Ryan M. Fryer

Effect of bradykinin metabolism inhibitors on evoked hypotension in rats: rank efficacy of enzymes associated with bradykinin-mediated angioedema

Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre‐clinical model of angioedema.

The clinically-tested S1P receptor agonists, FTY720 and BAF312, demonstrate subtype-specific bradycardia (S1P1) and hypertension (S1P3) in rat.

Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function in vivo. Indeed, FTY720 (non-selective S1PX receptor agonist) produces modest hypertension in patients (2–3 mmHg in 1-yr trial) as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, likely dependent upon the cardiovascular response in question (e.g. bradycardia, hypertension), and perhaps even species-dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, we characterized the S1P receptor subtype specificity for each compound in vitro and, in vivo, the cardiovascular effects of FTY720 and the more selective S1P1,5 agonist, BAF312, were tested during acute i.v. infusion in anesthetized rats and after oral administration for 10 days in telemetry-instrumented conscious rats. Acute i.v. infusion of FTY720 (0.1, 0.3, 1.0 mg/kg/20 min) or BAF312 (0.5, 1.5, 5.0 mg/kg/20 min) elicited acute bradycardia in anesthetized rats demonstrating an S1P1 mediated mechanism-of-action. However, while FTY720 (0.5, 1.5, 5.0 mg/kg/d) elicited dose-dependent hypertension after multiple days of oral administration in rat at clinically relevant plasma concentrations (24-hr mean blood pressure = 8.4, 12.8, 16.2 mmHg above baseline vs. 3 mmHg in vehicle controls), BAF312 (0.3, 3.0, 30.0 mg/kg/d) had no significant effect on blood pressure at any dose tested suggesting that hypertension produced by FTY720 is mediated S1P3 receptors. In summary, in vitro selectivity results in combination with studies performed in anesthetized and conscious rats administered two clinically tested S1P agonists, FTY720 or BAF312, suggest that S1P1 receptors mediate bradycardia while hypertension is mediated by S1P3 receptor activation.

G Protein–Coupled Bile Acid Receptor 1 Stimulation Mediates Arterial Vasodilation through a K Ca 1.1 (BK Ca )–Dependent Mechanism

Bile acids (BAs) and BA receptors, including G protein–coupled bile acid receptor 1 (GPBAR1), represent novel targets for the treatment of metabolic and inflammatory disorders. However, BAs elicit myriad effects on cardiovascular function, although this has not been specifically ascribed to GPBAR1. This study was designed to test whether stimulation of GPBAR1 elicits effects on cardiovascular function that are mechanism based that can be identified in acute ex vivo and in vivo cardiovascular models, to delineate whether effects were due to pathways known to be modulated by BAs, and to establish whether a therapeutic window between in vivo cardiovascular liabilities and on-target efficacy could be defined. The results demonstrated that the infusion of three structurally diverse and selective GPBAR1 agonists produced marked reductions in vascular tone and blood pressure in dog, but not in rat, as well as reflex tachycardia and a positive inotropic response, effects that manifested in an enhanced cardiac output. Changes in cardiovascular function were unrelated to modulation of the levothyroxine/thyroxine axis and were nitric oxide independent. A direct effect on vascular tone was confirmed in dog isolated vascular rings, whereby concentration-dependent decreases in tension that were tightly correlated with reductions in vascular tone observed in vivo and were blocked by iberiotoxin. Compound concentrations in which cardiovascular effects occurred, both ex vivo and in vivo, could not be separated from those necessary for modulation of GPBAR1-mediated efficacy, resulting in project termination. These results are the first to clearly demonstrate direct and potent peripheral arterial vasodilation due to GPBAR1 stimulation in vivo through activation of large conductance Ca2+ activated potassium channel KCa1.1.

A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 rat

Therapies that restore renal cGMP levels are hypothesized to slow the progression of diabetic nephropathy. We investigated the effect of BI 703704, a soluble guanylate cyclase (sGC) activator, on disease progression in obese ZSF1 rats. BI 703704 was administered at doses of 0.3, 1, 3, and 10 mg/kg/d to male ZSF1 rats for 15 weeks, during which mean arterial pressure (MAP), heart rate (HR), and urinary protein excretion (UPE) were determined. Histologic assessment of glomerular and interstitial lesions was also performed. Renal cGMP levels were quantified as an indicator of target modulation. BI 703704 resulted in sGC activation, as evidenced by dose-dependent increases in renal cGMP levels. After 15 weeks of treatment, sGC activation resulted in dose-dependent decreases in UPE (from 463 ± 58 mg/d in vehicle controls to 328 ± 55, 348 ± 23, 283 ± 45, and 108 ± 23 mg/d in BI 703704-treated rats at 0.3, 1, 3, and 10 mg/kg, respectively). These effects were accompanied by a significant reduction in the incidence of glomerulosclerosis and interstitial lesions. Decreases in MAP and increases in HR were only observed at the high dose of BI 703704. These results are the first demonstration of renal protection with sGC activation in a nephropathy model induced by type 2 diabetes. Importantly, beneficial effects were observed at doses that did not significantly alter MAP and HR.

Functional biomarkers of musculoskeletal syndrome (MSS) for early in vivo screening of selective MMP-13 inhibitors

INTRODUCTION Long-term administration of non-selective matrix metalloproteinase (MMP) inhibitors, such as marimastat, in humans elicits musculoskeletal syndrome (MSS), a syndrome characterized by joint damage including pain, stiffness, and inflammation. This pathology is a significant obstacle to the clinical development of MMP inhibitors and in pre-clinical models MSS can be verified only after terminal histopathology. Consequently, we devised a longitudinal and functional readout of MSS in conscious rats treated with marimastat that was validated against terminal histological assessment. METHODS MSS was induced by minipump infusion of marimastat (5-10mg/kg/day). In marimastat-treated or vehicle-control groups, three possible functional biomarkers were assessed: paw volume (PV), landing foot splay separation (LFSS), and rotarod performance (n=6 rats/group for each endpoint). RESULTS Histologically, fibrosis scores in the synovium and ligament increased from 0 on Day 1 (D1) to 4.6±0.2 and 4.7±0.1, respectively, on D15; growth plate thickness was also elevated from 215.0±6.3μm (D1) to 253.3±8.0μm (D15). While neither PV nor LFSS were correlative with MSS histopathology, marimastat (10mg/kg/day) reduced rotarod performance from 180±0s (D0) to 135±30s (D9) using a constant speed protocol (10rpm, 180s) and from 180±0s (D0) to 96±6s (D6) employing a variable speed protocol (increasing from 5 to 25rpm over 180s). DISCUSSION Results of the present study demonstrate that rotarod performance can be used as a predictive longitudinal, in vivo functional biomarker of MSS concomitant with histological evidence of joint damage to effectively facilitate compound selection during drug discovery. Moreover, for targets with a mechanistic risk for MSS, the model is also conducive to inclusion in secondary pharmacodynamic studies during lead optimization to identify the best (safest) compounds for advancement into clinical trials.

Quantification and Comparison of Anti-Fibrotic Therapies by Polarized SRM and SHG-Based Morphometry in Rat UUO Model

Renal interstitial fibrosis (IF) is an important pathologic manifestation of disease progression in a variety of chronic kidney diseases (CKD). However, the quantitative and reproducible analysis of IF remains a challenge, especially in experimental animal models of progressive IF. In this study, we compare traditional polarized Sirius Red morphometry (SRM) to novel Second Harmonic Generation (SHG)-based morphometry of unstained tissues for quantitative analysis of IF in the rat 5 day unilateral ureteral obstruction (UUO) model. To validate the specificity of SHG for detecting fibrillar collagen components in IF, co-localization studies for collagens type I, III, and IV were performed using IHC. In addition, we examined the correlation, dynamic range, sensitivity, and ability of polarized SRM and SHG-based morphometry to detect an anti-fibrotic effect of three different treatment regimens. Comparisons were made across three separate studies in which animals were treated with three mechanistically distinct pharmacologic agents: enalapril (ENA, 15, 30, 60 mg/kg), mycophenolate mofetil (MMF, 2, 20 mg/kg) or the connective tissue growth factor (CTGF) neutralizing antibody, EX75606 (1, 3, 10 mg/kg). Our results demonstrate a strong co-localization of the SHG signal with fibrillar collagens I and III but not non-fibrillar collagen IV. Quantitative IF, calculated as percent cortical area of fibrosis, demonstrated similar response profile for both polarized SRM and SHG-based morphometry. The two methodologies exhibited a strong correlation across all three pharmacology studies (r2 = 0.89–0.96). However, compared with polarized SRM, SHG-based morphometry delivered a greater dynamic range and absolute magnitude of reduction of IF after treatment. In summary, we demonstrate that SHG-based morphometry in unstained kidney tissues is comparable to polarized SRM for quantitation of fibrillar collagens, but with an enhanced sensitivity to detect treatment-induced reductions in IF. Thus, performing SHG-based morphometry on unstained kidney tissue is a reliable alternative to traditional polarized SRM for quantitative analysis of IF.

A soluble guanylate cyclase activator protects from diabetic nephropathy beyond standard of care in the ZSF1 rat.

The pathogenesis of diabetic nephropathy is associated with abnormalities of renal nitric oxide generation and signaling. We evaluated the effect of BI 684067, a soluble guanylate cyclase (sGC) activator, in combination with the current standard of care (SoC), on the progression of diabetic nephropathy. Male ZSF1 rats were administered enalapril (3 mg/kg in drinking water) for 10 days, after which they were randomized to either continue to receive enalapril alone or the combination of enalapril and one of three doses of BI 684067 (20, 40 and 80 mg/kg) in chow for 10 weeks. Weekly urinary protein to creatinine ratio (UPCR) as well as daily mean arterial pressure (MAP) and heart rate (HR) were measured. At study end, kidneys were assessed for glomerular lesions and α-SMA expression, a marker of myofibroblast activation. The combination of BI 684067 and enalapril resulted in significant dose-dependent decreases of the following when compared to enalapril alone: UPCR (BI 684067 at 20, 40, and 80 mg/kg : 27, 39, 48% reductions respectively), incidence of glomerulosclerosis (BI 684067 at 20, 40, and 80 mg/kg: 29, 32, 44% reductions, respectively) and α-SMA expression (BI 684067at 20, 40, and 80 mg/kg : 26, 40, 42% reductions, respectively). The MAP was significantly reduced by BI 684067 in combination with enalapril (- 3 mm Hg vs enalapril alone at the doses of 40 and 80 mg/kg), however there was no significant effect on HR. These results support the efficacy of an sGC activator in preventing the progression of diabetic nephropathy when combined with the SoC.

CCR1 Plays a Critical Role in Modulating Pain through Hematopoietic and Non-Hematopoietic Cells

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1−/− animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freunds adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1−/− mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

Engaging novel cell types, protein targets and efficacy biomarkers in the treatment of diabetic nephropathy.

Diabetic nephropathy (DN) is a well-known complication of diabetes and leading cause of chronic renal failure and end-stage renal disease (ESRD). Currently, angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARBs) are the standard of care for this disease although trials targeting the renin-angiotensin-aldosterone system demonstrate that they delay the progression to ESRD to only a limited extent [~18% relative risk reduction (Brenner et al., 2001; Lewis et al., 2001)]. Furthermore, dual-targeting of this pathway appears less-than-promising, if not deleterious, as evidenced by the failure of the recent ALTITUDE trial with the direct renin inhibitor, aliskiren, where in combination with losartan, therapy reduced albuminuria but failed to impact glomerular filtration rate (Parving et al., 2012). Thus, limited efficacy coupled with our incomplete understanding of this multifaceted disease highlights the importance of unraveling novel targets based on a greater understanding of the pathogenesis of renal impairment in diabetes. We must look beyond ACE-Is/ARBs and capitalize on blending new technologies, target-disease associations, physiology-based insight, and our enhanced ability to mine vast quantities of literature and data as a means to identify new targets and then deliver novel drug candidates into the clinic. Various signaling pathways encompassing inflammation, fibrosis, and oxidative stress are implicated in the pathogenesis of DN (Reidy et al., 2014) and perhaps the key to novel and efficacious therapies lays within the identification of druggable targets that encompass these diverse processes. Can a population of inflammatory cells be targeted to slow disease progression? Or, should we look more closely to podocyte-selective targets central to the regulation of glomerular filtration? Alternatively, do we set our sights within the complicated environment of the renal microvasculature? While early DN may represent an overtly angiogenic environment, advanced disease likely represents a condition of capillary loss and excess anti-angiogenic activity (Advani and Gilbert, 2012). If so, can we reasonably target the renal microvasculature in a manner expected to yield benefit across various stages of DN or might this type of targeted therapy treat only a smaller subset of the DN population? In this Research Topic, contributing authors explored four sub-areas within the realm of chronic kidney disease and DN including capitalizing on the discovery of novel mineralocorticoid receptors with potentially lesser risk for hyperkalemia (Orena et al., 2013), the potential for modulation of a secreted glycoprotein already recognized as a contributor to minimal change disease and now also as a target in DN (Chugh et al., 2014), use of an endogenous anti-fibrotic peptide in renal disease (Kanasaki et al., 2014), and basic science that investigated the expression of mitogen-activated protein kinases (MAPKs) in podocytes as potential new targets in DN (Badshah et al., 2014), an interesting area that may offer another pathway and novel players for targeted therapies in this disease. Indeed, results reported in this Research Topic by Orena et al. (2013), who investigated renal protection against aldosterone-mediated renal disease, in uninephrectomzied rats on high salt, demonstrate the anti-proteinuric and renal anti-fibrotic effect of PF-03882845 with a reduced risk of hyperkalemia compared to traditional mineralocorticoid (MR) antagonist. Should these findings translate to a greater safety profile in humans, this therapy might constitute a significant and beneficial add-on to the current standard of care. Chugh et al. (2014) describe, in detail, the discovery of angiopoietin-like protein 4 (ANGPTL4), secreted by podocytes, as a major player in human nephrotic syndrome and highlight the strategy used to identify and then selectively investigate genes and proteins that lay at the intersection of proteinuria, hyperlipidemia, and edema during disease progression. They suggest that sialylation-based, and recombinant mutated ANGPTL4-based, therapeutics could hold promise in the treatment of common forms of proteinuric kidney disease including DN. Moreover, it could be envisioned that others could capitalize on a strategy similar to that employed by the authors to identify additional novel genes and proteins that could be targeted for the treatment of human DN. Investigating endogenous molecules as potential means for identification of novel therapeutics, Kanasaki et al. (2014) discuss novel therapeutic possibilities for renal disease and detail work surrounding N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a substrate for ACE and theoretical candidate for anti-fibrotic therapeutics. A large body of published data suggests that podocytes play a major role in the development of proteinuria and kidney disease by their ability to regulate glomerular filtration. Additionally, analysis using samples from both type I and II diabetic patients indicated that podocyte number is highly correlated with proteinuria and appears to be one of the best disease predictors (White et al., 2002). However, the mechanisms of podocyte injury leading to foot process effacement and proteinuria are unclear. In the current research topic, Badshah et al. (2014) investigated the role of atypical MAP kinase Erk5 in mediating TGFβ1-induced podocyte damage. Indeed, the role of ERK5 in the proliferation, apoptosis, and motility of cultured human podocytes was explored using BIX02188, a novel Mek5 inhibitor that was experimentally shown to reduce TGFβ1-induced Erk5 phosphorylation. They confirmed the presence of three splice variants of Erk5 in human podocytes that were phosphorylated by Mek5 in a TGFβ1-dependent manner. TGFβ1-induced the proliferation and apoptosis of podocytes while decreasing motility. While proliferation was reduced by inhibiting Erk5 phosphorylation with BIX02188, no changes in apoptosis or motility were observed. Most importantly, inhibition of Mek5 with BIX02188 and subsequent loss of Erk5 phosphorylation, reduced TGFβ1-induced alterations of cellular phenotype, as determined by a reduction in the expression of P-cadherin and increased α-SMA, in addition to a reversal of TGFβ1-indcued loss of barrier function. These results describe for the first time the expression of Erk5 in podocytes and identify it as a potential target for the treatment of diabetic renal disease. Taken together, the findings highlighted in this research topic provide novel insight into new or pre-existing pathways implicated in disease. As researchers continue to seek and investigate mechanisms causal of disease, the importance of biomarkers predictors of efficacy should be emphasized. Novel biomarkers tailored toward the targeted pathways (such as inflammation, fibrosis, and endothelial function) and linked to disease are urgently needed to provide clear efficacy signals in phase II clinical trials and minimize further failures in phase III.

Cardiovascular safety pharmacology studies in dogs enabled for a poorly soluble molecule using spray-dried dispersion: Impact on lead selection.

The aim of this study was to identify an adequate formulation for a poorly soluble lead molecule (BI-A) that would achieve sufficiently high plasma concentrations after oral administration in dogs to enable a robust cardiovascular safety pharmacology assessment in telemetry-instrumented conscious dogs during lead optimization in drug discovery. A spray-dried dispersion of BI-A (BI-A-SDD) containing a 1:2 ratio of BI-A and hydroxypropyl methylcellulose acetate succinate-LF was prepared using a Büchi spray dryer B-90 (B-90). Physical form characterization, an in vitro dissolution test and a preliminary pharmacokinetic (PK) study following oral administration of BI-A-SDD were performed. Thereafter, effects on cardiovascular parameters in conscious, chronically-instrumented dogs were investigated for 24h after a single oral dose (5, 10, and 50mg/kg) using a modified Latin square cross-over study design. The BI-A-SDD powder was confirmed to be amorphous and was stable as an aqueous suspension for at least 4h. The BI-A-SDD suspension provided a greater rate and extent of dissolution than the crystalline BI-A suspension and the supersaturation was maintained for at least 4h. In PK studies the Cmax of the BI-A-SDD formulation (25.4μM; 77-fold the projected efficacious Cmax of 0.33μM) was 7.5-fold higher than the Cmax observed using oral administration of a 10% hydroxypropyl-β-cyclodextrin formulation at 100mg/kg in dogs (3.4μM). In conscious, chronically-instrumented dogs, the doses tested and plasma concentrations achieved were sufficient to enable a robust safety pharmacology evaluation. Multiple off-target hemodynamic effects were detected including acute elevations in aortic blood pressure (up to 22% elevation in systolic and diastolic blood pressure) and tachycardia (68% elevation in heart rate), results that were confirmed in other in vivo models. These results led to a deprioritization of BI-A. The study demonstrated that a spray-dried dispersion, prepared using the B-90 in drug discovery, enhanced the oral exposure of a poorly water-soluble molecule, BI-A, and thereby enabled its evaluation in safety pharmacology studies that ultimately resulted in deprioritization of BI-A from a pool of lead compounds.