Background and purpose: 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. Experimental approach: Rats were instrumented to record blood pressure and heart rate; inhibitors were infused for 35 min and bradykinin was infused during the last 5 min to elicit hypotension, as a functional marker of circulating bradykinin and relative angioedema risk. Key results: In the presence of omapatrilat bradykinin produced dose-dependent hypotension, an effect abolished by B 2 blockade. In the presence of lisinopril (ACE inhibitor), but not candoxatril (NEP inhibitor) or apstatin (APP inhibitor), bradykinin also elicited hypotension. Lisinopril-mediated hypotension was unchanged with concomitant blockade of NEP or NEP/DPPIV (candoxatril þ A-899301). However, hypotension was enhanced upon concomitant blockade of APP and further intensified in the presence of NEP inhibition to values not different from omapatrilat alone. Conclusions and implications:We demonstrated that bradykinin is degraded in vivo with an enzyme rank-efficacy of ACE4APPcNEP or DPPIV. These results suggest the effects of omapatrilat are mediated by inhibition of three BMEs, ACE/APP/ NEP. However, dual inhibition of ACE/NEP or ACE/NEP/DPPIV elicits no increased risk of angioedema compared to ACE inhibition alone. Thus, novel BME inhibitors must display no activity against APP to avoid angioedema risk due to high prevalence of ACE inhibitor therapy in patients with diabetes and cardiovascular disease.

The 5-tetrazole amide of L-N(6)-(1-iminoethyl)lysine (L-NIL), L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide (1), has been prepared and evaluated. In contrast to L-NIL, 1 is a stable, nonhygroscopic, crystalline solid. Unlike L-NIL, 1 has minimal inhibitory activity in vitro on human inducible nitric oxide synthase (iNOS). However, it is rapidly converted in vivo to L-NIL and produces dose-dependent inhibition of iNOS in acute and chronic models of inflammation in the rodent with efficacy comparable to L-NIL. In addition, both 1 and L-NIL exhibit significant and comparable in vivo selectivity for the inhibition of iNOS vs endothelial NOS. Doses approximately 80-fold greater than those that inhibited inflammation do not elevate systemic blood pressure. In summary, both the physical properties and the pharmacological profile of 1 make it an ideal molecule for preclinical and clinical studies on the role of selective iNOS inhibitors in mediating inflammatory disease processes.

In the reverse passive Arthus reaction in mouse skin and immune injury of mouse dermal basement membrane, neutrophil (PMN) infiltration in mast cell deficient WBB6F1-W/Wv (W/Wv) mice was only 40% of that in WBB6F1-(+)/+ (+/+) mice that had a normal mast cell repertoire. An anti-tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (mAb) decreased PMN infiltration by 35-80% in +/+ but not W/Wv mice. In addition, an anti-human interleukin-8 (IL-8) MAb, DM/C7, inhibited PMN infiltration of the skin induced by either intradermal administration of recombinant human IL-1 beta or immune complex deposition. In both models of immune complex injury, DM/C7 reduced PMN infiltration by 40-60% in +/+ mice but not W/Wv mice. PMN infiltration and the sensitivity of this infiltration to anti-TNF-alpha or DM/C7 MAb in W/Wv mice whose mast cell population had been restored was indistinguishable from the influx observed in +/+ mice. These data suggest that TNF-alpha, IL-8, and mast cells play a fundamental role in PMN recruitment following immune complex injury.

Background: Obese ZSF-1 rats display many features of human type II diabetes including nephropathy (DN). The study aimed to further understand the relevance of this model to DN, for which glomerular filtration rate (GFR), renal fibrosis and several urinary/tissue biomarkers was followed over 24 weeks in ZSF-1 rats. Methods: Intact/sham or uninephrectomized male and female ZSF-1 rats were studied. GFR was measured by transdermal clearance of fluorescein isothiocyanate-sinistrin. Urine was collected every 2-4 weeks for biomarker analysis. Renal tissue was examined histologically for fibrosis and for levels of inflammatory and fibrotic genes. Results: Male obese ZSF-1 rats demonstrated metabolic syndrome and proteinuria. Female counterparts were hyperlipidemic with delayed proteinuria, but were not hyperglycemic. Kidney hyperfiltration was observed in male obese rats in weeks 2-4 after surgery, and subsequently declined to levels significantly lower than controls. Tubulointerstitial/glomerular fibrosis in male obese rats was significantly elevated by week 12 post surgery and continued to expand in the ensuing weeks, particularly in uninephrectomized rats. Female rats had less severe fibrosis. Except for epidermal growth factor which decreased, the levels of several key inflammatory, injury and fibrotic factors were elevated in both tissue (mRNA) and urine (protein) of male obese rats. Conclusion: Male obese ZSF-1 rats represent an important DN model, manifesting key pathophysiological features including metabolic syndrome, proteinuria, progressive tubular and glomerular fibrosis, and transient hyperfiltration followed by progressive decline in renal function. Uninephrectomy significantly accelerated disease progression. Females were less severe in disease manifestation. Several urinary and tissue biomarkers were identified in the male obese rats that tracked with disease progression.

TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621–responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer. Significance: This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.

Calcium-sensing receptor (CaR) activation decreases serum parathyroid hormone (PTH) and Ca 2ϩ and, despite long-term reductions in mean arterial blood pressure (MAP), may produce acute hypertension in rats, an effect we hypothesized was mediated by constriction of multiple vascular beds. Rats were subjected to 5/6 nephrectomy (NX) or no surgery (Normal); at 7 to 8 weeks, uremia animals were anesthetized and instrumented to record MAP and regional blood flow (carotid, mesenteric, and hindlimb). Cinacalcet [N-(1-naphthalen-1-ylethyl)-3-[3-(trifluoromethyl)phenyl]-propan-1-amine; 1, 3, and 10 mg/ kg; 30 min/dose] was infused over 90 min. In NX rats, cinacalcet dose-dependently decreased ionized calcium (iCa 2ϩ ), elicited a 90% reduction in PTH, and produced dose-dependent selflimiting increases in MAP (from 119 Ϯ 6 to 129 Ϯ 5, 142 Ϯ 4, and 145 Ϯ 3 mm Hg at the end of each infusion). At 1 mg/kg, carotid vascular resistance (CVR) and mesenteric vascular resistance (MVR) increased to 16 Ϯ 6 and 18 Ϯ 6% above baseline, respectively. Hindlimb vascular resistance (HVR) also trended upward (13 Ϯ 8%). At 3 mg/kg, increases in CVR (38 Ϯ 10%), MVR (40 Ϯ 8%), and HVR (39 Ϯ 14%) were exacerbated; at 10 mg/kg, values remained at or near these levels. The effects of cinacalcet in Normal rats were similar to NX and were attenuated by ganglionic blockade with hexamethonium at low doses but remained significantly elevated at higher doses. Thus, CaR activation acutely increases MAP in uremic and nonuremic rats, responses that occur in parallel to vasoconstriction in multiple vascular beds through both a central and peripheral mechanism of action. Moreover, subsequent mechanistic studies suggest that increases in MAP produced by cinacalcet may be mediated by reduced tonic NO synthasedependent NO production subsequent to reductions in blood iCa 2ϩ .

ABT-869 is a novel multitargeted inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases (RTKs) with potent antiangiogenic properties that slow tumor progression. Vascular endothelial growth factor receptor blockade has been shown to produce hypertension. Atrasentan is a potent and selective endothelin (ETA) receptor antagonist that lowers blood pressure and affects tumor growth. To assess the utility of ETA receptor blockade in controlling hypertension with RTK inhibition, we evaluated the ability of atrasentan to block hypertension with ABT-869 in conscious, telemetry-instrumented rats. Changes in mean arterial pressure (MAP) and heart rate (HR) were evaluated using mean values and the area under the curve (AUC). Atrasentan (0.5, 1.5, and 5.0 mg kg(-1) d(-1) for 5 days) elicited dose-dependent decreases in MAP-AUC (-16.7 +/- 1.3, -20.94 +/- 3.68, and -30.12 +/- 3.57 mm Hg x day, respectively) compared with vehicle. ABT-869 (1, 3, 10, 30 mg kg(-1) d(-1) for 5 days) increased MAP compared with vehicle (MAP-AUC values of -5.52 +/- 3.75, 12.7 +/- 8.4, 37.5 +/- 4.4, and 63.8 +/- 3.3 mm Hg x day, respectively). Pretreatment with atrasentan (5 mg/kg for 5 days) prevented and abolished the hypertensive effects of ABT-869. Thus, ETA receptor blockade effectively alleviated hypertension with RTK inhibition and may serve a dual therapeutic role by preventing hypertension and slowing tumor progression.

Neutrophil (PMNL) infiltration of inflamed colonic tissue is a prominent feature of human inflammatory bowel disease (IBD). Colitis was established in New Zealand white rabbits by the intrarectal instillation of 1.5 mg/kg (in 10 ml 20% ethanol) phorbol-12-myristate-13-acetate (PMA) and assessed by visual grading of colonic inflammation, levels of the neutrophil marker enzyme myeloperoxidase (MPO), and histological examination. After 24 h there was a significant (P less than 0.001) increase in MPO levels in the PMA-treated colons compared to ethanol control. There was also increased inflammation based on visual scoring. Histologically, PMA-treated colons were necrotic with focal ulceration, heavy PMNL infiltration and edema at 24 h; by 96 h colitis was sustained with mild edema, crypt abscesses, and a staining pattern suggesting altered mucus quality. These results suggest that PMA-induced colitis in rabbits may be a new model of IBD in which to evaluate drugs known to mitigate the inflammatory process.