Paul C. Harrison

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.

Anti-LFA-1α reduces the dose of cyclosporin A needed to produce immunosuppression in heterotopic cardiac transplanted rats

BACKGROUND Monoclonal antibodies (MAb) against cell adhesion molecules prolong the time to acute rejection of transplanted organs in animals. Postulated mechanisms of action include blockade of trafficking of host leukocytes into or recognizing of effector/target cells within the allograft. We examined whether an anti-ICAM-1 (1A29), anti-LFA-1 alpha (WT.1), or anti-CD-18 (WT.3) could reduce the immunosuppressive dose of cyclosporin A (CsA) when used in combination. METHODS A rat heterotopic cardiac transplant model with ACI donors and Lewis recipients was used. MAb dose was 3 mg/kg, i.p. with treatment on Days -3 and -1 prior to transplant, followed by daily dosing for 10 days post-transplantation (Tx). Cyclosporin A doses were either 1.5 or 2.75 mg/kg, PO beginning the day of and for 10 days post-Tx. RESULTS Untreated allografted rats demonstrated a mean rejection time (MRT) +/- SEM of 8.8 +/- 0.6 days. Cyclosporin A at 1.5 and 2.75 mg/kg showed mean rejection times of 8.5 +/- 0.3 (NS) and 20.5 +/- 1.9 (p < 0.05) days, respectively. Monotherapy with 1A29 or WT.3 did not prolong MRT, whereas WT.1 increased MRT to 21.7 +/- 4.3 days (p < 0.05). MAb combination therapy did not extend MRT greater than that demonstrated by WT.1 alone. However, MAb and CsA combination therapy significantly increased MRT with WT.1 and CsA resulting in the greatest extension. WT.1 combination with CsA at 1.5 mg/kg and 2.75 mg/kg increased MRT to > 46.8 +/- 6.3 and > 44.2 +/- 9.4 days, respectively. CONCLUSIONS Anti-LFA-1 alpha and CsA combination therapy significantly extends the time to rejection of transplanted rat hearts. We conclude that combining an anti-LFA-1 alpha and CsA may be beneficial in prolonging allograft rejection times and in reducing the amount of CsA necessary for immune suppression, thereby minimizing its toxic effects.

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.

Mitigation of Off-Target Adrenergic Binding and Effects on Cardiovascular Function in the Discovery of Novel Ribosomal S6 Kinase 2 Inhibitors

We previously reported the discovery of a novel ribosomal S6 kinase 2 (RSK2) inhibitor, (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a] indole-8-carboxylic acid [1-(3-dimethylamino-propyl)-1H-benzoimidazol-2-yl]-amide (BIX 02565), with high potency (IC50 = 1.1 nM) targeted for the treatment of heart failure. In the present study, we report that despite nanomolar potency at the target, BIX 02565 elicits off-target binding at multiple adrenergic receptor subtypes that are important in the control of vascular tone and cardiac function. To elucidate in vivo the functional consequence of receptor binding, we characterized the cardiovascular (CV) profile of the compound in an anesthetized rat CV screen and telemetry-instrumented conscious rats. Infusion of BIX 02565 (1, 3, and 10 mg/kg) in the rat CV screen resulted in a precipitous decrease in both mean arterial pressure (MAP; to -65 ± 6 mm Hg below baseline) and heart rate (−93 ± 13 beats/min). In telemetry-instrumented rats, BIX 02565 (30, 100, and 300 mg/kg p.o. QD for 4 days) elicited concentration-dependent decreases in MAP after each dose (to −39 ± 4 mm Hg on day 4 at Tmax); analysis by Demming regression demonstrated strong correlation independent of route of administration and influence of anesthesia. Because of pronounced off-target effects of BIX 02565 on cardiovascular function, a high-throughput selectivity screen at adrenergic α1A and α2A was performed for 30 additional RSK2 inhibitors in a novel chemical series; a wide range of adrenergic binding was achieved (0–92% inhibition), allowing for differentiation within the series. Eleven lead compounds with differential binding were advanced to the rat CV screen for in vivo profiling. This led to the identification of potent RSK2 inhibitors (cellular IC50 <0.14 nM) without relevant α1A and α2A inhibition and no adverse cardiovascular effects in vivo.

Strategic integration of in vivo cardiovascular models during lead optimization: predictive value of 4 models independent of species, route of administration, and influence of anesthesia.

Abstract: The strategic integration of in vivo cardiovascular models is important during lead optimization to enable a wide therapeutic index for cardiovascular safety. However, under what conditions (eg, species, route of administration, anesthesia) studies should be performed to drive go/no-go is open to interpretation. Two compounds, torcetrapib and a novel steroid hormone mimetic (SHM-1121X), both with off-target cardiovascular liabilities, were profiled in 4 in vivo cardiovascular models. Overlapping plasma concentrations of torcetrapib were achieved in all models tested; values ranged from therapeutic to supratherapeutic. In anesthetized rats, intravenous torcetrapib elicited dose-dependent increases in mean arterial pressure (MAP; 2–18 mm Hg above vehicle during the low- and high-dose infusion), and in anesthetized dogs, torcetrapib increased MAP from 4 to 22 mm Hg. In conscious rats, a single oral dose of torcetrapib increased MAP from 10 to 18 mm Hg in the low-dose and high-dose groups, respectively, whereas in conscious dogs, MAP increased from 3 to 12 mm Hg. SHM-1121X produced marked hypotension in the same models. Pharmacokinetic–pharmacodynamic analysis demonstrated strong correlation across the models tested for both compounds. Results suggest that equivalency across models allows for flexibility to address key issues and enable go/no-go during lead optimization without concern for discordant results. The predictive value of each model was validated with torcetrapib and, when put into practice, led to a decisive no-go for SHM-1121X.

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.

Protective Effects of Monoclonal Antibodies to Cell Adhesion Glycoproteins in Models of Cardiac Reperfusion Injury and Leukocyte/Vascular Responses

Prompt reperfusion of an ischemic tissue is necessary for restoration of function but can be associated, paradoxically, with the progressive deterioration of reversibly damaged cells resulting in dysfunction and necrosis (Braunwald 1985; Heras et al. 1988). This reperfusion injury has been studied extensively in models of myocardial ischemia (Lehr et al. 1993; Lefer et al. 1994) but has also been implicated in delayed graft function and rejection following cardiac allograft transplantation (Land 1994). The cause of reperfusion injury is no doubt multifactorial and is believed to involve an inappropriate infiltration of leukocytes and the subsequent release of their cytotoxic mediators into the previously ischemic tissue (Pinckard et al. 1983; Mullane et al. 1984). The leukocytic infiltrate has been implicated in the alteration of tissue function which can lead to cell death and organ failure. Leukocyte adhesion to the vascular endothelium, and subsequent migration into the ischemic myocardium or into a transplanted heart is orchestrated by a series of ligand-receptor events between leukocyte and endothelial cell surface glycoproteins (Lipsky et al. 1993). The importance of the various cell adhesion glycoproteins in mediating tissue damage in preclinical models of these disease states has been addressed with the administration of monoclonal antibodies (MAbs) which recognize and neutralize the function of the particular glycoproteins.