched control immunoglobulin and stored at 4°C in University of Wisconsin solution for 6 hr before transplantation.CD47mAb perfusion of donor kidneys resulted in marked improvement in posttransplant survival, lower levels of serum creatinine, blood urea nitrogen, phosphorus and magnesium, and less histological evidence of injury. In contrast, control groups did not survive more than 5 days, had increased biochemical indicators of renal injury, and exhibited severe pathological injury with tubular atrophy and necrosis. Recipients of CD47mAb-treated kidneys showed decreased levels of plasma biomarkers of renal injury including Cystatin C, Osteopontin, Tissue Inhibitor of Metalloproteinases-1 (TIMP1), β2-Microglobulin, Vascular Endothelial Growth Factor A (VEGF-A), and clusterin compared to the control group. Furthermore, laser Doppler assessment showed higher renal blood flow in the CD47mAb-treated kidneys.These results provide strong evidence for the use of CD47 antibody-mediated blockade to reduce IRI and improve organ preservation for renal transplantation.

Recent success in cancer immunotherapy has targeted immune checkpoints such as PD-1, PDL-1, and CTLA-4 to enhance the cytotoxic activity of the adaptive T-cell immune response. While the clinical response to these therapies has been dramatic for some, many others have shown partial or even no response highlighting the need for alternative or synergistic approaches that activate innate immunity. Disruption of the interaction between SIRP alpha and CD47, an innate checkpoint inhibitor, using anti-CD47 antibodies, for example, is known to enhance innate immunity by increasing the phagocytosis of tumor cells by macrophages and dendritic cells (DCs) leading to processing and presentation of tumor antigens. Recently, we described AO-176, a next generation anti-CD47 antibody that blocks the CD47/SIRP alpha interaction, induces phagocytosis and causes a direct tumor cell-autonomous death while negligibly binding RBCs. Herein, we characterize the ability of our CD47 antibodies such as AO-176 to induce immunogenic cell death (ICD) and damage-associated molecular patterns (DAMPs) in tumor cells and to potentiate chemotherapy-induced ICD/DAMPs. ICD is a process whereby an agent induces cell surface exposure and release of DAMPs from dying cells which stimulates DCs and adaptive immune responses. Tumor cells were treated in vitro with our CD47 antibodies either alone or in combination with chemotherapeutics followed by assessment of ICD/DAMPs using flow cytometry and biochemical assays. RNAseq was also performed on cells undergoing CD47 antibody mediated ICD/DAMP induction to better understand how CD47 inhibition may regulate ICD. AO-176 and other CD47 antibodies, developed by Arch Oncology, caused mitochondrial stress and loss of outer-membrane integrity, typically observed prior to cells undergoing apoptosis. In addition, CD47 antibody treatment induced a significant ER stress response at the genetic level resulting in the surface exposure of ER chaperone proteins calreticulin, Hsp90, and PDIA3. Concomitantly, our CD47 antibodies increased autophagy and JAK/STAT signaling, which resulted in both ATP and HMGB1 release, respectively. Finally, we demonstrated that in combination, our antibodies potentiated the effects of ICD/DAMP-inducing chemotherapy (e.g., doxorubicin). Here, we describe the unique ability of a specific subset of next generation CD47 antibodies, such as AO-176 to induce ICD/DAMPs. RNAseq analysis of treated cells also revealed alteration of several pathways, including those where DAMPs play a role. In summary, next-generation CD47 antibodies such as AO-176 may provide a novel approach to enhancing the current landscape of checkpoint immunotherapy by enhancing both the innate and adaptive immune responses against tumors. Citation Format: Daniel S. Pereira, Benjamin J. Capoccia, Ronald R. Hiebsch, Michael J. Donio, Alun J. Carter, Robyn J. Puro, W. Casey Wilson, Pamela T. Manning, Robert W. Carr. AO-176, a next-generation anti-CD47 antibody, induces immunogenic cell death [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A147.

Despite the efficacy of orthotopic liver transplantation in the treatment of end-stage liver diseases, its therapeutic utility is severely limited by the availability of donor organs. The ability to rehabilitate marginal organs, such as steatotic allografts, has the potential to address some of the supply limitations of available organs for transplantation. Steatotic livers are more susceptible to ischemia-reperfusion injury (IRI), which is exacerbated by the thrombospondin-1/CD47 pathway through inhibition of nitric oxide signaling. We postulated that CD47 blockade with a monoclonal antibody specific to CD47, clone 400 (CD47mAb400) may reduce the extent of IRI in steatotic liver allografts.Orthotopic liver transplantation was performed using steatotic liver grafts from Zucker rats transplanted into lean recipients. Control IgG or the CD47mAb400 was administered to the donor livers at procurement. Serum transaminases, histological changes, and animal survival were assessed. Hepatocellular damage, oxidative and nitrosative stress, and inflammation were also quantified.Administration of CD47mAb400 to donor livers increased recipient survival and resulted in significant reduction of serum transaminases, bilirubin, triphosphate nick-end labeling staining, caspase-3 activity, oxidative and nitrosative stresses, and proinflammatory cytokine expression of TNF-α, IL-6 and IL-1β.We conclude that administration of CD47mAb400 to donor grafts may reduce IRI through CD47 blockade to result in improved function of steatotic liver allografts and increased survival of recipients and represent a novel strategy to allow the use of livers with higher levels of steatosis.

Inhibitors of adaptive immune checkpoints have shown promise as cancer treatments. CD47 is an innate immune checkpoint receptor broadly expressed on normal tissues and over-expressed on several tumors. Binding of tumor CD47 to signal regulatory protein alpha (SIRPalpha) on macrophages and dendritic cells triggers a "don't eat me" signal that inhibits phagocytosis enabling escape of innate immune surveillance. Blocking CD47/SIRPα interaction promotes phagocytosis reducing tumor burden in numerous xenograft and syngeneic animal models. We have developed a next generation humanized anti-CD47 antibody, AO-176, that not only blocks the CD47/SIRPalpha interaction and induces phagocytosis of hematologic and solid tumor cells, but also exhibits several unique functional properties. The first property is the ability of AO-176 to induce direct tumor cytotoxic cell death in hematologic (ex. Jurkat, Raji and Molt-4) as well as solid human tumor cell lines by a cell autonomous mechanism (not ADCC). Secondly, AO-176 exhibits preferential binding to tumor versus normal cells, including red blood cells (RBCs), T cells, endothelial cells, skeletal muscle cells and epithelial cells. A0-176 also does not affect the function of any of these primary cells when assayed ex vivo. The negligible binding of AO-176 to RBCs versus hematologic (ex. Jurkat, Raji or Molt-4) or solid tumor cells is particularly profound and different from other reported anti-CD47 antibodies. AO-176 also does not induce hemagglutination of RBCs. These properties are expected not only to decrease the antigen sink, but also to minimize on-target clinical adverse effects observed following treatment with other reported RBC-binding anti-CD47 antibodies. Consistent with this attribute, AO-176 was well tolerated in cynomolgus monkeys with no adverse effects in general nor with respect to RBCs which was consistent with ex vivo results. A third novel property of AO-176 is its enhanced binding to tumor cells at acidic pH. Because the microenvironment of leukemic bone marrow and solid tumors has an acidic pH, this enhanced binding of AO-176 at low pH has the potential added advantage of tumor-specific targeting. Lastly, we show that AO-176 demonstrates dose-dependent anti-tumor activity in hematologic and solid tumor xenograft models. Taken together, the unique properties and anti-tumor activity of our next generation anti-CD47 antibody, AO-176, distinguishes it from other CD47/SIRPalpha axis targeting agents as it progresses to clinical development. Disclosures No relevant conflicts of interest to declare.

CD47 is a cell surface glycoprotein that interacts with signal regulatory protein alpha (SIRPα) on macrophages and dendritic cells triggering a “don't eat me” signal that inhibits phagocytosis. Many tumors evade immune surveillance by overexpressing CD47, thereby preventing their recognition by phagocytes. Blocking the interaction of SIRPα/CD47 promotes phagocytosis and tumor cell destruction leading to a reduction in tumor burden. We have developed a humanized anti-CD47 antibody, AO-176, that blocks the interaction between CD47 and SIRPα and exhibits several additional novel functional characteristics. These characteristics include the induction of cell death in multiple human tumor cell lines in a cell autonomous manner (not ADCC), assessed by an increase in phosphatidylserine/7AAD positive staining. A second novel characteristic is enhanced binding to tumor cells at acidic pH. AO-176 binds to human tumor cell lines in the high pM to low nM range at physiologic pH, however, binding is enhanced up to 20-fold at an acidic pH of 6.5. The acidic pH of the tumor microenvironment which ranges from 6.4-7.2 is characteristic of solid tumors and correlates with tumor progression and metastasis. As a result of this enhanced binding at acidic pH, AO-176 has the potential added advantage of tumor-specific targeting. A third novel characteristic exhibited by AO-176 is its selective binding to tumor cells while exhibiting reduced binding to normal cells including red blood cells (cynomolgus monkey and human), endothelial, epithelial and skeletal muscle cells. In addition to these novel characteristics, AO-176 also exhibits dose-dependent efficacy in multiple mouse tumor models. Taken together, the unique combination of functional characteristics of AO-176, including induction of cell-autonomous killing, enhanced binding to tumor cells at acidic pH, significantly reduced binding to normal cells and potent in vivo efficacy provides the preclinical rationale for further development. Citation Format: Robyn Puro, Katherine Liu, Benjamin Capoccia, Michael Donio, Ronald Hiebsch, Myriam Bouchlaka, Alun Carter, Pamela Manning, Kathleen Crowley, Robert Karr. A humanized anti-CD47 monoclonal antibody that directly kills human tumor cells and has additional unique functional characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1765.

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival times. The efficacy of current systemic therapies for HCC is limited. In this study, we used xenograft tumor models to investigate the use of antibodies that block CD47 and inhibit HCC tumor growth. Immunostaining of tumor tissue and HCC cell lines demonstrated CD47 over-expression in HCC as compared to normal hepatocytes. Macrophage phagocytosis of HCC cells was increased after treatment with CD47 antibodies (CD47mAbs) that block CD47 binding to SIRPα. Further, CD47 blockade inhibited tumor growth in both heterotopic and orthotopic models of HCC, and promoted the migration of macrophages into the tumor mass. Our results demonstrate that targeting CD47 by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Following experimental, transient, retinal ischemia in the rat, there is loss of retinal neurons, which occurs over several weeks. Retinal ganglion cells (RGCs) are particularly susceptible and there is early, massive degeneration of these neurons after ischemia. We have determined the early mechanisms by which RGCs are killed following ischemia. Retinal ischemia/reperfusion was produced in rats by transient unilateral elevation of intraocular pressure above systolic blood pressure. Retinas were studied by immunohistochemistry for the presence of inducible nitric oxide synthase (NOS-2) at several time points post-ischemia and specific cell types were identified. Rats were also treated orally with L -N(6) -(1-iminoethyl)lysine 5-tetrazole amide (SC-51), a prodrug of an inhibitor of NOS-2 or with aminoguanidine (AG) for a period of 14 days. Retrograde labelling with Fluoro-Gold quantitated the loss of RGCs. NOS-2 was not present in the normal retina and was not present in the eyes that were contralateral to the ischemic eyes. Within 24hr after ischemia, polymorphonuclear leukocytes containing NOS-2 had entered the ganglion cell layer and surrounded RGCs. Within 5 days after ischemia, NOS-2 was present in many inner retina cells and in invading monocytes in the vitreous. Between 7 and 14 days post-ischemia, there were few hematogenous cells in the retina but NOS-2 was sparsely detectable in microglia and other cells of the inner retina. Two weeks after ischemia, rat eyes lost approximately 50% of the RGCs. Treatment with AG for 14 days following ischemia was partially neuroprotective; approximately 28% of the RGCs were lost. Treatment with SC-51 for 14 days following ischemia almost completely prevented the loss of RGCs. Thus, within 24hr following ischemia, polymorphonuclear leukocytes containing NOS-2 attack and kill neurons in the ganglion cell layer. For 2 weeks after ischemia, NOS-2 appears transiently in the retina in several different cell types at different times. Continuous pharmacological treatment with inhibitors of NOS-2 activity during the 2 weeks post-ischemia period provides significant neuroprotection against the loss of RGCs.

International audience; OBJECTIVE:To investigate in situ the relationship between 2 key mediators implicated in osteoarthritic (OA) cartilage: nitric oxide (NO) and interleukin-1-converting enzyme (ICE). Interleukin-18 (IL-18) was also studied and served as reference for the effects of ICE.METHODS:An OA model was created in dogs by sectioning (stab wound) the anterior cruciate ligament of the right stifle joint. Three experimental groups were studied: unoperated untreated dogs, operated untreated dogs (OA), and OA dogs treated with oral N-iminoethyl-L-lysine (L-NIL), a specific inhibitor of inducible nitric oxide synthase (iNOS) (10 mg/kg twice a day starting immediately after surgery). At 12 weeks after surgery, cartilage from the femoral condyles and tibial plateaus were processed for immunohistochemistry for ICE, IL-18, and protease inhibitor 9 (PI-9), a natural inhibitor of ICE, followed by morphometric analysis. Cartilage specimens from the femoral condyles of untreated OA dogs were dissected and incubated with specific inhibitors of different signaling pathways likely to be involved in the OA process: SB 202190 (10 microM; a p38 mitogen-activated protein kinase [MAPK] inhibitor), PD 98059 (100 microM; a MAPK kinase 1/2 [MEK-1/2] inhibitor), NS-398 (10 ng/ml; a specific cyclooxygenase 2 [COX-2] inhibitor), and L-NIL (50 microM).RESULTS:Both ICE and IL-18 were present in situ in the canine cartilage, with a significant increase in the level of these 2 proteins in OA cartilage. In contrast, the level of PI-9 was lower in OA than in normal cartilage (difference not statistically significant). Compared with untreated OA cartilage, oral treatment with L-NIL significantly decreased ICE and IL-18 levels in cartilage from the femoral condyles and tibial plateaus, to values similar to those in normal dogs. L-NIL also increased the PI-9 level in normal dogs compared with OA dogs, reaching statistical significance for femoral condyle cartilage. Interestingly, in vitro experiments demonstrated significant inhibition of ICE levels by p38, MEK-1/2, and COX-2 inhibitors, but not by the iNOS inhibitor.CONCLUSION:This study demonstrated that in situ in OA cartilage, the stimulation of chondrocytes by NO is at least partly responsible for the up-regulation of ICE and IL-18 synthesis while decreasing the level of the ICE inhibitor PI-9. The ICE level is controlled by the activation of at least 2 MAPK pathways, p38 and MEK-1/2. Interestingly, it appears that ICE synthesis is not regulated by the endogenous production of NO. These data highlight the role played by iNOS in regulating the synthesis of major catabolic factors involved in OA cartilage degradation.

To test the hypothesis that nitric oxide, synthesized by inducible nitric oxide synthase, causes degeneration of retinal ganglion cells in an animal model of glaucoma.Rats with unilateral, chronic, moderately elevated intraocular pressure were treated orally with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of an inhibitor of inducible nitric oxide synthase. The loss of retinal ganglion cells was quantitated as an indicator of glaucomatous damage.At the end of seven months, rat eyes with chronic, moderately elevated intraocular pressure lost approximately 20,000 retinal ganglion cells. Treatment with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide for seven months completely prevented the loss of retinal ganglion cells in eyes with chronic, moderately elevated intraocular pressure. When treatment with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide was delayed and started after three months of chronic, moderately elevated intraocular pressure, further loss of retinal ganglion cells was prevented.Pharmacological neuroprotection with a selective inhibitor of inducible nitric oxide synthase may be useful for the treatment of glaucoma.

The efficient syntheses of two new types of conformationally constrained S-[2-[(1-iminoethyl)amino]ethyl]homocysteine derivatives, 1-amino-3-[2[(1-iminoethyl)amino]ethylthio]cyclobutane carboxylic Acid (5) and (4S)-4-[[2-[(1-Iminoethyl)amino]ethyl]thio]-L-proline (6), are reported. These molecules represent the first attempts to probe conformational constraint near the α-amino acid moiety of known homocysteine-based inhibitors of nitric oxide synthase. Targets 5 and 6 were evaluated as potential inhibitors of the three human isoforms of nitric oxide synthase. © 2002 John Wiley & Sons, Inc. Heteroatom Chem 13:77–83, 2002; DOI 10.1002/hc.1109

TPS3109 Background: The cell surface protein CD47 is expressed or over-expressed on many tumor types. CD47 binds to signal regulatory protein alpha (SIRPα) on macrophages resulting in a “don’t eat me” signal that blocks host cell phagocytosis of the tumor cells, thus allowing them to escape removal by the innate immune system. Recent data indicate that anti-CD47 antibodies also contribute to an effective anti-tumor T cell response in immune-competent mice. Therefore, anti-CD47 antibodies are a new class of immune checkpoint inhibitors that modulate both the innate and adaptive immune systems. Ti-061 is a novel IgG4 humanized monoclonal antibody that specifically binds to CD47 with Kd values range from 100 – 500 pM. Ti-061 exhibits cross-species binding to cynomolgus monkey, mouse and rat CD47, enabling efficacy and toxicity testing across species. Ti-061 binds to CD47 on RBCs; however, it does not cause agglutination of RBCs in vitro from any of the species tested. Ti-061 exhibits anti-tumor activity in several in vivo mouse tumor models. This ongoing Phase 1-2 study will assess the safety, efficacy, pharmacokinetics (PK) and pharmacodynamics (PD) of Ti-061 alone and in combination with other anti-cancer agents in patients with advanced malignancies. Methods: Part A is an open-label, dose-escalation study of Ti-061 administered as a weekly 1-hour IV infusion at doses ranging from 1 to 20 mg/kg. Once the MTD/RP2D or “active dose” is determined, patients with specific solid tumors and high CD47 expression will be enrolled in 4 or more expansion cohorts. Up to 160 patients with histologically confirmed solid tumors, ECOG PS 0-1, adequate blood counts (Hb≥ 10 g/dL), organ function, and archival or fresh tumor tissue will be enrolled in Part A, and will be treated until disease progression, unacceptable toxicity, or withdrawal. Primary endpoint is safety, which will be assessed using NCI-CTCAE v4.03. Secondary endpoints include PK, PD, objective response rate (ORR) and progression-free survival (PFS), which will be assessed using RECIST v1.1. The results of this study will support further development of Ti-061 in combination with checkpoint inhibitors (Part B) and other anti-cancer agents.

Objective To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase, on the progression of structural lesions in the experimental canine model of osteoarthritis (OA), and to explore the effect of L-NIL on the level of chondrocyte apoptosis and of important proteins involved in the apoptotic phenomenon, i.e., caspase 3 (inducer) and Bcl-2 (inhibitor). Methods The OA model was created by sectioning the anterior cruciate ligament. Dogs were placed into 4 experimental groups: unoperated dogs that received no treatment (controls), operated (OA) dogs that received placebo treatment, OA dogs that received oral L-NIL at 10 mg/kg/day, and OA dogs that received oral L-NIL at 1.0 mg/kg/day. In both L-NIL groups, treatment started immediately after surgery. The OA dogs were killed at 12 weeks after surgery. Results OA dogs treated with L-NIL showed a reduction in the size of osteophytes and a significant decrease in the severity of macroscopic and histologic cartilage lesions on both condyles and plateaus, compared with untreated OA dogs. L-NIL treatment also significantly decreased metalloprotease activity in cartilage. Immunohistochemical analysis revealed that the levels of chondrocyte apoptosis, caspase 3, and Bcl-2 were markedly increased in OA cartilage (P < 0.0001). A positive correlation between the levels of chondrocyte apoptosis and levels of caspase 3 was found (r = 0.54, P < 0.0001). OA dogs treated with the higher dosage L-NIL showed significantly reduced levels of chondrocyte apoptosis (P < 0.003) and caspase 3 (P < 0.04), but no effect on the increased level of Bcl-2 was demonstrated. Conclusion This study shows that L-NIL reduces the progression of experimental OA. This effect could be related to a reduced level of chondrocyte apoptosis and is likely mediated by a decrease in the level of caspase 3 activity. A sparing effect of L-NIL on the increased level of Bcl-2 may also be a contributing factor.

The efficient syntheses of three new racemic NG-(1-iminoethyl)phosphalysine derivatives are reported where the lysine carboxylate group is systematically replaced by phosphonic acid, 4, methyl phosphinic acid, 5, and phosphinic acid, 6. These compounds were evaluated as potential inhibitors of the three isoforms of human nitric oxide synthase. © 2000 John Wiley & Sons, Inc. Heteroatom Chem 11:505–511, 2000

A series of substituted 2-iminopyrrolidines has been prepared and shown to be potent and selective inhibitors of the human inducible nitric oxide synthase (hiNOS) isoform versus the human endothelial nitric oxide synthase (heNOS) and the human neuronal nitric oxide synthase (hnNOS). Simple substitutions at the 3-, 4-, or 5-position afforded more potent analogues than the parent 2-iminopyrrolidine 1. The effect of ring substitutions on both potency and selectivity for the different NOS isoforms is described. Substitution at the 4- and 5-positions of the 2-iminopyrrolidine yielded both potent and selective inhibitors of hiNOS. In particular, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine, monohydrochloride (20), displayed potent inhibition of hiNOS (IC50 = 0.25 microM) and selectivities of 897 (heNOS IC50/hiNOS IC50) and 13 (hnNOS IC50/hiNOS IC50). Example 20 was shown to be an efficacious inhibitor of NO production in the mouse endotoxin assay. Furthermore, 20 displayed in vivo selectivity, versus heNOS isoform, by not elevating blood pressure at multiples of the effective dose in the mouse.

Objective To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase (iNOS), on the progression of lesions in an experimental osteoarthritis (OA) dog model. The effect of L-NIL on metalloprotease activity, levels of interleukin-1beta (IL-1beta), prostaglandin E2 (PGE2), and nitrite/nitrate in synovial fluid was determined. Methods The OA model was created by sectioning the anterior cruciate ligament of the right stifle joint of mongrel dogs by a stab wound. Dogs were separated into experimental groups: Group 1 was made up of unoperated dogs that received no treatment, group 2 were operated dogs with no treatment, and group 3 were operated dogs that received oral L-NIL (10 mg/kg/twice daily) starting immediately after surgery. The OA dogs were killed at 10 weeks after surgery. Results Experiments showed that dog OA cartilage explants in culture produced an increased amount of NO (nitrite). Immunohistochemical study demonstrated that this was due to an increased level of iNOS in chondrocytes. OA dogs treated with L-NIL showed a reduction in the incidence of osteophytes compared with the untreated OA dogs (58% versus 92%) as well as in their size (mean +/- SEM 1.92 +/- 0.58 mm versus 5.08 +/- 0.66 mm). Macroscopically, L-NIL decreased the size of the cartilage lesions by approximately 50% both on condyles and plateaus. The histologic severity of both the cartilage lesions and synovial inflammation was significantly decreased in the L-NIL-treated dogs. Treatment with L-NIL also significantly decreased both collagenase and general metalloprotease activity in the cartilage and the levels of IL-1beta, PGE2, and nitrite/nitrate in synovial fluid. Conclusion This study demonstrated the effectiveness of a selective inhibitor of iNOS, L-NIL, in attenuating the progression of experimental OA. The data suggest that L-NIL may act by reducing the activity of metalloproteases in cartilage and the production of IL-1beta by synovium, both of which are known to play a major role in the pathophysiology of OA structural changes.

An attractive approach to the treatment of inflammatory conditions such as osteo- and rheumatoid arthritis, inflammatory bowel disease, and sepsis is through the selective inhibition of human inducible nitric oxide synthase (hiNOS) since localized excess nitric oxide (NO) release has been implicated in the pathology of these diseases. A series of monosubstituted iminohomopiperidinium salts possessing lipophilic functionality at ring positions 3, 5, 6, and 7 has been synthesized, and series members have demonstrated the ability to inhibit the hiNOS isoform with an IC50 as low as 160 nM (7). Compounds were found that selectively inhibit hiNOS over the human endothelial constitutive enzyme (heNOS) with a heNOS/hiNOS IC50 ratio in excess of 100 and as high as 314 (9). Potencies for inhibition of hiNOS and the human neuronal constitutive enzyme (hnNOS) are comparable. Substitution in the 3 and 7 positions provides compounds that exhibit the greatest degree of selectivity for hiNOS and hnNOS over heNOS. Submicromolar potencies for 6 and 7 in a mouse RAW cell assay demonstrated the ability of these compounds to inhibit iNOS in a cellular environment. These latter compounds were also found to be orally bioavailable and efficacious due to their ability to inhibit the increase in plasma nitrite/nitrate levels in a rat LPS model.

A series of analogues of 2-iminopiperidine have been prepared and shown to be potent inhibitors of the human nitric oxide synthase (NOS) isoforms. Methyl substitutions on the 4-position (3) or 4- and 6-positions (8) afforded the most potent analogues. These compounds exhibited IC50 values of 0.1 and 0.08 microM, respectively, for hiNOS inhibition. Substitution with cyclohexylmethyl at the 6-position (13) afforded an inhibitor that showed the best selectivity for hiNOS versus heNOS (heNOS IC50/hiNOS IC50 = 64). Following oral administration, inhibitors were found to decrease serum nitrite/nitrate levels in an in vivo rat endotoxin assay. This series of 2-iminopiperidines were prepared via the described synthetic methodologies. The effect of ring substitutions on potency and selectivity for this class of cyclic amidines as NOS inhibitors is described.

Peroxynitrite, which is generated by the reaction of nitric oxide (NO) with superoxide, is a strong oxidant that can damage subcellular organelles, membranes and enzymes through its actions on proteins, lipids, and DNA, including the nitration of tyrosine residues of proteins. Detection of nitrotyrosine (NT) serves as a biochemical marker of peroxynitrite-induced damage. In the present studies, NT was detected by immunohistochemistry in CNS tissues from mice with acute experimental autoimmune encephalomyelitis (EAE). NT immunoreactivity was displayed by many mononuclear inflammatory cells, including CD4+ cells. It was also observed in astrocytes near EAE lesions. Immunostaining for the inducible isoform of NO synthase (iNOS) was also observed, particularly during acute EAE. These data strongly suggest that peroxynitrite formation is a major consequence of NO produced via iNOS, and implicate this powerful oxidant in the pathogenesis of EAE.

Abstract: Nitric oxide is synthesized enzymatically from arginine in numerous tissues and cell types by three_distinct isoforms of the enzyme nitric oxide synthase (NOS). Two of these isoforms are expressed in a constitutive manner (cNOS) predominantly in the vascular endothelium (eNOS, type III NOS) and in the nervous system (nNOS, type I NOS) and function in the maintenance of normal homeostasis. Under normal physiological conditions, these constitutive isoforms of NOS generate low levels of nitric oxide in response to increases in intracellular calcium concentrations. The expression of the third form (iNOS, type II NOS) is induced by endotoxin and/or inflammatory cytokines and generates high levels of nitric oxide over long periods of time. The excessive production of nitric oxide, generated either by iNOS or by the sustained activation of nNOS, elicits cellular cytotoxicity and tissue damage and is thought to contribute to the pathophysiology of human disease states. The development of selective inhibitors of iNOS and/or nNOS offers the potential of blocking the synthesis of a major injurious agent, nitric oxide, and ultimately reducing tissue damage during states of chronic inflammation or prolonged elevations in cytosolic calcium. In this review, the selective inhibition of the various isoforms of NOS is examined by structure activity relationships as unique targets for drug research and therapeutic intervention in a variety of disease states.

Messenger RNA encoding inducible NO synthase (iNOS) was measured by competitive reverse transcriptase polymerase chain reaction (cRT-PCR) and ribonuclease protection assays in spinal cords from mice at varying stages of experimental allergic encephalomyelitis (EAE) and from control mice. iNOS mRNA was increased in spinal cords from mice with acute EAE. cRT-PCR assays revealed a 10-20-fold increase in iNOS mRNA in spinal cords during acute EAE compared with the level observed in normal mouse spinal cords. Functional iNOS activity, as assessed by assay of calcium-independent citrulline production, was also significantly increased in spinal cords from mice with acute EAE in comparison to normal controls. The correlation of functional iNOS expression with active disease in EAE in consistent with a pathogenic role for excess NO in this model of cell-mediated central nervous system autoimmunity.

1 The role of nitric oxide (NO) derived from constitutive and inducible nitric oxide synthase (cNOS and iNOS) and its relationship to oxygen-derived free radicals and prostaglandins (PG) was investigated in a carrageenan-induced model of acute hindpaw inflammation. 2 The intraplantar injection of carrageenan elicited an inflammatory response that was characterized by a time-dependent increase in paw oedema, neutrophil infiltration, and increased levels of nitrite/nitrate (NO2-/NO3-) and prostaglandin E2(PGE2) in the paw exudate. 3 Paw oedema was maximal by 6 h and remained elevated for 10 h following carrageenan administration. The non-selective cNOS/iNOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME) given intravenously (30-300 mg kg-1) 1 h before or after carrageenan administration, inhibited paw oedema at all time points. 4 The selective iNOS inhibitors, N-iminoethyl-L-lysine (L-NIL) or aminoguanidine (AG), failed to inhibit carrageenan-induced paw oedema during the first 4 h following carrageenan administration, but inhibited paw oedema at subsequent time points (from 5-10 h). iNOS mRNA was detected between 3 to 10 h following carrageenan administration using ribonuclease protection assays. iNOS protein was first detected 6 h and was maximal 10 h following carrageenan administration as shown by Western blot analysis. Administration of the iNOS inhibitors 5 h after carrageenan (a time point where iNOS was expressed) inhibited paw oedema at all subsequent time points. Infiltrating neutrophils were not the source of iNOS since pretreatment with colchicine (2 mg kg-1) suppressed neutrophil infiltration, but did not inhibit the iNOS mRNA expression or the elevated NO2-/NO3- levels in the paw exudate. 5 Inhibition of paw oedema by the NOS inhibitors was associated with attenuation of both the NO2-/NO3- and PGE2 levels in the paw exudate. These inhibitors also reduced the neutrophil infiltration at the site of inflammation. 6 Recombinant human Cu/Zn superoxide dismutase coupled to polyethyleneglycol (PEGrhSOD; 12 x 10(3) u kg-1), administered intravenously either 30 min prior to or 1 h after carrageenan injection, inhibited paw oedema and neutrophil infiltration, but had no effect on NO2-/NO3- or PGE2 production in the paw exudate. The administration of catalase (40 x 10(3) u kg-1), given intraperitoneally 30 min before carrageenan administration, had no effect on paw oedema. Treatment with desferrioxamine (300 mg kg-1), given subcutaneously 1 h before carrageenan, inhibited paw oedema during the first 2 h after carrageenan administration, but not at later times. 7 These results suggest that the NO produced by cNOS is involved in the development of inflammation at early time points following carrageenan administration and that NO produced by iNOS is involved in the maintenance of the inflammatory response at later time points. The potential interactions of NO with superoxide anion and PG is discussed.

A series of 2-iminoazaheterocycles have been prepared and shown to be potent inhibitors of human nitric oxide synthase (NOS) isoforms. This series includes cyclic amidines ranging from five- to nine-membered rings, of which 2-iminopiperidine and 2-iminohomopiperidine were the most potent inhibitors, with IC50 values of 1.0 and 2.0 microM, respectively, for human inducible nitric oxide synthase. This series of cyclic inhibitors was further expanded to include analogs with heteroatoms in the 3-position of the six-membered ring. This modification was tolerated for sulfur and oxygen, but nitrogen reduced the inhibitory potency. The oral administration of 2-iminopiperidine in lipopolysaccharide (LPS)-treated rats inhibited the LPS-induced increase in plasma nitrite/nitrate levels in a dose-dependent manner, demonstrating its ability to inhibit inducible NOS activity in vivo. These cyclic amidines represent a new class of potent NOS inhibitors and the foundation for potential therapeutic agents.

l -N6-(1-Iminoethyl)-lysine is a novel inhibitor of nitric oxide (NO) synthase, which similar to aminoguanidine but unlike NG-monomethyl- l -arginine is 30-fold more selective for the inducible than for the constitutive isoform of the enzyme. Here, we characterized this inhibitor for the first time in intact cells and during infection of mice with a NO-sensitive parasite (Leishmania major). l -N6-(1-Iminoethyl)-lysine potently inhibited the activity of inducible NO-synthase in primary macrophages. After stimulation by interferon-γ the IC50 of l -N6-(1-iminoethyl)-lysine was 0.4 ± 0.1 μM and 10- or 30-fold lower than that of NG-monomethyl- l -arginine or aminoguanidine, respectively. In vivo, l -N6-(1-imino-ethyl)-lysine (0.4–9 mM in the drinking water) suppressed inducible NO-synthase activity and caused a dramatic exacerbation of leishmaniasis, despite a counterregulatory increase of inducible NO-synthase protein in the tissue. In contrast, considerably higher concentrations of NG-monomethyl- l -arginine (20–50 mM) were required in order to achieve comparable effects. NG-Monomethyl- l -arginine, but not l -N6-(1-imino-ethyl)-lysine led to weight loss, reduced water and food consumption. We conclude that l -N6-(1-iminoethyl)-lysine should be used instead of NG-monomethyl- l -arginine for potent suppression of inducible NO-synthase in vitro and in vivo.

The role of nitric oxide in the immune response to allogeneic tissue was explored in an in vivo cardiac transplant model in the rat. Nitric oxide production during organ rejection was demonstrated by elevations in systemic serum nitrite/nitrate levels and by electron paramagnetic resonance spectroscopy. Messenger RNA for the inducible nitric oxide synthase enzyme was detected in the rejecting allografted heart, but not in the nonrejecting isografted heart. The enzyme was demonstrated to be biologically active by the in vitro conversion of L-arginine to L-citrulline and was immunohistochemically localized to the infiltrating inflammatory cells. Treatment with aminoguanidine, a preferential inhibitor of the inducible nitric oxide synthase isoform, prevented the increased nitric oxide production in the transplanted organ and significantly attenuated the pathogenesis of acute rejection. Aminoguanidine treatment prolonged graft survival, improved graft contractile function, and significantly reduced the histologic grade of rejection. These results suggest an important role for nitric oxide in mediating the immune response to allogeneic tissue. Inhibition of inducible nitric oxide synthase may provide a novel therapeutic modality in the management of acute transplant rejection and of other immune-mediated processes.

Adjuvant-induced arthritis is a model of chronic inflammation that exhibits several pathological changes similar to those occurring in rheumatoid arthritis, an autoimmune disease in humans characterized by chronic inflammation of the joints. We have examined the role of inducible nitric oxide synthase in producing the pathological changes associated with adjuvant-induced arthritis. Plasma nitrite concentrations were maximally elevated 14 days following adjuvant administration compared to untreated control animals. Arthritic changes in the paw were first observed between days 10-12 and were maximally elevated 21 days following adjuvant administration. Inducible nitric oxide synthase immunoreactivity was found localized in the synovial tissue from adjuvant-treated rats, while untreated controls exhibited no inducible nitric oxide synthase staining. Two selective inducible nitric oxide synthase inhibitors, aminoguanidine and N-iminoethyl-L-lysine, suppressed the increase in plasma nitrite levels and joint inflammation associated with adjuvant-induced arthritis in a dose-dependent manner. N-Iminoethyl-L-lysine attenuated the inducible nitric oxide synthase immunoreactivity in adjuvant-treated rats. Blood pressure was not affected by the highest dose of N-iminoethyl-L-lysine administered in the drinking water, indicating a lack of inhibition of constitutive nitric oxide synthase.

Described is a method of introducing trifluoroalkyl groups at C-6 of lysine. This chemistry has the potential to introduce a variety of functionality at C-6 of lysine.

Objective To determine if inhibition of inducible nitric oxide synthase (iNOS) with cindunistat hydrochloride maleate slows progression of osteoarthritis (OA) Methods This 2-year, multinational, double-blind, placebo-controlled trial enrolled patients with symptomatic knee OA (Kellgren and Lawrence Grade (KLG) 2 or 3). Standard OA therapies were permitted throughout. Patients were randomly assigned to cindunistat (50 or 200 mg/day) or placebo. Randomisation was stratified by KLG. Radiographs to assess joint space narrowing (JSN) were acquired using the modified Lyon-schuss protocol at baseline, week 48 and 96. Results Of 1457 patients (50 mg/day, n=485; 200 mg/day, n=486; placebo, n=486), 1048 (71.9%) completed the study. Patients were predominantly women; 56% had KLG3. The primary analysis did not demonstrate superiority of cindunistat versus placebo for rate of change in JSN. In KLG2 patients, JSN after 48 weeks was lower with cindunistat 50 mg/day versus placebo (p=0.032). Least-squares mean±SE JSN with cindunistat 50 mg/day ( −0.048±0.028 mm) and 200 mg/day (−0.062±0.028 mm) were 59.9% (95% CI 6.8% to 106.9%) and 48.7% (95% CI -8.4% to 93.9%) of placebo, improvement was not maintained at 96 weeks. No improvement was observed for KLG3 patients at either time-point. Cindunistat did not improve joint pain or function, but was generally well tolerated. Conclusions Cindunistat (50 or 200 mg/day) did not slow the rate of JSN versus placebo. After 48-weeks, KLG2 patients showed less JSN; however, the improvement was not sustained at 96-weeks. iNOS inhibition did not slow OA progression in KLG3 patients. Clinical trial listing NCT00565812

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammation in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-directed antiinflammatory drugs.

The potency and selectivity of a series of 5-hetero-2-iminohexahydroazepines were examined as inhibitors of the three human NOS isoforms. The effect of ring substitution of the 5-carbon for a heteroatom is presented. Potencies (IC50's) for these inhibitors are in the low micromolar range for hi-NOS with some examples exhibiting a 500× selectivity versus hec-NOS.

The potency and selectivity of a series of 5-hetero-2-iminohexahydroazepines were examined as inhibitors of the three human NOS isoforms. The effect of ring substitution of the 5-carbon for a heteroatom is presented. Potencies (IC50's) for these inhibitors are in the low micromolar range for hi-NOS with some examples exhibiting a 500× selectivity versus hec-NOS.

Objective To evaluate the in-vivo therapeutic efficacy of N -iminoethyl-L-Lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase (iNOS) in a dose response study, on the progression of lesions in the experimental osteoarthritic (OA) dog model. Design The sectioning of the anterior cruciate ligament of the right stifle joint of mongrel dogs was done by a stab wound. Dogs were separated into experimental groups: Group 1 received no treatment, Groups 2, 3, and 4 received oral L-NIL (0.3, 1 or 10mg/kg/day, respectively) starting immediately after surgery. The OA dogs were killed at 12 weeks after surgery. Results Macroscopically, L-NIL decreased the size of the cartilage lesions on condyles and plateaus. The histologic severity of the cartilage lesions was decreased in the L-NIL-treated dogs. This effect was more pronounced at the highest dosage tested (3 and 10mg/kg/day). Conclusions This study confirms the effectiveness of L-NIL, a selective inhibitor of iNOS, in attenuating the progression of experimental OA. It also clearly shows that the effect is dose-dependent.

Antibodies vs CD47 (CD47mAbs) that block the CD47-SIRPalpha interaction promote the phagocytosis of cancer cells and have efficacy in several tumor models. A select few CD47mAbs also directly kill cancer cells by lowering cellular cAMP levels leading to mitochondrial damage and cell death. These CD47mAbs are thus referred to as “dual-function” mAbs. Activation of protein kinase A prevents CD47mAb-mediated death suggesting that phosphorylation of one or more target proteins in the cancer cell can block the death mechanism. BNIP3, a member of the BH3-only family, is induced by hypoxia and oncogenes and is necessary for induction of cell death by dual-function CD47mAbs. BNIP3 can activate autophagy, a pro-survival function, and can also induce cell death by damaging mitochondria. The role of BNIP3 in cancer is controversial and context-dependent with some cancers over-expressing BNIP3 compared to low levels of expression in normal tissue, while other cancers cannot tolerate BNIP3 expression and silence the gene, often by methylation of the BNIP3 promoter. This suggests that cancers that tolerate BNIP3 expression employ an as yet unknown mechanism to protect themselves from its toxic effects. The C-terminal transmembrane (TM) domain of BNIP3 (residues 164-184) penetrates the outer mitochondrial membrane allowing the extreme C-terminal ten residue tail of BNIP3 (RRLTTSTSTF, residues 185-194) to extend across the intermembrane space to bind OPA1 on the inner mitochondrial membrane, a key step in the death mechanism. We used a phosphosite-specific antibody to detect phosphorylation of BNIP3 immunoprecipitated from Jurkat leukemia cells at residue T188 which resides in a canonical protein kinase A site (RRLT, amino acids 185-188). Using mass spectrometry of 6His-tagged BNIP3 isolated from HEK293 cells treated with 8BrcAMP, we identified as many as 4 additional phosphorylated sites in the C-terminal tail sequence (residues 189-194, TSTSTF). We generated phosphomimetic (S/T to D) and unphosphorylated (S/T to A or N) mutations at these residues and expressed the mutant BNIP3 proteins in 293 cells. All of the BNIP3 mutants associated with mitochondria but only the phosphomimetic mutants prevented BNIP3-induced mitochondrial damage and cell death. In contrast, mutation of the phosphorylated S/T residues to unphosphorylated residues resulted in rapid and extensive cell death. Importantly, phosphomimetic C-terminal BNIP3 residues blocked cell death without preventing autophagy, providing evidence that the two roles of BNIP3 can be regulated independently. We replicated these results in one lung cancer and three breast cancer cell lines. These findings suggest that phosphorylation at the C-terminus of BNIP3 is a switch that determines the pro-survival vs pro-death effects of BNIP3. Dual-function CD47mAbs may act by dephosphorylating BNIP3 expressed in tumor cells thus unleashing its killing potential. Citation Format: William Frazier, Katherine Liu, Julie Dimitry, Benjamin Capoccia, Pamela Manning, Robert Karr. Phosphorylation of BNIP3 is a switch between life and death in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2015-2

5(S)-Fluoro-N6-(iminoethyl)-l-lysine (14), an analogue of the potent, selective induced nitric oxide synthase (iNOS) inhibitor iminoethyl-l-lysine (1), was synthesized and found to be a selective iNOS inhibitor.