SGLT2 inhibition potentiates the cardiovascular, renal, and metabolic effects of sGC stimulation in hypertensive rats with prolonged exposure to high-fat diet.

Prolonged high-fat diet (HFD) accelerates the cardiovascular, renal, and metabolic dysfunction in hypertensive rats with altered renal development (ARDev). Soluble guanylate cyclase (sGC) stimulation or sodium-glucose cotransporter 2 (SGLT2) inhibition may improve cardiovascular, renal, and metabolic function in settings of hypertension and obesity. This study examined whether 6 wk treatment with an SGLT2 inhibitor (empagliflozin, 7 mg/kg/day) enhances the cardiovascular, renal, and metabolic effects of a sGC stimulator (praliciguat, 10 mg/kg/day) in hypertensive rats with ARDev and prolonged exposure to HFD. Arterial pressure (AP), renal vascular resistance (RVR), fat abdominal volume (FAV), insulin resistance, leptin and triglycerides levels, and intrarenal infiltration of inflammatory cells were higher, but cardiac output and creatinine clearance were lower in hypertensive rats (n = 15) than in normotensive rats (n = 7). Praliciguat administration (n = 10) to hypertensive rats reduced (P < 0.05) AP, FAV, plasma concentrations of leptin and triglycerides, and increased (P < 0.05) cardiac output and creatinine clearance. Empagliflozin administration (n = 8) only increased (P < 0.05) glucosuria and creatinine clearance and decreased (P < 0.05) plasma leptin and triglycerides concentrations in hypertensive rats. Simultaneous administration of praliciguat and empagliflozin (n = 10) accelerated the decrease in AP, improved glucose tolerance, reduced (P < 0.05) incremental body weight gain, and decreased (P < 0.05) insulin resistance index, RVR, and the infiltration of T-CD3 lymphocytes in renal cortex and renal medulla. In summary, the combined administration of praliciguat and empagliflozin leads to a greater improvement of the cardiovascular, renal, and metabolic dysfunction secondary to prolonged exposure to HFD in hypertensive rats with ARDev than the treatment with either praliciguat or empagliflozin alone.NEW & NOTEWORTHY This is the first study, to our knowledge, showing that SGLT2 inhibition potentiates the beneficial cardiovascular, renal, and metabolic effects elicited by sGC stimulation in hypertensive rats with prolonged high-fat diet. The effects of the simultaneous administration of praliciguat and empagliflozin are greater than those elicited by either one alone. The effects of the simultaneous treatment may be related to a greater reduction in the inflammatory status.

Olinciguat, a stimulator of soluble guanylyl cyclase, attenuates inflammation, vaso-occlusion and nephropathy in mouse models of sickle cell disease.

BACKGROUND AND PURPOSE: Reduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso-occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP-amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα-induced inflammation in wild-type C57BL/6 mice and in 'humanised' mouse models of SCD. EXPERIMENTAL APPROACH: Effects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα-induced and systemic inflammation associated with SCD. KEY RESULTS: Acute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte-endothelial cell interactions in TNFα-challenged mice. Co-treatment with hydroxyurea, an FDA-approved SCD therapeutic agent, further augmented the anti-inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα-induced vaso-occlusive crisis, a single dose of olinciguat attenuated leukocyte-endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle-treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat- than in vehicle-treated mice. In addition, kidney mass, water consumption, 24-h urine excretion, plasma levels of cystatin C and urinary excretion of N-acetyl-ß-d-glucosaminidase and neutrophil gelatinase-associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle-treated mice. CONCLUSION AND IMPLICATIONS: Our results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso-occlusion and kidney injury in mouse models of SCD and systemic inflammation.

Stimulation of soluble guanylate cyclase exerts antiinflammatory actions in the liver through a VASP/NF-κB/NLRP3 inflammasome circuit.

Soluble guanylate cyclase (sGC) catalyzes the conversion of guanosine triphosphate into cyclic guanosine-3',5'-monophosphate, a key second messenger in cell signaling and tissue homeostasis. It was recently demonstrated that sGC stimulation is associated with a marked antiinflammatory effect in the liver of mice with experimental nonalcoholic steatohepatitis (NASH). Here, we investigated the mechanisms underlying the antiinflammatory effect of the sGC stimulator praliciguat (PRL) in the liver. Therapeutic administration of PRL exerted antiinflammatory and antifibrotic actions in mice with choline-deficient l-amino acid-defined high-fat diet-induced NASH. The PRL antiinflammatory effect was associated with lower F4/80- and CX3CR1-positive macrophage infiltration into the liver in parallel with lower Ly6CHigh- and higher Ly6CLow-expressing monocytes in peripheral circulation. The PRL antiinflammatory effect was also associated with suppression of hepatic levels of interleukin (IL)-1ß, NLPR3 (NACHT, LRR, and PYD domain-containing protein 3), ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), and active cleaved-caspase-1, which are components of the NLRP3 inflammasome. In Kupffer cells challenged with the classical inflammasome model of lipopolysaccharide plus adenosine triphosphate, PRL inhibited the priming (expression of Il1b and Nlrp3) and blocked the release of mature IL-1ß. Mechanistically, PRL induced the protein kinase G (PKG)-mediated phosphorylation of the VASP (vasodilator-stimulated phosphoprotein) Ser239 residue which, in turn, reduced nuclear factor-κB (NF-κB) activity and Il1b and Nlrp3 gene transcription. PRL also reduced active cleaved-caspase-1 levels independent of pannexin-1 activity. These data indicate that sGC stimulation with PRL exerts antiinflammatory actions in the liver through mechanisms related to a PKG/VASP/NF-κB/NLRP3 inflammasome circuit.

Praliciguat inhibits progression of diabetic nephropathy in ZSF1 rats and suppresses inflammation and apoptosis in human renal proximal tubular cells.

Praliciguat, a clinical-stage soluble guanylate cyclase (sGC) stimulator, increases cGMP via the nitric oxide-sGC pathway. Praliciguat has been shown to be renoprotective in rodent models of hypertensive nephropathy and renal fibrosis. In the present study, praliciguat alone and in combination with enalapril attenuated proteinuria in the obese ZSF1 rat model of diabetic nephropathy. Praliciguat monotherapy did not affect hemodynamics. In contrast, enalapril monotherapy lowered blood pressure but did not attenuate proteinuria. Renal expression of genes in pathways involved in inflammation, fibrosis, oxidative stress, and kidney injury was lower in praliciguat-treated obese ZSF1 rats than in obese control rats; fasting glucose and cholesterol were also lower with praliciguat treatment. To gain insight into how tubular mechanisms might contribute to its pharmacological effects on the kidneys, we studied the effects of praliciguat on pathological processes and signaling pathways in cultured human primary renal proximal tubular epithelial cells (RPTCs). Praliciguat inhibited the expression of proinflammatory cytokines and secretion of monocyte chemoattractant protein-1 in tumor necrosis factor-α-challenged RPTCs. Praliciguat treatment also attenuated transforming growth factor-ß-mediated apoptosis, changes to a mesenchyme-like cellular phenotype, and phosphorylation of SMAD3 in RPTCs. In conclusion, praliciguat improved proteinuria in the ZSF1 rat model of diabetic nephropathy, and its actions in human RPTCs suggest that tubular effects may contribute to its renal benefits, building upon strong evidence for the role of cGMP signaling in renal health.

Pharmacokinetics, mass balance, tissue distribution, metabolism, and excretion of praliciguat, a clinical-stage soluble guanylate cyclase stimulator in rats.

The pharmacokinetics (PK), metabolism, excretion, mass balance, and tissue distribution of [14 C]praliciguat were evaluated following oral administration of a 3-mg/kg dose in Sprague-Dawley rats and in a quantitative whole-body autoradiography (QWBA) study conducted in male Long-Evans rats. Plasma Tmax was 1 hour and the t1/2 of total plasma radioactivity was 23.7 hours. Unchanged praliciguat accounted for 87.4%, and a minor metabolite (N-dealkylated-praliciguat) accounted for 7.6% of the total radioactivity in plasma through 48 hours (AUC0-48 ). Tissues with the highest exposure ratios relative to plasma were liver, intestines, adrenal gland, and adipose, and those with the lowest values were seminal vesicle, blood, CNS tissues, lens of the eye, and bone. Most of the [14 C]praliciguat-derived radioactivity was excreted within 48 hours after oral administration. Mean cumulative recovery of the administered radioactivity in urine and feces over 168 hours was 3.7% and 95.7%, respectively. Unchanged praliciguat was not quantifiable in urine or bile of cannulated rats; however, based on the total radioactivity in these fluids, a minimum of approximately 82% of the orally administered dose was absorbed. [14 C]Praliciguat was metabolized via oxidative and glucuronidation pathways and the most abundant metabolites recovered in bile were praliciguat-glucuronide and hydroxy-praliciguat-glucuronide. These results indicate that praliciguat had rapid absorption, high bioavailability, extensive tissue distribution, and elimination primarily via hepatic metabolism.

Olinciguat, an Oral sGC Stimulator, Exhibits Diverse Pharmacology Across Preclinical Models of Cardiovascular, Metabolic, Renal, and Inflammatory Disease.

Nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic 3',5' GMP (cGMP) signaling plays a central role in regulation of diverse processes including smooth muscle relaxation, inflammation, and fibrosis. sGC is activated by the short-lived physiologic mediator NO. sGC stimulators are small-molecule compounds that directly bind to sGC to enhance NO-mediated cGMP signaling. Olinciguat, (R)-3,3,3-trifluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl)pyrimidin-4-yl)amino)methyl)-2-hydroxypropanamide, is a new sGC stimulator currently in Phase 2 clinical development. To understand the potential clinical utility of olinciguat, we studied its pharmacokinetics, tissue distribution, and pharmacologic effects in preclinical models. Olinciguat relaxed human vascular smooth muscle and was a potent inhibitor of vascular smooth muscle proliferation in vitro. These antiproliferative effects were potentiated by the phosphodiesterase 5 inhibitor tadalafil, which did not inhibit vascular smooth muscle proliferation on its own. Olinciguat was orally bioavailable and predominantly cleared by the liver in rats. In a rat whole body autoradiography study, olinciguat-derived radioactivity in most tissues was comparable to plasma levels, indicating a balanced distribution between vascular and extravascular compartments. Olinciguat was explored in rodent models to study its effects on the vasculature, the heart, the kidneys, metabolism, and inflammation. Olinciguat reduced blood pressure in normotensive and hypertensive rats. Olinciguat was cardioprotective in the Dahl rat salt-sensitive hypertensive heart failure model. In the rat ZSF1 model of diabetic nephropathy and metabolic syndrome, olinciguat was renoprotective and associated with lower circulating glucose, cholesterol, and triglycerides. In a mouse TNFα-induced inflammation model, olinciguat treatment was associated with lower levels of endothelial and leukocyte-derived soluble adhesion molecules. The pharmacological features of olinciguat suggest that it may have broad therapeutic potential and that it may be suited for diseases that have both vascular and extravascular pathologies.

Soluble guanylate cyclase stimulator praliciguat attenuates inflammation, fibrosis, and end-organ damage in the Dahl model of cardiorenal failure.

Reduced nitric oxide (NO) and a decrease in cGMP signaling mediated by soluble guanylate cyclase (sGC) has been linked to the development of several cardiorenal diseases. Stimulation of sGC is a potential means for enhancing cGMP production in conditions of reduced NO bioavailability. The purpose of our studies was to determine the effects of praliciguat, a clinical-stage sGC stimulator, in a model of cardiorenal failure. Dahl salt-sensitive rats fed a high-salt diet to induce hypertension and organ damage were treated with the sGC stimulator praliciguat to determine its effects on hemodynamics, biomarkers of inflammation, fibrosis, tissue function, and organ damage. Praliciguat treatment reduced blood pressure, improved cardiorenal damage, and attenuated the increase in circulating markers of inflammation and fibrosis. Notably, praliciguat affected markers of renal damage at a dose that had minimal effect on blood pressure. In addition, liver fibrosis and circulating markers of tissue damage were attenuated in praliciguat-treated rats. Stimulation of the NO-sGC-cGMP pathway by praliciguat attenuated or normalized indicators of chronic inflammation, fibrosis, and tissue dysfunction in the Dahl salt-sensitive rat model. Stimulation of sGC by praliciguat may present an effective mechanism for treating diseases linked to NO deficiency, particularly those associated with cardiac and renal failure. Praliciguat is currently being evaluated in patients with diabetic nephropathy and heart failure with preserved ejection fraction.

sGC stimulator praliciguat suppresses stellate cell fibrotic transformation and inhibits fibrosis and inflammation in models of NASH.

Endothelial dysfunction and reduced nitric oxide (NO) signaling are a key element of the pathophysiology of nonalcoholic steatohepatitis (NASH). Stimulators of soluble guanylate cyclase (sGC) enhance NO signaling; have been shown preclinically to reduce inflammation, fibrosis, and steatosis; and thus have been proposed as potential therapies for NASH and fibrotic liver diseases. Praliciguat, an oral sGC stimulator with extensive distribution to the liver, was used to explore the role of this signaling pathway in NASH. We found that sGC is expressed in hepatic stellate cells and stellate-derived myofibroblasts, but not in hepatocytes. Praliciguat acted directly on isolated hepatic stellate cells to inhibit fibrotic and inflammatory signaling potentially through regulation of AMPK and SMAD7. Using in vivo microdialysis, we demonstrated stimulation of the NO-sGC pathway by praliciguat in both healthy and fibrotic livers. In preclinical models of NASH, praliciguat treatment was associated with lower levels of liver fibrosis and lower expression of fibrotic and inflammatory biomarkers. Praliciguat treatment lowered hepatic steatosis and plasma cholesterol levels. The antiinflammatory and antifibrotic effects of praliciguat were recapitulated in human microtissues in vitro. These data provide a plausible cellular basis for the mechanism of action of sGC stimulators and suggest the potential therapeutic utility of praliciguat in the treatment of NASH.

Pharmacological Characterization of IW-1973, a Novel Soluble Guanylate Cyclase Stimulator with Extensive Tissue Distribution, Antihypertensive, Anti-Inflammatory, and Antifibrotic Effects in Preclinical Models of Disease.

Soluble guanylate cyclase (sGC), a key signal-transduction enzyme, increases the conversion of guanosine-5'-triphosphate to cGMP upon binding of nitric oxide (NO). Endothelial dysfunction and/or reduced NO signaling have been implicated in cardiovascular disease pathogenesis and complications of diabetes and have been associated with other disease states and aging. Soluble guanylate cyclase (sGC) stimulators are small-molecule drugs that bind sGC and enhance NO-mediated cGMP signaling. The pharmacological characterization of IW-1973 [1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl) pyrimidin-4-yl)amino)methyl)propan-2-ol], a novel clinical-stage sGC stimulator under clinical investigation for treatment of heart failure with preserved ejection fraction and diabetic nephropathy, is described. In the presence of NO, IW-1973 stimulated sGC in a human purified enzyme assay and a HEK-293 whole cell assay. sGC stimulation by IW-1973 in cells was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. IW-1973, at doses of 1-10 mg/kg, significantly lowered blood pressure in normotensive and spontaneously hypertensive rats. In a Dahl salt-sensitive hypertension model, IW-1973 significantly reduced blood pressure, inflammatory cytokine levels, and renal disease markers, including proteinuria and renal fibrotic gene expression. The results were affirmed in mouse lipopolysaccharide-induced inflammation and rat unilateral ureteral obstruction renal fibrosis models. A quantitative whole-body autoradiography study of IW-1973 revealed extensive tissue distribution and pharmacokinetic studies showed a large volume of distribution and a profile consistent with predicted once-a-day dosing in humans. In summary, IW-1973 is a potent, orally available sGC stimulator that exhibits renoprotective, anti-inflammatory, and antifibrotic effects in nonclinical models.

The soluble guanylate cyclase stimulator IW-1973 prevents inflammation and fibrosis in experimental non-alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Non-alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation and fibrosis. Soluble guanylate cyclase (sGC) stimulation reduces inflammation and fibrosis in experimental models of lung, kidney and heart disease. Here, we tested whether sGC stimulation is also effective in experimental NASH. EXPERIMENTAL APPROACH: NASH was induced in mice by feeding a choline-deficient, l-amino acid-defined, high-fat diet. These mice received either placebo or the sGC stimulator IW-1973 at two different doses (1 and 3 mg·kg-1 ·day-1 ) for 9 weeks. IW-1973 was also tested in high-fat diet (HFD)-induced obese mice. Steatosis, inflammation and fibrosis were assessed by Oil Red O, haematoxylin-eosin, Masson's trichrome, Sirius Red, F4/80 and α-smooth muscle actin staining. mRNA expression was assessed by quantitative PCR. Levels of IW-1973, cytokines and cGMP were determined by LC-MS/MS, Luminex and enzyme immunoassay respectively. KEY RESULTS: Mice with NASH showed reduced cGMP levels and sGC expression, increased steatosis, inflammation, fibrosis, TNF-α and MCP-1 levels and up-regulated collagen types I α1 and α2, MMP2, TGF-ß1 and tissue metallopeptidase inhibitor 1 expression. IW-1973 restored hepatic cGMP levels and sGC expression resulting in a dose-dependent reduction of hepatic inflammation and fibrosis. IW-1973 levels were ≈40-fold higher in liver tissue than in plasma. IW-1973 also reduced hepatic steatosis and adipocyte hypertrophy secondary to enhanced autophagy in HFD-induced obese mice. CONCLUSIONS AND IMPLICATIONS: Our data indicate that sGC stimulation prevents hepatic steatosis, inflammation and fibrosis in experimental NASH. These findings warrant further evaluation of IW-1973 in the clinical setting.

The Soluble Guanylate Cyclase Stimulator IWP-953 Increases Conventional Outflow Facility in Mouse Eyes.

PURPOSE: The nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway regulates aqueous humor outflow and therefore, intraocular pressure. We investigated the pharmacologic effects of the soluble guanylate cyclase (sGC) stimulator IWP-953 on primary human trabecular meshwork (HTM) cells and conventional outflow facility in mouse eyes. METHODS: Cyclic GMP levels were determined in vitro in HEK-293 cells and four HTM cell strains (HTM120/HTM123: predominantly myofibroblast-like phenotype, HTM130/HTM141: predominantly endothelial-like phenotype), and in HTM cell culture supernatants. Conventional outflow facility was measured following intracameral injection of IWP-953 or DETA-NO using a computerized pressure-controlled perfusion system in enucleated mouse eyes ex vivo. RESULTS: IWP-953 markedly stimulated cGMP production in HEK-293 cells in the presence and absence of DETA-NO (half maximal effective concentrations: 17 nM, 9.5 µM). Similarly, IWP-953 stimulated cGMP production in myofibroblast-like HTM120 and HTM123 cells, an effect that was greatly amplified by the presence of DETA-NO. In contrast, IWP-953 stimulation of cGMP production in endothelial-like HTM130 and HTM141 cells was observed, but was markedly less prominent than in HTM120 and HTM123 cells. Notably, cGMP was found in all HTM culture supernatants, following IWP-953/DETA-NO stimulation. In paired enucleated mouse eyes, IWP-953 at 10, 30, 60, and 100 µM concentration-dependently increased outflow facility. This effect (89.5%) was maximal at 100 µM (P = 0.002) and in magnitude comparable to DETA-NO at 100 µM (97.5% increase, P = 0.030). CONCLUSIONS: These data indicate that IWP-953, via modulation of the sGC-cGMP pathway, increases aqueous outflow facility in mouse eyes, suggesting therapeutic potential for sGC stimulators as novel ocular hypotensive drugs.

Discovery and Characterization of a Potent Interleukin-6 Binding Peptide with Neutralizing Activity In Vivo.

Interleukin-6 (IL-6) is an important member of the cytokine superfamily, exerting pleiotropic actions on many physiological processes. Over-production of IL-6 is a hallmark of immune-mediated inflammatory diseases such as Castleman's Disease (CD) and rheumatoid arthritis (RA). Antagonism of the interleukin IL-6/IL-6 receptor (IL-6R)/gp130 signaling complex continues to show promise as a therapeutic target. Monoclonal antibodies (mAbs) directed against components of this complex have been approved as therapeutics for both CD and RA. To potentially provide an additional modality to antagonize IL-6 induced pathophysiology, a peptide-based antagonist approach was undertaken. Using a combination of molecular design, phage-display, and medicinal chemistry, disulfide-rich peptides (DRPs) directed against IL-6 were developed with low nanomolar potency in inhibiting IL-6-induced pSTAT3 in U937 monocytic cells. Targeted PEGylation of IL-6 binding peptides resulted in molecules that retained their potency against IL-6 and had a prolongation of their pharmacokinetic (PK) profiles in rodents and monkeys. One such peptide, PN-2921, contained a 40 kDa polyethylene glycol (PEG) moiety and inhibited IL-6-induced pSTAT3 in U937 cells with sub-nM potency and possessed 23, 36, and 59 h PK half-life values in mice, rats, and cynomolgus monkeys, respectively. Parenteral administration of PN-2921 to mice and cynomolgus monkeys potently inhibited IL-6-induced biomarker responses, with significant reductions in the acute inflammatory phase proteins, serum amyloid A (SAA) and C-reactive protein (CRP). This potent, PEGylated IL-6 binding peptide offers a new approach to antagonize IL-6-induced signaling and associated pathophysiology.

Renal effects induced by prolonged mPGES1 inhibition.

The importance of membrane-bound PGE synthase 1 (mPGES1) in the regulation of renal function has been examined in mPGES1-deficient mice or by evaluating changes in its expression. However, it is unknown whether prolonged mPGES1 inhibition induces significant changes of renal function when Na(+) intake is normal or low. This study examined the renal effects elicited by a selective mPGES1 inhibitor (PF-458) during 7 days in conscious chronically instrumented dogs with normal Na(+) intake (NSI) or low Na(+) intake (LSI). Results obtained in both in vitro and in vivo studies have strongly suggested that PF-458 is a selective mPGES1 inhibitor. The administration of 2.4 mg·kg(-1)·day(-1) PF-458 to dogs with LSI did not induce significant changes in renal blood flow (RBF) and glomerular filtration rate (GFR). A larger dose of PF-458 (9.6 mg·kg(-1)·day(-1)) reduced RBF (P < 0.05) but not GFR in dogs with LSI and did not induce changes of renal hemodynamic in dogs with NSI. Both doses of PF-458 elicited a decrease (P < 0.05) in PGE2 and an increase (P < 0.05) in 6-keto-PGF1α. The administration of PF-458 did not induce significant changes in renal excretory function, plasma renin activity, and plasma aldosterone and thromboxane B2 concentrations in dogs with LSI or NSI. The results obtained suggest that mPGES1 is involved in the regulation of RBF when Na(+) intake is low and that the renal effects elicited by mPGES1 inhibition are modulated by a compensatory increment in PGI2. These results may have some therapeutical implications since it has been shown that prolonged mPGES1 inhibition has lower renal effects than those elicited by nonsteroidal anti-inflammatory drugs or selective cyclooxygenase-2 inhibitors.

Highly specific and sensitive measurements of human and monkey interleukin 21 using sequential protein and tryptic peptide immunoaffinity LC-MS/MS.

A highly specific and sensitive immunoaffinity LC-MS/MS assay for quantification of human and cynomolgus monkey interleukin 21 (IL-21) was developed, qualified, and implemented. The workflow includes offline enrichment of IL-21 using an anti-IL-21 capture antibody, followed by isolation using magnetic beads, trypsin digestion, online enrichment of IL-21 derived tryptic peptides using antipeptide antibodies, and quantification using nanoflow LC-MS/MS. This assay was developed and qualified in human and cynomolgus monkey serum and tissues with a lower limit of quantitation of 0.78 pg/mL based on the intact cytokine. Both intra- and interbatch precision and accuracy, as well as stability and recovery, were found to be acceptable. IL-21 was not detected in serum from normal healthy volunteers or from autoimmune disease patients. However, IL-21 levels were quantified in cynomolgus monkey spleen and colon tissue and normal and inflammatory bowel disease (IBD) human colon tissue as well as hyperplasia human tonsils.

pSTAT3: a target biomarker to study the pharmacology of the anti-IL-21R antibody ATR-107 in human whole blood.

BACKGROUND: IL-21 has been shown to play an important role in autoimmune diseases. ATR-107 is an antibody which directly targets the IL-21 receptor (IL-21R). To aid the clinical development of ATR-107, there is a need for understanding the mechanism of action (MOA) of this antibody when assessing target engagement in human subjects. METHODS: To determine ATR-107 biological activity and potency in human blood, its inhibitory function against IL-21 induced STAT3 phosphorylation in human peripheral T and B cells was measured. RESULTS: The data show that IL-21 induces STAT3 phosphorylation in a concentration-dependent manner, consistent with its migration to the nuclear. Using a flow cytometry based functional whole blood assay, ATR-107 is demonstrated to be a potent IL-21 pathway inhibitor. It competes with IL-21 for receptor binding in a competitive manner, but once it binds to the receptor it behaves like a non-competitive inhibitor, most probably due to the long observed k(off). The concentration-dependent inhibition observed with ATR-107 correlates inversely with the levels of receptor occupancy, both in ex vivo whole blood assays and directly in human blood when ATR-107 was given to healthy volunteers. CONCLUSIONS: IL-21 induced phosphorylation of STAT3 in T and B cells can be used as a biomarker to evaluate the target engagement of ATR-107 in human whole blood. The antibody behaves like a potent non-competitive inhibitor blocking IL-21 induced STAT3 phosphorylation for a long period of time. These results may help with the translation of preclinical information and dose selection towards ATR-107 clinical efficacy.

Leukotrienes, but not angiotensin II, are involved in the renal effects elicited by the prolonged cyclooxygenase-2 inhibition when sodium intake is low.

It is known that cyclooxygenase-2 (COX-2) inhibition elicits significant renal hemodynamics alterations when sodium intake is low. However, the mechanisms involved in these renal changes are not well known. Our objective was to evaluate the role of angiotensin II and 5-lipooxygenase-derived metabolites in the renal effects induced by prolonged COX-2 inhibition when sodium intake is low. Conscious dogs were treated during 7 days with a COX-2 inhibitor (1 mg·kg·d, SC75416), and either a vehicle, an AT1 receptor antagonist (0.4 mg · kg · d, candesartan) or a selective 5-lipooxygenase inhibitor (PF-150, 20 and 60 mg · kg · d). The administration of SC75416 alone induced significant changes in renal blood flow (219 ± 14 to 160 ± 10 mL/min), glomerular filtration rate (51 ± 2 to 42 ± 3 mL/min), and plasma potassium (pK) (4.3 ± 0.1 to 4.6 ± 0.1 mEq/L). Similar decrements in renal blood flow (27%) and glomerular filtration rate (20%) and a similar increment in pK (7%) were found when SC75416 was administered in candesartan-pretreated dogs. However, SC75416 administration did not elicit significant changes in renal hemodynamics and pK in dogs pretreated with each dose of PF-150. Our data suggest that leukotrienes but not angiotensin II are involved in the renal effects induced by COX-2 inhibition when sodium intake is low.

Discovery and SAR of PF-4693627, a potent, selective and orally bioavailable mPGES-1 inhibitor for the potential treatment of inflammation.

Inhibition of mPGES-1, the terminal enzyme in the arachidonic acid/COX pathway to regulate the production of pro-inflammatory prostaglandin PGE2, is considered an attractive new therapeutic target for safe and effective anti-inflammatory drugs. The discovery of a novel series of orally active, selective benzoxazole piperidinecarboxamides as mPGES-1 inhibitors is described. Structure-activity optimization of lead 5 with cyclohexyl carbinols resulted in compound 12, which showed excellent in vitro potency and selectivity against COX-2, and reasonable pharmacokinetic properties. Further SAR studies of the benzoxazole ring substituents lead to a novel series of highly potent compounds with improved PK profile, including 23, 26, and 29, which were effective in a carrageenan-stimulated guinea pig air pouch model of inflammation. Based on its excellent in vitro and in vivo pharmacological, pharmacokinetic and safety profile and ease of synthesis, compound 26 (PF-4693627) was advanced to clinical studies.

Multiplex transcriptional analysis of paraffin-embedded liver needle biopsy from patients with liver fibrosis.

BACKGROUND: The possibility of extracting RNA and measuring RNA expression from paraffin sections can allow extensive investigations on stored paraffin samples obtained from diseased livers and could help with studies of the natural history of liver fibrosis and inflammation, and in particular, correlate basic mechanisms to clinical outcomes. RESULTS: To address this issue, a pilot study of multiplex gene expression using branched-chain DNA technology was conducted to directly measure mRNA expression in formalin-fixed paraffin-embedded needle biopsy samples of human liver. Twenty-five genes were selected for evaluation based on evidence obtained from human fibrotic liver, a rat BDL model and in vitro cultures of immortalized human hepatic stellate cells. The expression levels of these 25 genes were then correlated with liver fibrosis and inflammation activity scores. Statistical analysis revealed that three genes (COL3A1, KRT18, and TUBB) could separate fibrotic from non-fibrotic samples and that the expression of ten genes (ANXA2, TIMP1, CTGF, COL4A1, KRT18, COL1A1, COL3A1, ACTA2, TGFB1, LOXL2) were positively correlated with the level of liver inflammation activity. CONCLUSION: This is the first report describing this multiplex technique for liver fibrosis and has provided the proof of concept of the suitability of RNA extracted from paraffin sections for investigating the modulation of a panel of proinflammatory and profibrogenic genes. This pilot study suggests that this technique will allow extensive investigations on paraffin samples from diseased livers and possibly from any other tissue. Using identical or other genes, this multiplex expression technique could be applied to samples obtained from extensive patient cohorts with stored paraffin samples in order to correlate gene expression with valuable clinically relevant information. This method could be used to provide a better understanding of the mechanisms of liver fibrosis and inflammation, its progression, and help development of new therapeutic approaches for this indication.

Pharmacologic evaluation of sulfasalazine, FTY720, and anti-IL-12/23p40 in a TNBS-induced Crohn's disease model.

BACKGROUND: 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis has been used as an inflammatory bowel disease (IBD), Crohn's disease (CD), preclinical model. However, published data on pharmacologic and therapeutic efficacy testing of this model are limited. FTY720 inhibits lymphoid cell trafficking in inflammatory conditions and is of interest to treat IBD. AIM: We investigated the pharmacologic therapeutic efficacy of sulfasalazine, FTY720, and anti-IL-12/23p40, in a TNBS CD model. METHODS: Female, 7-week-old, BALB/c mice were given sulfasalazine orally (PO) and intraperitoneally (IP) at 10 mg/kg, FTY720 at 3 mg/kg PO, and mouse anti-IL-12/23p40 at 25 mg/kg IP. Vehicle groups given PO either phosphate-buffered saline/water or 40% ethanol served as controls. Pharmacologic efficacy was assessed using body weight loss, clinical scores of diarrhea and intestinal gross pathology, and colon weight parameters. RESULTS: Sulfasalazine and FTY720 treatment did not prevent body weight loss or reduce clinical scores of diarrhea or intestinal gross pathology, when compared with vehicle treatment. However, anti-IL-12/23p40 treatment showed significant efficacy by preventing body weight loss, reducing clinical scores of diarrhea, and reducing intestinal gross pathologic lesions, when compared with vehicle-treated animals. Sulfasalazine, anti-IL-12/23p40, and FTY720 were not effective in reducing colon weight. CONCLUSION: With the exception of anti-IL-12/23p40, sulfasalazine, and FTY720 did not demonstrate full pharmacologic efficacy in our TNBS CD model.

High-throughput screening assay for sphingosine kinase inhibitors in whole blood using RapidFire® mass spectrometry.

To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire(®) mass spectrometry system.