The Novel, Clinical-Stage Soluble Guanylate Cyclase Activator BI 685509 Protects from Disease Progression in Models of Renal Injury and Disease.

Activation of soluble guanylate cyclase (sGC) to restore cyclic guanosine monophosphate (cGMP) and improve functionality of nitric oxide (NO) pathways impaired by oxidative stress is a potential treatment of diabetic and chronic kidney disease. We report the pharmacology of BI 685509, a novel, orally active small molecule sGC activator with disease-modifying potential. BI 685509 and human sGC α1/ß1 heterodimer containing a reduced heme group produced concentration-dependent increases in cGMP that were elevated modestly by NO, whereas heme-free sGC and BI 685509 greatly enhanced cGMP with no effect of NO. BI 685509 increased cGMP in human and rat platelet-rich plasma treated with the heme-oxidant ODQ; respective EC50 values were 467 nM and 304 nM. In conscious telemetry-instrumented rats, BI 685509 did not affect mean arterial pressure (MAP) or heart rate (HR) at 3 and 10 mg/kg (p.o.), whereas 30 mg/kg decreased MAP and increased HR. Ten days of BI 685509 at supratherapeutic doses (60 or 100 mg/kg p.o., daily) attenuated MAP and HR responses to a single 100 mg/kg challenge. In the ZSF1 rat model, BI 685509 (1, 3, 10, and 30 mg/kg per day, daily) coadministered with enalapril (3 mg/kg per day) dose-dependently reduced proteinuria and incidence of glomerular sclerosis; MAP was modestly reduced at the higher doses versus enalapril. In the 7-day rat unilateral ureteral obstruction model, BI 685509 dose-dependently reduced tubulointerstitial fibrosis (P < 0.05 at 30 mg/kg). In conclusion, BI 685509 is a potent, orally bioavailable sGC activator with clear renal protection and antifibrotic activity in preclinical models of kidney injury and disease. SIGNIFICANCE STATEMENT: BI 685509 is a novel small soluble guanylate cyclase (sGC) molecule activator that exhibits an in vitro profile consistent with that of an sGC activator. BI 685509 reduced proteinuria and glomerulosclerosis in the ZSF1 rat, a model of diabetic kidney disease (DKD), and reduced tubulointerstitial fibrosis in a rat 7-day unilateral ureteral obstruction model. Thus, BI 685509 is a promising new therapeutic agent and is currently in phase II clinical trials for chronic kidney disease and DKD.

Selectivity of BI 689648, a Novel, Highly Selective Aldosterone Synthase Inhibitor: Comparison with FAD286 and LCI699 in Nonhuman Primates.

The mineralocorticoid aldosterone is an important regulator of blood pressure, volume, and electrolyte balance. However, excess aldosterone can be deleterious as a driver of vascular remodeling and tissue fibrosis associated with cardiometabolic diseases. Aldosterone synthase (AS) inhibitors (ASI) attenuate the production of aldosterone directly and have been proposed as an alternative to mineralocorticoid receptor antagonists for blocking the pathologic effects of excess aldosterone. Discovery of selective ASIs has been challenging because of the high sequence identity (93%) AS shares with cortisol synthase (CS), and the low identity of rodent AS compared with human (63%). Using cynomolgus (cyno) monkey-based models, we identified BI 689648 [6-(5-methoxymethyl-pyridin-3-yl)-3,4-dihydro-2H-[1,8]naphthyridine-1-carboxylic acid amide], a novel, highly selective ASI that exhibits an in vitro IC50 of 2 nM against AS and 300 nm against CS (150-fold selectivity) compared with the recently described ASIs FAD286 [4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-5-yl)benzonitrile] (3 nM AS; 90 nM CS; 40-fold) and LCI699 (4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluorobenzonitrile) (10 nM AS; 80 nM CS; 8-fold). After oral administration in cyno monkeys, BI 689648 (5 mg/kg) exhibits a peak plasma concentration of ∼500 nM. For in vivo profiling we used an adrenocorticotropin-challenge model in which BI 689648 was >20-fold more selective compared with FAD286 and LCI699. Because both FAD286 and LCI699 failed to provide adequate selectivity for CS when tested in patients, the desire for more selective molecules to test the ASI hypothesis remains high. Therefore, highly selective aldosterone synthase inhibitors such as BI 689648 represent an important step forward toward developing ASIs with greater potential for clinical success in cardiometabolic diseases.

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 (IC(50) = 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 T(max)); 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 α(1A) and α(2A) 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 IC(50) <0.14 nM) without relevant α(1A) and α(2A) 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.

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.

Tumour-selective antivascular effects of the novel anti-mitotic compound A-318315: An in vivo rat regional haemodynamic study.

1. It has been shown that tubulin-binding agents can destabilize cellular microtubules and suppress tumour growth; but it has also become apparent that some compounds can exert anti-vascular effects within the neovasculature of a solid tumour. To date, the difficulty with these targets has been the ability to selectivity induce vascular damage to the tumour while leaving normal vasculature unaffected. The data presented here characterizes the in vivo, tumour selective, anti-vascular effects of the novel tubulin-binding agent A-318315. 2. To that purpose, we have used an anaesthetized in vivo rat model designed to quantify acute changes in regional vascular resistance (VR) in both tumour and non-tumour vascular beds, simultaneously. Tissue-isolated tumours (approximately 1.25 gm) with blood flow supplied by a single epigastric artery were grown in the hindlimb of adult male rats. Blood flow to the tumour, mesenteric, renal and normal (non-tumour epigastric) arteries was measured pre-dose and post-dose under anaesthesia. 3. A-318315 was tested at 3, 10 and 30 mg/kg, i.v. These doses produced modest, transient increases in mean arterial pressure with little to no effect on heart rate. At peak effect, tumour VR increased to 175 +/- 47, 337 +/- 77 and 751 +/- 151% above the baseline, for the 3, 10 and 30 mg/kg doses, respectively, whereas VR was only modestly and transiently increased in normal epigastric (88 +/- 19%), mesenteric (33 +/- 3.3%) and renal arteries (17 +/- 8.6%). 4. These data demonstrate that A-318315 produces marked reductions in tumour blood flow in the rat at doses that exert minor effects on normal vascular function.

Differential inhibition of renin mRNA expression by paricalcitol and calcitriol in C57/BL6 mice.

BACKGROUND/AIMS: Vitamin D receptor activators (VDRAs) may suppress renin expression and VDR-mediated renin inhibitors may offer a novel mechanism to control the RAS. METHODS: We delineated the effects of paricalcitol and calcitriol on PTH, renin, and iCa(2+) in C57/BL6 mice administered vehicle, paricalcitol, or calcitriol (0.01, 0.03, 0.10, 0.33, 1.0 microg/kg s.c.) 3 days/week for 9 days. RESULTS: Paricalcitol produced PTH suppression from 0.03 to 1.0 microg/kg (values between 9.7 +/- 3.3 and 20.7 +/- 4.7 pg/ml; vehicle = 88.0 +/- 16.9) and elicited dose-dependent reductions in renin/GAPDH expression at 0.33 and 1.0 microg/kg (0.037 +/- 0.002, 0.027 +/- 0.003; vehicle = 0.054 +/- 0.003) but produced no increases iCa(2+) at any dose tested. Calcitrol produced PTH suppression at all doses tested (between 6.4 +/- 1.2 and 29.5 +/- 17.2 pg/ml) and renin suppression at 0.10, 0.33, and 1.0 microg/kg (0.029 +/- 0.002, 0.031 +/- 0.003, and 0.038 +/- 0.02). However, at 0.33 and 1.0 mg/kg, calcitriol produced increases iCa(2+) (1.31 +/- 0.03 and 1.48 +/- 0.02 mmol/l; vehicle = 1.23 +/- 0.02 mmol/l). CONCLUSIONS: Paricalcitol produces significant, dose-dependent suppression of renin expression in the absence of hypercalcemia at doses 10-fold above those necessary for PTH suppression. Calcitriol also produced suppression of renin at doses at least 10-fold above those required for PTH suppression, but increases in iCa(2+) were observed at doses only 3-fold above those necessary to elicit renin suppression.

Predictive, non-GLP models of secondary pharmacodynamics: putting the best compounds forward.

Secondary pharmacodynamic studies of new chemical entities (NCEs) play a critical role in support of efficient drug discovery. In an era in which speed and efficiency are the norm for pharmaceutical discovery, the need to identify NCEs with greater patient tolerability continues to increase. Early use of secondary pharmacodynamic models (in vivo and in vitro) provides the foundation for critical, early decisions regarding lead molecules. Scientifically robust, non-GLP (good laboratory practices) secondary pharmacodynamic studies can eliminate compounds or structural series with undesirable profiles early, and may prove useful in defining structure-activity relationships (SARs) with regards to off-target effects.

Discovery of ((4R,5S)-5-amino-4-(2,4,5- trifluorophenyl)cyclohex-1-enyl)-(3- (trifluoromethyl)-5,6-dihydro- [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone (ABT-341), a highly potent, selective, orally efficacious, and safe dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP4) deactivates glucose-regulating hormones such as GLP-1 and GIP, thus, DPP4 inhibition has become a useful therapy for type 2 diabetes. Optimization of the high-throughput screening lead 6 led to the discovery of 25 (ABT-341), a highly potent, selective, and orally bioavailable DPP4 inhibitor. When dosed orally, 25 dose-dependently reduced glucose excursion in ZDF rats. Amide 25 is safe in a battery of in vitro and in vivo tests and may represent a new therapeutic agent for the treatment of type 2 diabetes.

Screening for cardiovascular safety: a structure-activity approach for guiding lead selection of melanin concentrating hormone receptor 1 antagonists.

An inactin-anesthetized rat cardiovascular (CV) assay was employed in a screening mode to triage multiple classes of melanin-concentrating hormone receptor 1 (MCHr1) antagonists. Lead identification was based on a compound profile producing high drug concentration in both plasma (>40 microM) and brain (>20 microg/g) with <15% change in cardiovascular endpoints. As a result of these stringent requirements, lead optimization activities on multiple classes of MCHr1 antagonists were terminated. After providing evidence that the cardiovascular liabilities were not a function of MCHr1 antagonism, continued screening identified the chromone-substituted aminopiperidine amides as a class of MCHr1 antagonists that demonstrated a safe cardiovascular profile at high drug concentrations in both plasma and brain. The high incidence of adverse cardiovascular effects associated with an array of MCHr1 antagonists of significant chemical diversity, combined with the stringent safety requirements for antiobesity drugs, highlight the importance of incorporating cardiovascular safety assessment early in the lead selection process.

Optimization of chromone-2-carboxamide melanin concentrating hormone receptor 1 antagonists: assessment of potency, efficacy, and cardiovascular safety.

Evaluation of multiple structurally distinct series of melanin concentrating hormone receptor 1 antagonists in an anesthetized rat cardiovascualar assay led to the identification of a chromone-2-carboxamide series as having excellent safety against the chosen cardiovascular endpoints at high drug concentrations in the plasma and brain. Optimization of this series led to considerable improvements in affinity, functional potency, and pharmacokinetic profile. This led to the identification of a 7-fluorochromone-2-carboxamide (22) that was orally efficacious in a diet-induced obese mouse model, retained a favorable cardiovascular profile in rat, and demonstrated dramatic improvement in effects on mean arterial pressure in our dog cardiovascular model compared to other series reported by our group. However, this analogue also led to prolongation of the QT interval in the dog that was linked to affinity for hERG channel and unexpectedly potent functional blockade of this ion channel.

Discovery of 2-[4-{{2-(2S,5R)-2-cyano-5-ethynyl-1-pyrrolidinyl]-2-oxoethyl]amino]- 4-methyl-1-piperidinyl]-4-pyridinecarboxylic acid (ABT-279): a very potent, selective, effective, and well-tolerated inhibitor of dipeptidyl peptidase-IV, useful for the treatment of diabetes.

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are poised to be the next major drug class for the treatment of type 2 diabetes. Structure-activity studies of substitutions at the C5 position of the 2-cyanopyrrolidide warhead led to the discovery of potent inhibitors of DPP-IV that lack activity against DPP8 and DPP9. Further modification led to an extremely potent (Ki(DPP)(-)(IV) = 1.0 nM) and selective (Ki(DPP8) > 30 microM; Ki(DPP9) > 30 microM) clinical candidate, ABT-279, that is orally available, efficacious, and remarkably safe in preclinical safety studies.

Cardiovascular disease in chronic kidney failure: the role of VDR activators.

Vitamin D3 is modified by vitamin D3 25-hydroxylase in the liver, and by 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) in the kidney, to form the active metabolite 1alpha,25-dihydroxyvitamin D3. Several vitamin D receptor (VDR) activators, including paricalcitol and calcitriol, are currently available for the treatment of hyperparathyroidism secondary to chronic kidney disease (CKD). CKD patients encounter a much higher risk of cardiovascular disease than do members of the general public, and recent clinical observations have shown that VDR activator therapy provides survival benefit for CKD patients in the rank order of paricalcitol > calcitriol > no VDR activator therapy, independent of parathyroid hormone, phosphorus and calcium. One possible explanation for this observation is that VDR activators exert a positive impact on cardiovascular functions. Studies in animals with disrupted genes involved in the vitamin D signaling pathway have provided some interesting data. For example, in mice lacking VDR or CYP27B1, it was found that in addition to the expected phenotype (hypocalcemia, secondary hyperparathyroidism and osteomalacia), expression of renin or atrial natriuretic peptide was elevated. The mice also developed hypertension and cardiac hypertrophy. Gene expression profiling studies have revealed that VDR may play a role in regulating smooth-muscle-cell (SMC) proliferation, thrombosis, fibrinolysis and vessel relaxation. Paricalcitol and calcitriol are equally potent at suppressing plasminogen activator inhibitor-1 synthesis and inhibiting cellular proliferation in human coronary artery SMCs. The effect of VDR activators on the modulation of renin expression and vascular functions may be factors that contribute to reduced mortality and morbidity risk in VDR-activator-treated CKD patients. In this review, we discuss recent preclinical and clinical data regarding the role of VDR and its ligands in the cardiovascular system.

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.

Discovery and characterization of aminopiperidinecoumarin melanin concentrating hormone receptor 1 antagonists.

4-(1-Benzo[1,3]dioxol-5-ylmethylpiperidine-4-ylmethyl)-6-chlorochromen-2-one (7) is a potent, orally bioavailable melanin concentrating hormone receptor 1 (MCHr1) antagonist that causes dose-dependent weight loss in diet-induced obese mice. Further evaluation of 7 in an anesthetized dog model of cardiovascular safety revealed adverse hemodynamic effects at a plasma concentration comparable to the minimally effective therapeutic concentration. These results highlight the need for scrutiny of the cardiovascular safety profile of MCHr1 antagonists.

Long-term stability, reproducibility, and statistical sensitivity of a telemetry-instrumented dog model: A 27-month longitudinal assessment.

INTRODUCTION: ICH guidelines, as well as best-practice and ethical considerations, provide strong rationale for use of telemetry-instrumented dog colonies for cardiovascular safety assessment. However, few studies have investigated the long-term stability of cardiovascular function at baseline, reproducibility in response to pharmacologic challenge, and maintenance of statistical sensitivity to define the usable life of the colony. These questions were addressed in 3 identical studies spanning 27months and were performed in the same colony of dogs. METHODS: Telemetry-instrumented dogs (n=4) received a single dose of dl-sotalol (10mg/kg, p.o.), a ß1 adrenergic and IKr blocker, or vehicle, in 3 separate studies spanning 27months. Systemic hemodynamics, cardiovascular function, and ECG parameters were monitored for 18h post-dose; plasma drug concentrations (Cp) were measured at 1, 3, 5, and 24h post-dose. RESULTS: Baseline hemodynamic/ECG values were consistent across the 27-month study with the exception of modest age-dependent decreases in heart rate and the corresponding QT-interval. dl-Sotalol elicited highly reproducible effects in each study. Reductions in heart rate after dl-sotalol treatment ranged between -22 and -32 beats/min, and slight differences in magnitude could be ascribed to variability in dl-sotalol Cp (range=3230-5087ng/mL); dl-sotalol also reduced LV-dP/dtmax 13-22%. dl-Sotalol increased the slope of the PR-RR relationship suggesting inhibition of AV-conduction. Increases in the heart-rate corrected QT-interval were not significantly different across the 3 studies and results of a power analysis demonstrated that the detection limit for QTc values was not diminished throughout the 27month period and across a range of power assumptions despite modest, age-dependent changes in heart rate. DISCUSSION: These results demonstrate the long-term stability of a telemetry dog colony as evidenced by a stability of baseline values, consistently reproducible response to pharmacologic challenge and no diminished statistical sensitivity over time.

Vitamin D analogs: novel therapeutic agents for cardiovascular disease?

Vitamin D3 plays a key role in regulating calcium and mineral homeostasis in support of normal development and maintenance of bone. The classic effects of vitamin D3 include promoting absorption of dietary calcium in the gut and, through its actions as a steroid endocrine hormone, regulating the synthesis and secretion of parathyroid hormone. The effects of the vitamin D3 system are mediated through the highly regulated generation of the potent, active metabolite 1,25-dihydroxyvitamin D3 (calcitriol). Vitamin D3 exerts its effects through the vitamin D3 receptor (VDR), a ligand-activated nuclear receptor expressed in a wide array of tissue and cell types. Studies performed in mice rendered deficient for VDR suggest that calcitriol and VDR may inhibit the renin-angiotensin system and reduce blood pressure in the long-term. Clinical studies suggest that administration of vitamin D3 analogs produces differential benefit with regards to mortality in dialysis patients; other studies suggest that vitamin D3 analogs may provide cardiovascular benefit in both dialysis and nondialysis patients. This paper reviews clinical and preclinical studies, which suggest that vitamin D3 analogs may provide therapeutic utility in the treatment of cardiovascular disease independent of those mechanisms typically associated with the vitamin D3 endocrine system.

Tumor selective antivascular effects of the novel antimitotic compound ABT-751: an in vivo rat regional hemodynamic study.

Selective induction of vascular damage within a growing tumor is a potentially important approach in the search for potent anticancer therapeutics. Tubulin-binding (antimitotic) agents destabilize cellular microtubules, suppress tumor growth, and exert antivascular effects with varying degrees of tumor selectivity in preclinical models. The tumor-selective, antivascular effects of ABT-751, a novel, orally active antimitotic agent, currently in phase II clinical development, were characterized in vivo in the present study. We developed an in vivo rat model designed to quantify acute changes in regional vascular resistance (VR) in both tumor and non-tumor vascular beds simultaneously. Tissue-isolated tumors (1 g) with blood flow supplied by a single epigastric artery were grown in rats. Subsequently, tumor blood flow was measured under anesthesia in solid tumors and also in mesenteric, renal, and normal epigastric arteries. Phenylephrine-induced (1 micromol/kg) increases in VR were not different between tumor and non-tumor epigastric arteries, suggesting that tumor vessels possess relatively normal vasoconstrictive function. ABT-751 (3, 10, and 30 mg/kg; i.v.) produced modest transient increases in mean arterial pressure with no effect on heart rate. Tumor VR increased to 75+/-36, 732+/-172, and 727+/-125% above baseline, respectively (P<0.05 for the 10 and 30 mg/kg doses), whereas VR in normal epigastric arteries was not significantly affected. Administration of ABT-751 produced transient modest ( P<0.05) increases in mesenteric VR and no effect on renal VR. These results demonstrate that ABT-751 produces marked reductions in tumor blood flow in the intact rat at doses that exert negligible effects on normal vascular function.

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 S1P(X) 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.

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.

Comparative effects of levosimendan, OR-1896, OR-1855, dobutamine, and milrinone on vascular resistance, indexes of cardiac function, and O2 consumption in dogs.

Levosimendan enhances cardiac contractility via Ca(2+) sensitization and induces vasodilation through the activation of ATP-dependent K(+) and large-conductance Ca(2+)-dependent K(+) channels. However, the hemodynamic effects of levosimendan, as well as its metabolites, OR-1896 and OR-1855, relative to plasma concentrations achieved, are not well defined. Thus levosimendan, OR-1896, OR-1855, or vehicle was infused at 0.01, 0.03, 0.1, and 0.3 mumol.kg(-1).30 min(-1), targeting therapeutic to supratherapeutic concentrations of total levosimendan (62.6 ng/ml). Results were compared with those of the beta(1)-agonist dobutamine and the phosphodiesterase 3 inhibitor milrinone. Peak concentrations of levosimendan, OR-1896, and OR-1855 were 455 +/- 21, 126 +/- 6, and 136 +/- 6 ng/ml, respectively. Levosimendan and OR-1896 produced dose-dependent reductions in mean arterial pressure (-31 +/- 2 and -42 +/- 3 mmHg, respectively) and systemic resistance without affecting pulse pressure, effects paralleled by increases in heart rate; OR-1855 produced no effect at any dose tested. Dobutamine, but not milrinone, increased mean arterial pressure and pulse pressure (17 +/- 2 and 23 +/- 2 mmHg, respectively). Regarding potency to elicit reductions in time to peak pressure and time to systolic pressure recovery: OR-1896 > levosimendan > milrinone > dobutamine. Levosimendan and OR-1896 elicited dose-dependent increases in change in pressure over time (118 +/- 10 and 133 +/- 13%, respectively), concomitant with reductions in left ventricular end-diastolic pressure and ejection time. However, neither levosimendan nor OR-1896 produced increases in myocardial oxygen consumption at inotropic and vasodilatory concentrations, whereas dobutamine increased myocardial oxygen consumption (79% above baseline). Effects of the levosimendan and OR-1896 were limited to the systemic circulation; neither compound produced changes in pulmonary pressure, whereas dobutamine produced profound increases (74 +/- 13%). Thus levosimendan and OR-1896 are hemodynamically active in the anesthetized dog at concentrations observed clinically and elicit cardiovascular effects consistent with activation of both K(+) channels and Ca(2+) sensitization, whereas OR-1855 is inactive on endpoints measured in this study.