Therapeutic Assessment of Combination Therapy with a Neprilysin Inhibitor and Angiotensin Type 1 Receptor Antagonist on Angiotensin II-Induced Atherosclerosis, Abdominal Aortic Aneurysms, and Hypertension

Abstract

Combined neprilysin (NEP) inhibition (sacubitril) and angiotensin type 1 receptor (AT1R) antagonism (valsartan) is used in the treatment of congestive heart failure and is gaining interest for other angiotensin II (AngII)-related cardiovascular diseases. In addition to heart failure, AngII promotes hypertension, atherosclerosis, and abdominal aortic aneurysms (AAAs). Similarly, NEP substrates or products have broad effects on the cardiovascular system. In this study, we examined NEP inhibition (with sacubitril) and AT1R antagonism (with valsartan) alone or in combination on AngII-induced hypertension, atherosclerosis, or AAAs in male low-density lipoprotein receptor-deficient mice. Preliminary studies assessed drug delivery via osmotic minipumps for simultaneous release of sacubitril and/or valsartan with AngII over 28 days. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of sacubitril (1, 6, or 9 mg/kg per day), valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), or the combination thereof (1 and 0.3, or 9 or 0.5 mg/kg per day of sacubitril and valsartan, respectively). Plasma AngII and renin concentrations increased 4-fold at higher valsartan doses, indicative of removal of AngII negative feedback on renin. Sacubitril doubled plasma AngII concentrations at lower doses (1 mg/kg per day). Valsartan dose-dependently decreased systolic blood pressure, aortic atherosclerosis, and AAAs of AngII-infused mice, whereas sacubitril had no effect on atherosclerosis or AAAs but reduced blood pressure of AngII-infused mice. Combination therapy with sacubitril and valsartan did not provide additive benefits. These results suggest limited effects of combination therapy with NEP inhibition and AT1R antagonism against AngII-induced hypertension, atherosclerosis, or AAAs. SIGNIFICANCE STATEMENT: The combination of valsartan (angiotensin type 1 receptor antagonist) and sacubitril (neprilysin inhibitor) did not provide benefit above valsartan alone on AngII-induced hypertension, atherosclerosis, or abdominal aortic aneurysms in low-density lipoprotein receptor-deficient male mice. These results do not support this drug combination in therapy of these AngII-induced cardiovascular diseases.

Fig. 1.

Aortic neprilysin (NEP) mRNA abundance and immunostaining. (A) Male Ldlr^−/− mice fed a Western diet were infused with saline or AngII for 5 days. NEP mRNA abundance in abdominal aorta was increased by AngII compared with saline controls. Data are expressed as means ± S.D. (n = 5 mice per group). *P < 0.05 by Student’s t test. (B) NEP immunostaining in normal and AAA aorta tissue sections. Control aortic sections were incubated with secondary antibody only. Box indicates magnified area in bottom panel for each section. Scale bar, 100 µm. CT, cycle threshhold.

Fig. 2.

Plasma AngII (A), renin (B), and systolic blood pressures (C) of mice infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), sacubitril (1, 6, or 9 mg/kg per day), or combinations thereof (0.3/1; 0.5/9 valsartan/sacubitril). (A) Plasma AngII concentrations increased significantly at higher doses of valsartan. Sacubitril had no effect on plasma AngII concentrations, and the drug combinations tested did not differ from effects observed with either drug alone. (B) Plasma renin concentrations increased significantly at higher doses of valsartan or sacubitril and remained evident when drugs were administered in combination. (C) Systolic blood pressures decreased significantly with higher valsartan or sacubitril doses, but the effectiveness of sacubitril (9 mg/kg per day) to decrease blood pressure was not evident when combined with valsartan (0.5 mg/kg per day). Data are individual mice with means ± S.D. indicated by horizontal lines from vehicle (total of 51 mice, of which 31 mice survived the infusion protocol), valsartan 0.3 (total of 19 mice, of which measurements were obtained from n = 5 mice), valsartan 0.5 (total of eight mice, of which six mice survived the infusion protocol), valsartan 1 (total of eight mice, of which seven mice survived the infusion protocol), valsartan 6 (total of eight mice, of which eight mice survived the infusion protocol), valsartan 20 (total of eight mice, of which eight mice survived the infusion protocol), sacubitril 1 (total of 19, of which 11 mice survived the infusion protocol), sacubitril 6 (total of 10, of which seven mice survived the infusion protocol), valsartan 0.3/sacubitril 1 (total of nine mice, of which five mice survived the infusion protocol), valsartan 0.5/sacubitril 9 (total of eight mice, of which six mice survived the infusion protocol). *P < 0.05 compared with vehicle by one-way ANOVA and Kruskal-Wallis test. Closed circle: vehicle closed square: valsartan 0.3 mg/kg/day closed triangle: valsartan 0.5 mg/kg/day closed inverted triangle: valsartan 1 mg/kg/day closed diamond: valsartan 6 mg/kg/day open circle: valsartan 20 mg/kg/day open square: sacubitril 1 mg/kg/day open triangle: sacubitril 6 mg/kg/day open inverted triangle: sacubitril 9 mg/kg/day open diamond: valsartan 0.3 mg/kg/day and sacubitril 1 mg/kg/day closed diamond: valsartan 0.5 mg/kg/day and sacubitril 9 mg/kg/day

Fig. 3.

Effects of valsartan and sacubitril, alone or in combination, on AngII-induced AAAs. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), sacubitril (1, 6, or 9 mg/kg per day), or combinations thereof (0.3/1; 0.5/9 valsartan/sacubitril). (A) Abdominal aortic internal lumen diameters (day 28 of infusions) were decreased dose-dependently by valsartan, whereas sacubitril had no effect alone or in combination with valsartan. (B) AAA external diameters were decreased dose-dependently by valsartan, with no effect of sacubitril alone or in combination with valsartan. For (A) and (B), data are individual mice with means ± S.D. indicated by horizontal lines from vehicle (total of 51 mice, of which 31 mice survived the infusion protocol), valsartan 0.3 (total of 19 mice, of which 11 mice survived the infusion protocol), valsartan 0.5 (total of eight mice, of which six mice survived the infusion protocol), valsartan 1 (total of eight mice, of which seven mice survived the infusion protocol), valsartan 6 (total of eight mice, of which eight mice survived the infusion protocol), valsartan 20 (total of eight mice, of which eight mice survived the infusion protocol), sacubitril 1 (total of 19, of which 11 survived the infusion protocol), sacubitril 6 (total of 10, of which seven mice survived the infusion protocol), valsartan 0.3/sacubitril 1 (total of nine mice, of which five mice survived the infusion protocol), and valsartan 0.5/sacubitril 9 (total of eight mice of which six mice survived the infusion protocol). (C) AAA incidence (percentage) was decreased dose-dependently by valsartan, with no effect of sacubitril alone or in combination with valsartan. Data are individual mice with means ± S.D. indicated by horizontal lines from vehicle (n = 51), valsartan 0.3 (n = 19), valsartan 0.5 (n = 8), valsartan 1 (n = 8), valsartan 6 (n = 8), valsartan 20 (n = 8), sacubitril 1 (n = 19), sacubitril 6 (n = 10), valsartan 0.3/sacubitril 1 (n = 9), and valsartan 0.5/sacubitril 9 (n = 8). (D) Representative aortas from mice of each treatment group. *P < 0.05 compared with vehicle by one-way ANOVA, Kruskal-Wallis, or Fisher’s exact test.Sacubitril, Sac; Valsartan, Val.

Fig. 4.

Effects of valsartan and sacubitril, alone or in combination, on serum lipids and atherosclerotic lesion surface areas of AngII-infused mice. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), sacubitril (1, 6, or 9 mg/kg per day), or combinations thereof (0.3/1; 0.5/9 valsartan/sacubitril). (A) Serum cholesterol concentrations were not influenced by valsartan or sacubitril alone. However, when drugs were combined at the higher dose (0.5/9 valsartan/sacubitril), serum cholesterol decreased significantly. (B) Serum triglyceride concentrations were not influenced by valsartan or sacubitril alone. However, when drugs were combined at the higher dose (0.5/9 valsartan/sacubitril), serum triglyceride decreased significantly. (C) Valsartan or sacubitril had no significant effect on atherosclerosis in the aortic arch (percent lesion surface area) when administered alone. However, the lower-dose drug combination (0.3/1, valsartan/sacubitril) significantly increased atherosclerosis. (D) Oil Red O–stained representative aortas from mice of each treatment group. For (A–C), data are individual mice with means ± S.D. indicated by horizontal lines from vehicle (total of 51 mice, of which 31 mice survived the infusion protocol), valsartan 0.3 (total of 19 mice, of which 11 mice survived the infusion protocol), valsartan 0.5 (total of eight mice, of which six mice survived the infusion protocol), valsartan 1 (total of eight mice, of which seven mice survived the infusion protocol), valsartan 6 (total of eight mice, of which eight mice survived the infusion protocol), valsartan 20 (total of eight mice, of which eight mice survived the infusion protocol), sacubitril 1 (total of 19, of which 11 survived the infusion protocol), sacubitril 6 (total of 10, of which seven mice survived the infusion protocol), valsartan 0.3/sacubitril 1 (total of nine mice, of which five mice survived the infusion protocol), and valsartan 0.5/sacubitril 9 (total of eight mice, of which six mice survived the infusion protocol). *P < 0.05 compared with vehicle by one-way ANOVA.