Rationale and design of the SOluble guanylate Cyclase stimulatoR in heArT failurE Studies (SOCRATES).

AIMS: The clinical outcomes for patients with worsening chronic heart failure (WCHF) remain exceedingly poor despite contemporary evidence-based therapies, and effective therapies are urgently needed. Accumulating evidence supports augmentation of cyclic guanosine monophosphate (cGMP) signalling as a potential therapeutic strategy for HF with reduced or preserved ejection fraction (HFrEF and HFpEF, respectively). Direct soluble guanylate cyclase (sGC) stimulators target reduced cGMP generation due to insufficient sGC stimulation and represent a promising method for cGMP enhancement. METHODS: The phase II SOluble guanylate Cyclase stimulatoR in heArT failurE Study (SOCRATES) programme consists of two randomized, parallel-group, placebo-controlled, double-blind, multicentre studies, SOCRATES-REDUCED (in patients with LVEF <45%) and SOCRATES-PRESERVED (in those with LVEF ≥ 45%), that will explore the pharmacodynamic effects, safety and tolerability, and pharmacokinetics of four dose regimens of the once-daily oral sGC stimulator vericiguat (BAY 1021189) over 12 weeks compared with placebo. These studies will enrol patients stabilized during hospitalization for HF at the time of discharge or within 4 weeks thereafter. The primary endpoint in SOCRATES-REDUCED is change in NT-proBNP at 12 weeks. The primary endpoints in SOCRATES-PRESERVED are change in NT-proBNP and left atrial volume at 12 weeks. PERSPECTIVES: SOCRATES will be the first programme to enrol specifically both inpatients and outpatients with WCHF and patients with reduced or preserved ejection fraction. Results will inform the benefits of pursuing subsequent event-driven clinical outcome trials with sGC stimulators in this patient population.

AAV-mediated gene therapy in the guanylate cyclase (RetGC1/RetGC2) double knockout mouse model of Leber congenital amaurosis.

Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.

Long-term preservation of cones and improvement in visual function following gene therapy in a mouse model of leber congenital amaurosis caused by guanylate cyclase-1 deficiency.

Leber congenital amaurosis (LCA) is a severe retinal dystrophy manifesting from early infancy as poor vision or blindness. Loss-of-function mutations in GUCY2D cause LCA1 and are one of the most common causes of LCA, accounting for 20% of all cases. Human GUCY2D and mouse Gucy2e genes encode guanylate cyclase-1 (GC1), which is responsible for restoring the dark state in photoreceptors after light exposure. The Gucy2e(-/-) mouse shows partially diminished rod function, but an absence of cone function before degeneration. Although the cones appear morphologically normal, they exhibit mislocalization of proteins involved in phototransduction. In this study we tested the efficacy of an rAAV2/8 vector containing the human rhodopsin kinase promoter and the human GUCY2D gene. Following subretinal delivery of the vector in Gucy2e(-/-) mice, GC1 protein was detected in the rod and cone outer segments, and in transduced areas of retina cone transducin was appropriately localized to cone outer segments. Moreover, we observed a dose-dependent restoration of rod and cone function and an improvement in visual behavior of the treated mice. Most importantly, cone preservation was observed in transduced areas up to 6 months post injection. To date, this is the most effective rescue of the Gucy2e(-/-) mouse model of LCA and we propose that a vector, similar to the one used in this study, could be suitable for use in a clinical trial of gene therapy for LCA1.

Lineage-specific T-cell responses to cancer mucosa antigen oppose systemic metastases without mucosal inflammatory disease.

Cancer mucosa antigens are emerging as a new category of self-antigens expressed normally in immunologically privileged mucosal compartments and universally by their derivative tumors. These antigens leverage the established immunologic partitioning of systemic and mucosal compartments, limiting tolerance opposing systemic antitumor efficacy. An unresolved issue surrounding self-antigens as immunotherapeutic targets is autoimmunity following systemic immunization. In the context of cancer mucosa antigens, immune effectors to self-antigens risk amplifying mucosal inflammatory disease promoting carcinogenesis. Here, we examined the relationship between immunotherapy for systemic colon cancer metastases targeting the intestinal cancer mucosa antigen guanylyl cyclase C (GCC) and its effect on inflammatory bowel disease and carcinogenesis in mice. Immunization with GCC-expressing viral vectors opposed nascent tumor growth in mouse models of pulmonary metastasis, reflecting systemic lineage-specific tolerance characterized by CD8(+), but not CD4(+), T-cell or antibody responses. Responses protecting against systemic metastases spared intestinal epithelium from autoimmunity, and systemic GCC immunity did not amplify chemically induced inflammatory bowel disease. Moreover, GCC immunization failed to promote intestinal carcinogenesis induced by germ-line mutations or chronic inflammation. The established role of CD8(+) T cells in antitumor efficacy, but CD4(+) T cells in autoimmunity, suggests that lineage-specific responses to GCC are particularly advantageous to protect against systemic metastases without mucosal inflammation. These observations support the utility of GCC-targeted immunotherapy in patients at risk for systemic metastases, including those with inflammatory bowel disease, hereditary colorectal cancer syndromes, and sporadic colorectal cancer.

Antinociception synergy between the peripheral and spinal sites of the heme oxygenase-carbon monoxide pathway.

We have shown that the peripheral and spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase-cGMP pathways play an important role in antinociception in the rat experimental formalin model. Our objective was to determine if there is synergism between peripheral (paw) and spinal HO-CO pathways in nociception. Rats were handled and adapted to the experimental environment for a few days before the formalin test, in which 50 microL of a 1% formalin was injected subcutaneously into the dorsal surface of the right hind paw. The animals were then observed for 1 h and the frequency of flinching behavior was taken to represent the nociceptive response. Thirty minutes before the test, rats were pretreated with intrathecal injections of the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is a substrate of the HO pathway. The paw treatments took place 20 min before the test. Low doses of ZnDPBG did not increase nociception, while a low heme-lysinate dose did not change flinching behavior after paw or spinal injections. Combined subactive spinal (50 nmol) and peripheral (40 nmol) low doses of ZnDPBG induced hypernociception (increase of 80% in the first and 25% in the second phase flinching), whereas combined spinal-peripheral heme-lysinate (50 and 30 nmol) led to second phase antinociception (40% reduction in flinching). These findings suggest a synergy between the peripheral and spinal HO-CO pathways. Local activation of the HO system probably regulates the nociception initiation in peripheral tissue and participates in buffering the emerging nociceptive signals at the peripheral and spinal sites of action. In short, an antinociceptive synergy exists between peripheral and spinal HO pathways, which may reduce the doses required and side effects.

Antinociception synergy between the peripheral and spinal sites of the heme oxygenase-carbon monoxide pathway

We have shown that the peripheral and spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase-cGMP pathways play an important role in antinociception in the rat experimental formalin model. Our objective was to determine if there is synergism between peripheral (paw) and spinal HO-CO pathways in nociception. Rats were handled and adapted to the experimental environment for a few days before the formalin test, in which 50 µL of a 1 percent formalin was injected subcutaneously into the dorsal surface of the right hind paw. The animals were then observed for 1 h and the frequency of flinching behavior was taken to represent the nociceptive response. Thirty minutes before the test, rats were pretreated with intrathecal injections of the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is a substrate of the HO pathway. The paw treatments took place 20 min before the test. Low doses of ZnDPBG did not increase nociception, while a low heme-lysinate dose did not change flinching behavior after paw or spinal injections. Combined subactive spinal (50 nmol) and peripheral (40 nmol) low doses of ZnDPBG induced hypernociception (increase of 80 percent in the first and 25 percent in the second phase flinching), whereas combined spinal-peripheral heme-lysinate (50 and 30 nmol) led to second phase antinociception (40 percent reduction in flinching). These findings suggest a synergy between the peripheral and spinal HO-CO pathways. Local activation of the HO system probably regulates the nociception initiation in peripheral tissue and participates in buffering the emerging nociceptive signals at the peripheral and spinal sites of action. In short, an antinociceptive synergy exists between peripheral and spinal HO pathways, which may reduce the doses required and side effects.

Different roles of two nitric oxide activated pathways in spinal long-term potentiation of C-fiber-evoked field potentials.

There is accumulating evidence implicating the involvement of nitric oxide (NO) in spinal central sensitization. The long-term potentiation (LTP) of spinal C-fiber-evoked field potentials is considered as a fundamental mechanism of sensitization of nociceptive neurons in the spinal cord. The present study examined the roles of soluble guanylate cyclase (sGC) or ADP-ribosyltransferase (ADPRT), two potential NO targets, in spinal LTP. The results showed that (1) administration of sGC inhibitors, methyl blue (MB, 4mM, 20 microl) or 1H-[1,2,4]oxadiazolo[4,3-a]-quiloxalin-1-one (ODQ, 10 microM, 20 microl) before tetanic stimulation, significantly inhibited the induction of spinal LTP, and this was reversed by 8-Br-cGMP, a membrane-permeable cGMP analog. However, the maintenance of spinal LTP was not changed when application of ODQ 2h after tetanic stimulation. (2) Although our previous experiments have identified a key role for NO in the induction of spinal LTP, NO synthase (NOS) inhibitor, L-NAME (1mM, 20 microl) or hemoglobin (2mg/ml, 20 microl), a scavenger of NO, had no effect on established spinal LTP when applied 2h after the induction of spinal LTP. (3) The mono-ADPRT inhibitor, nicotinamide (10mM, 20 microl), had no effect on the induction and maintenance of spinal LTP. However, the poly-ADPRT inhibitor, benzamide (100 microM, 20 microl), inhibited its maintenance, but not its induction. The results suggest that NO-stimulated guanylyl cyclase activity plays a critical role in the induction of LTP of C-fiber-evoked field potentials in the spinal cord, whereas NO-related poly-ADPRT activity contributes to the maintenance of spinal LTP.

Utilidad de sildenafilo en disfunciones sexuales secundarias a patología psiquiátrica y psicofármacos / Usefulness of sildenafil in sexual dysfunctions secondary to psychiatric disorders and psychotropic drugs

No disponible