Previously, we had shown that a vasopeptidase inhibitor drug containing ACE and neprilysin inhibitors was an effective treatment for diabetic vascular and neural complications. However, side effects prevented further development. This led to the development of sacubitril/valsartan, a drug containing angiotensin II receptor blocker and neprilysin inhibitor that we hypothesized would be an effective treatment for diabetic peripheral neuropathy. Using early and late intervention protocols (4 and 12 weeks posthyperglycemia, respectively), type 2 diabetic rats were treated with valsartan or sacubitril/valsartan for 12 weeks followed by an extensive evaluation of vascular and neural end points. The results demonstrated efficacy of sacubitril/valsartan in improving vascular and neural function was superior to valsartan alone. In the early intervention protocol, sacubitril/valsartan treatment was found to slow progression of these deficits and, with late intervention treatment, was found to stimulate restoration of vascular reactivity, motor and sensory nerve conduction velocities, and sensitivity/regeneration of sensory nerves of the skin and cornea in a rat model of type 2 diabetes. These preclinical studies suggest that sacubitril/valsartan may be an effective treatment for diabetic peripheral neuropathy, but additional studies will be needed to investigate these effects further.

Peripheral neuropathy is a devastating complication affecting ∼50% of the population with diabetes (1). Good glycemic control and improved lifestyle are cited as the only options for treatment; however, in subjects with type 2 diabetes, this has provided little benefit (1). Preclinical studies of diabetic animal models have identified multiple factors that can lead to damage of neurons, Schwann cells, and the vasculature (2,3). Given its complex etiology, a successful treatment for diabetic peripheral neuropathy will likely require a combination of early detection, lifestyle changes, and pharmaceutical interventions targeting the mechanisms deemed most responsible for the pathogenesis.

Previously, we have reported that the vasopeptidase inhibitor ilepatril was an effective treatment for vascular dysfunction and peripheral neuropathy in rodent models of types 1 and 2 diabetes (49). Vasopeptidase inhibitors are a combination drug consisting of an inhibitor of neprilysin and ACE (10). However, undesirable side effects of this class of drugs in clinical trials prevented further development (11). This led to the development of angiotensin receptor neprilysin inhibitors, which combine the beneficial effects of angiotensin II receptor blockers with neprilysin inhibition while reducing the risk of angioedema (11). LCZ696 was the first representative of this class of drug and combines the dual action of sacubitril and valsartan (11). Sacubitril is a neprilysin inhibitor, and valsartan is angiotensin II receptor antagonist. In a recent clinical trial, sacubitril/valsartan was shown to be an effective treatment for heart failure, with reduced ejection fraction and side effects comparable to ACE inhibitor enalapril (12). Sacubitril/valsartan has subsequently obtained U.S. Food and Drug Administration approval. Because of the targeting similarities of vasopeptidase inhibitors and sacubitril/valsartan, we were interested in examining the efficacy of sacubitril/valsartan on diabetic vascular and neural impairment. We had previously demonstrated that treating diabetic rats with an ACE inhibitor or angiotensin receptor antagonist provided similar benefits on vascular and neural complications (13). We had also previously demonstrated that pharmaceutically or genetically blocking neprilysin activity improved vascular and neural deficits in diabetic rodents (49,14). Neprilysin is a protease that degrades a number of peptides that have neuroprotective and vasoactive properties for which expression is increased by hyperglycemia and diabetes (14). Increased expression of neprilysin could compromise vascular and neural function, and blocking activity of both angiotensin II and neprilysin would be expected to reduce oxidative stress and protect biologically active peptides necessary for normal vascular and neural function (14). Thus, we hypothesized that treating a rat model of type 2 diabetes with sacubitril/valsartan would prevent or reverse vascular and neural dysfunction and possibly provide a new source of treatment for diabetic neuropathy.

Sigma-Aldrich (St. Louis, MO) was the primary source of all chemicals used in these studies. Valsartan and sacubitril/valsartan were provided by Novartis (East Hanover, NJ).

Animals

Sprague-Dawley (Harlan Sprague Dawley, Indianapolis, IN) male rats 11 weeks of age were housed in an Association for Assessment and Accreditation of Laboratory Animal Care–certified animal care facility, and food (#7001; Harlan Teklad, Madison, WI) and water were provided ad libitum. All institutional (ACORP #5081487) guidelines for use of animals were followed. At 12 weeks of age, rats were separated into six groups. Three groups remained on the control diet (#7001; 4.25% kcal as fat and 3.0 kcal/g; Envigo Teklad, Madison, WI), and the other three groups were fed a high-fat diet (D12451 [45% kcal as fat and 4.7 kcal/g]; Research Diets, New Brunswick, NJ). Rats were maintained on these diets for 8 weeks. Afterward, the rats fed the high-fat diet were treated with streptozotocin (30 mg/kg in 0.1 mol/L citric acid buffer, pH 4.5, i.p.; EMD/Millipore, Billerica, MA) to induce hyperglycemia (7). The rats on the control diet were treated with vehicle. Blood glucose was evaluated 96 h later using glucose-oxidase reagent strips (Aviva Accu-Chek; Roche, Mannheim, Germany), and rats having a blood glucose level of ≥250 mg/dL (13.8 mmol/L) were considered to be diabetic. These rats are a model for late-stage type 2 diabetes and not hyperinsulinemic (6). At 4 (early intervention) and 16 (late intervention) weeks posthyperglycemia, a group of the control and diabetic rats were treated with valsartan (31 mg/kg body wt daily by oral gavage) or sacubitril/valsartan (68 mg/kg body wt daily by oral gavage). The dose of sacubitril/valsartan used was based upon a previous study examining effect of the drug on cardiac remodeling and dysfunction after myocardial infarction (15). Other pharmacokinetic and pharmacodynamic studies have shown sacubitril/valsartan to be safe and well tolerated (16). Valsartan was first dissolved at 12-fold higher than dosing concentration in 1 N sodium hydroxide. This stock solution was diluted with distilled water, and its pH was adjusted to 8.5 with 1 N hydrochloric acid. The solution volume was further increased with distilled water to generate final concentration of 15.5 mg/mL, and the dosing volume was 2 mL/kg body wt. Sacubitril/valsartan was dissolved in distilled water at 34 mg/mL, and the dosing volume was 2 mL/kg body wt. Untreated control and diabetic rats received vehicle. The treatment phase for each of the early and late intervention groups was 12 weeks. A group of control and diabetic rats was examined prior to beginning treatment of the early intervention group to establish a baseline for the pathology present after 12 weeks of high-fat diet and 4 weeks of untreated hyperglycemia.

Glucose Utilization

Glucose utilization was determined by injecting rats with a saline solution containing 2 g/kg glucose, i.p., after an overnight fast (5).

Thermal Nociceptive Response and Corneal Reactivity

Thermal nociceptive response in the hind paw was measured using the Hargreaves method (7). Data were reported in seconds. Corneal sensation was determined using two separate methods. First, a Cochet-Bonnet filament esthesiometer was used in unanesthetized rats (Luneau Ophtalmologie, Prunay le Gillon, France) (4). Rats were gently restrained by hand and the 6-cm filament advanced to touch the eye. If the rat blinked, the length of the filament was recorded. If the rat did not blink, the filament was shortened by 0.5 cm and the procedure repeated. This process was continued until the rat blinked. Each eye was evaluated. The data were reported in centimeters.

Corneal sensation was also measured by applying buffered isotonic (290 mOsm/L) and hypertonic eye drops (5% sodium solution; Muro 128, Bausch and Lomb; Valeant Pharmaceuticals, Bridgewater, NJ) to the eye of unanesthetized rats (17). For this method, rats were placed in a custom-made restraining apparatus, and 10 min was allowed for the animal to acclimate to the restraint device and lighting. Six complementary metal-oxide semiconductor cameras (Imaging Development Systems, Obersulm, Germany), three per each eye, were positioned in order to observe both eyes simultaneously. Custom software was used to synchronize video streams and obtain images (MATLAB R2012a; The MathWorks Inc., Natick, MA). Video recording was started 30 s before the first epoch. An image from this recording period was used to establish a baseline area of eyelid opening. The first recording epoch began with the addition of 20 µL isotonic solution to the right eye. The recording period was 3 min followed by a 5-min washout and recovery period. The next epoch began with the addition of the hypertonic solution and recorded for 3 min. An image collector was used offline to retrieve video frames from each epoch. Fiji image analysis software was used by a masked technician to measure the visible surface area of both eyes between the upper and lower eyelids. These areas were expressed as a percentage of the baseline area of eyelid opening. These data were plotted versus time, and the area under the curve (AUC) used to quantify corneal sensitivity to topical isotonic and hypertonic saline, based on narrowing of the palpebral fissure in response to corneal nerve reactivity.

Corneal Innervation

On the day of the terminal studies, rats were weighed and anesthetized with Nembutal (50 mg/kg, i.p.; Abbott Laboratories, North Chicago, IL). Subepithelial corneal nerves were imaged using the Rostock cornea module of the Heidelberg Retina Tomograph confocal microscope (4). The investigator acquiring these images was masked with respect to identity of the animal condition. Corneal nerve fiber length was defined as the total length of all nerve fibers and branches (in millimeters) present in the acquired images standardized for area of the image (in square millimeters). A minimum of six images was acquired for each animal. The corneal fiber length for each animal was the mean value obtained from the acquired images and expressed as millimeters per square millimeter.

Motor and Sensory Nerve Conduction Velocity

Motor and sensory nerve conduction velocity was determined as previously described using a noninvasive procedure in the sciatic-posterior tibial conducting system and digital nerve, respectively (7). Motor and sensory nerve conduction velocities were reported in meters per second.

Vascular Reactivity in Epineurial Arterioles

Videomicroscopy was used to investigate in vitro vasodilatory responsiveness of epineurial arterioles vascularizing the region of the sciatic nerve (5). The arterioles used in this study should be regarded as epineurial rather than perineurial vessels. Following isolation and suspension of the vessels, cumulative concentration-response relationships were evaluated for acetylcholine (10−8–10−4 mol/L) and calcitonin gene-related peptide (10−11–10−8 mol/L). At the end of each dose-response curve, papaverine (10−5 mol/L) was added to determine maximal vasodilation.

Intraepidermal Nerve Fiber Density in the Hind Paw

Immunoreactive nerve fiber profiles innervating the skin from the hind paw were visualized using standard confocal microscopy combined with immunohistochemistry (7). Profiles were counted by two individual investigators that were masked to the sample identity. All immunoreactive profiles were counted and normalized to length, and the data are presented as profiles per millimeter.

Physiological Markers

Nonfasting blood glucose was determined with Aviva Accu-Chek strips (Roche). Serum was collected for determining levels of free fatty acid, triglyceride, free cholesterol, and activity of ACE using commercial kits from Roche Diagnostics (Mannheim, Germany), Sigma-Aldrich, BioVision (Mountain View, CA), and Bühlmann Laboratories (Schönenbuch, Switzerland), respectively. Serum was also used to determine thiobarbituric acid–reactive substances, nitrotyrosine, and soluble intercellular adhesion molecule-1 (ICAM-1; using the Quantikine rat sICAM-1/CD54 ELISA kit [R&D Systems, Inc., Minneapolis, MN], according to the manufacturer’s instructions) (18). Liver was used to determine activity of neprilysin (19).

Data Analysis

Results are presented as mean ± SEM. Comparisons between groups were conducted using a one-way ANOVA and Bonferroni test for multiple comparisons (Prism; GraphPad Software, San Diego, CA). A P value of <0.05 was considered significant.

The experimental design consisted of an early and late intervention protocol, and data for weight change, blood glucose, and serum lipids and oxidative/inflammatory stress markers are provided in Tables 1 and 2, respectively. For all data to be presented, the results shown for the control and untreated diabetic rats for the early intervention are the condition of the control and diabetic rats at start of treatment for the late intervention.

Table 1

Effect of early intervention of sacubitril/valsartan or valsartan on weight gain, blood glucose, serum free fatty acids, triglycerides, cholesterol, thiobarbituric acid–reactive substances, 3-nitrotyrosine, soluble ICAM-1, and cornea sensitivity in type 2 diabetic Sprague-Dawley rats

DeterminationControl (12)Control + valsartan (6)Control + sacubitril/valsartan (6)Diabetes (12)Diabetes + valsartan (10)Diabetes + sacubitril/valsartan (11)
Start weight (g) 391 ± 3 379 ± 6 380 ± 5 381 ± 4 381 ± 3 390 ± 4 
Final weight (g) 537 ± 9 512 ± 13 524 ± 20 458 ± 15a 458 ± 11a 462 ± 12a 
Blood glucose (mg/dL) 145 ± 4 139 ± 10 137 ± 11 395 ± 41a 397 ± 39a 446 ± 34a 
Triglycerides (mg/mL) 0.88 ± 0.09 0.73 ± 0.07 0.74 ± 0.08 3.26 ± 1.28a 4.83 ± 1.27a 3.10 ± 0.59a 
Free fatty acids (mmol/L) 0.12 ± 0.01 0.11 ± 0.02 0.10 ± 0.01 0.39 ± 0.07a 0.39 ± 0.10a 0.39 ± 0.09a 
Cholesterol (mg/mL) 1.51 ± 0.11 1.19 ± 0.04 1.17 ± 0.15 3.13 ± 0.72a 3.81 ± 0.74a 3.43 ± 0.64a 
Thiobarbituric acid–reactive substances (µg/mL) 0.54 ± 0.04 0.53 ± 0.04 0.49 ± 0.02 0.62 ± 0.08 0.60 ± 0.03 0.70 ± 0.03 
3-Nitrotyrosine (pmol/mg protein) 5.2 ± 0.3 4.5 ± 0.5 4.5 ± 0.7 7.0 ± 0.5a 4.4 ± 0.5b 5.3 ± 0.4b 
sICAM-1 (ng/mL) 17.8 ± 1.3 16.4 ± 2.2 14.6 ± 1.1 25.1 ± 5.0 19.9 ± 1.3 22.8 ± 1.4 
Cochet-Bonnet filament esthesiometer (cm) 5.8 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 4.7 ± 0.2a 5.8 ± 0.1b 5.9 ± 0.1b 
DeterminationControl (12)Control + valsartan (6)Control + sacubitril/valsartan (6)Diabetes (12)Diabetes + valsartan (10)Diabetes + sacubitril/valsartan (11)
Start weight (g) 391 ± 3 379 ± 6 380 ± 5 381 ± 4 381 ± 3 390 ± 4 
Final weight (g) 537 ± 9 512 ± 13 524 ± 20 458 ± 15a 458 ± 11a 462 ± 12a 
Blood glucose (mg/dL) 145 ± 4 139 ± 10 137 ± 11 395 ± 41a 397 ± 39a 446 ± 34a 
Triglycerides (mg/mL) 0.88 ± 0.09 0.73 ± 0.07 0.74 ± 0.08 3.26 ± 1.28a 4.83 ± 1.27a 3.10 ± 0.59a 
Free fatty acids (mmol/L) 0.12 ± 0.01 0.11 ± 0.02 0.10 ± 0.01 0.39 ± 0.07a 0.39 ± 0.10a 0.39 ± 0.09a 
Cholesterol (mg/mL) 1.51 ± 0.11 1.19 ± 0.04 1.17 ± 0.15 3.13 ± 0.72a 3.81 ± 0.74a 3.43 ± 0.64a 
Thiobarbituric acid–reactive substances (µg/mL) 0.54 ± 0.04 0.53 ± 0.04 0.49 ± 0.02 0.62 ± 0.08 0.60 ± 0.03 0.70 ± 0.03 
3-Nitrotyrosine (pmol/mg protein) 5.2 ± 0.3 4.5 ± 0.5 4.5 ± 0.7 7.0 ± 0.5a 4.4 ± 0.5b 5.3 ± 0.4b 
sICAM-1 (ng/mL) 17.8 ± 1.3 16.4 ± 2.2 14.6 ± 1.1 25.1 ± 5.0 19.9 ± 1.3 22.8 ± 1.4 
Cochet-Bonnet filament esthesiometer (cm) 5.8 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 4.7 ± 0.2a 5.8 ± 0.1b 5.9 ± 0.1b 

Data are presented as the mean ± SEM. Numbers in parentheses are the number of experimental animals.

sICAM-1, soluble ICAM-1.

aP < 0.05 compared with control;

bP < 0.05 compared with diabetes.

Table 2

Effect of late intervention of sacubitril/valsartan or valsartan on weight gain, blood glucose, serum free fatty acids, triglycerides, cholesterol, thiobarbituric acid–reactive substances, 3-nitrotyrosine, soluble ICAM-1, and cornea sensitivity in type 2 diabetic Sprague-Dawley rats

DeterminationControl (12)Control + valsartan (6)Control + sacubitril/valsartan (6)Diabetes (12)Diabetes + valsartan (12)Diabetes + sacubitril/valsartan (12)
Weight at treatment (g) 530 ± 11 514 ± 6 506 ± 13 481 ± 13 477 ± 17 476 ± 17 
Final weight (g) 578 ± 11 550 ± 8 536 ± 18 518 ± 23 510 ± 23 504 ± 28 
Blood glucose (mg/dL) 137 ± 5 130 ± 9 137 ± 9 446 ± 35a 452 ± 42a 445 ± 46a 
Triglycerides (mg/mL) 0.71 ± 0.16 0.45 ± 0.13 0.66 ± 0.26 6.39 ± 1.10a 2.27 ± 0.30a,b 2.68 ± 0.53a,b 
Free fatty acids (mmol/L) 0.12 ± 0.02 0.12 ± 0.01 0.11 ± 0.01 0.35 ± 0.05a 0.44 ± 0.08a 0.55 ± 0.11a 
Cholesterol (mg/mL) 1.85 ± 0.33 2.01 ± 0.49 1.77 ± 0.41 8.03 ± 1.16a 3.23 ± 0.50b 4.44 ± 0.62a,b 
Thiobarbituric acid–reactive substances (µg/mL) 0.78 ± 0.04 0.80 ± 0.05 0.83 ± 0.05 1.34 ± 0.15a 1.06 ± 0.07 1.05 ± 0.09 
3-Nitrotyrosine (pmol/mg protein) 4.4 ± 0.3 4.1 ± 0.7 5.0 ± 0.5 6.8 ± 0.5a 6.2 ± 0.5a 6.3 ± 0.7a 
sICAM-1 (ng/mL) 22.4 ± 1.4 19.0 ± 2.1 17.3 ± 0.6 33.2 ± 4.5a 26.0 ± 1.6 24.7 ± 1.4 
Cochet-Bonnet filament esthesiometer (cm) 5.7 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 4.6 ± 0.2a 5.3 ± 0.2b 5.9 ± 0.1b,c 
DeterminationControl (12)Control + valsartan (6)Control + sacubitril/valsartan (6)Diabetes (12)Diabetes + valsartan (12)Diabetes + sacubitril/valsartan (12)
Weight at treatment (g) 530 ± 11 514 ± 6 506 ± 13 481 ± 13 477 ± 17 476 ± 17 
Final weight (g) 578 ± 11 550 ± 8 536 ± 18 518 ± 23 510 ± 23 504 ± 28 
Blood glucose (mg/dL) 137 ± 5 130 ± 9 137 ± 9 446 ± 35a 452 ± 42a 445 ± 46a 
Triglycerides (mg/mL) 0.71 ± 0.16 0.45 ± 0.13 0.66 ± 0.26 6.39 ± 1.10a 2.27 ± 0.30a,b 2.68 ± 0.53a,b 
Free fatty acids (mmol/L) 0.12 ± 0.02 0.12 ± 0.01 0.11 ± 0.01 0.35 ± 0.05a 0.44 ± 0.08a 0.55 ± 0.11a 
Cholesterol (mg/mL) 1.85 ± 0.33 2.01 ± 0.49 1.77 ± 0.41 8.03 ± 1.16a 3.23 ± 0.50b 4.44 ± 0.62a,b 
Thiobarbituric acid–reactive substances (µg/mL) 0.78 ± 0.04 0.80 ± 0.05 0.83 ± 0.05 1.34 ± 0.15a 1.06 ± 0.07 1.05 ± 0.09 
3-Nitrotyrosine (pmol/mg protein) 4.4 ± 0.3 4.1 ± 0.7 5.0 ± 0.5 6.8 ± 0.5a 6.2 ± 0.5a 6.3 ± 0.7a 
sICAM-1 (ng/mL) 22.4 ± 1.4 19.0 ± 2.1 17.3 ± 0.6 33.2 ± 4.5a 26.0 ± 1.6 24.7 ± 1.4 
Cochet-Bonnet filament esthesiometer (cm) 5.7 ± 0.1 5.9 ± 0.1 5.9 ± 0.1 4.6 ± 0.2a 5.3 ± 0.2b 5.9 ± 0.1b,c 

Data are presented as the mean ± SEM. Numbers in parentheses are the number of experimental animals.

sICAM-1, soluble ICAM-1.

aP < 0.05 compared with control;

bP < 0.05 compared with diabetes;

cP < 0.05 compared with diabetes + valsartan.

All animals weighed the same at the beginning of the study. At the end of the early intervention protocol, diabetic rats weighed less than control rats, and this was not altered by treatment. Weight was not statistically different between untreated or treated control and diabetic rats in the late intervention protocol. All diabetic rats were hyperglycemic at the end of the studies, and this was not changed with treatment. Data in Supplementary Fig. 1 demonstrate that glucose clearance, a marker of insulin resistance, was impaired in type 2 diabetic rats at the time of treatment for the early intervention protocol. Early or late intervention treatment of control or diabetic rats with valsartan or sacubitril/valsartan did not improve glucose utilization (data not shown).

Serum triglycerides, free fatty acids, and cholesterol were significantly increased in diabetic rats, and this was not changed with early intervention with valsartan or sacubitril/valsartan treatment (Table 1). With late intervention, treatment with valsartan or sacubitril/valsartan reduced serum triglyceride and cholesterol levels compared with untreated diabetic rats (Table 2). Serum triglyceride and cholesterol levels were increased with duration of hyperglycemia (compare untreated diabetic rats between Tables 1 and 2). Treatment of diabetic rats in the late intervention protocol prevented the further increase in serum triglyceride or cholesterol levels.

Serum biomarkers of oxidative and inflammatory stress were significantly increased in diabetic rats after 36 weeks of a high-fat diet and 28 weeks of hyperglycemia (end of late intervention) (Table 2). Treatment of diabetic rats with valsartan or sacubitril/valsartan slowed the increase in thiobarbituric acid–reactive substances and soluble ICAM-1 levels. Serum nitrotyrosine levels remained elevated in treated with valsartan or sacubitril/valsartan diabetic rats. In the early intervention study, only serum nitrotyrosine levels were significantly increased in untreated diabetic rats compared with control rats. Treating diabetic rats early with valsartan or sacubitril/valsartan prevented the increase.

For these studies, we also measured activity of ACE in serum and neprilysin activity in liver in rats prior to early intervention. Serum ACE activity was increased ∼65% in untreated diabetic rats (70 ± 6 and 114 ± 6 units/mL in control and diabetic rats, respectively; P < 0.01 compared with control rats; n = 6). Liver neprilysin activity in these same rats was 2.2 ± 0.1 and 3.5 ± 0.3 mmol 7-amido-3-methylcoumarin/min/mg protein for control and diabetic rats, respectively (P < 0.01 compared with control rats; n = 6).

Determination of motor and sensory nerve conduction velocities has been a standard neurological end point for preclinical and clinical studies. Data in Fig. 1 demonstrate that motor and sensory nerve conduction velocities are decreased in untreated diabetic rats in both early and late intervention protocols with duration of diabetes having little impact on the final measurement. At the time of treatment for early intervention, motor and sensory nerve conduction velocities were 55.4 ± 1.7 and 34.4 ± 0.6 vs. 37.7 ± 1.6 and 28.9 ± 0.8 m/s for control and diabetic rats, respectively (P < 0.05 compared with control rats; n = 12). Early intervention with valsartan did not significantly improve motor or sensory nerve conduction velocity deficit (Fig. 1A). In contrast, early intervention with sacubitril/valsartan interrupted the progression of slowing of both motor and sensory nerve conduction velocity with significant improvement versus untreated diabetic rats observed for motor nerve conduction velocity. In the late intervention study, valsartan treatment and to a greater extent treatment with sacubitril/valsartan significantly improved both motor and sensory nerve conduction velocity (Fig. 1B).

Figure 1

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on motor and sensory nerve conduction velocity (MNCV and SNCV, respectively) in type 2 diabetic rats. Motor and sensory nerve conduction velocity was examined after early (A) or late (B) intervention as described in the researchdesign andmethods. Data are presented as the mean ± SEM in meters per second. At the time of treatment for early intervention, the baseline motor and sensory nerve conduction velocity for control and diabetic rats was 55.4 ± 1.7 and 34.4 ± 0.6 vs. 37.7 ± 1.6 and 28.9 ± 0.8, respectively (P < 0.05 compared with control rats; n = 12). Baseline values for the late intervention group were the motor and sensory nerve conduction velocity for control and untreated diabetic rats at the end of the early intervention study. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats.

Figure 1

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on motor and sensory nerve conduction velocity (MNCV and SNCV, respectively) in type 2 diabetic rats. Motor and sensory nerve conduction velocity was examined after early (A) or late (B) intervention as described in the researchdesign andmethods. Data are presented as the mean ± SEM in meters per second. At the time of treatment for early intervention, the baseline motor and sensory nerve conduction velocity for control and diabetic rats was 55.4 ± 1.7 and 34.4 ± 0.6 vs. 37.7 ± 1.6 and 28.9 ± 0.8, respectively (P < 0.05 compared with control rats; n = 12). Baseline values for the late intervention group were the motor and sensory nerve conduction velocity for control and untreated diabetic rats at the end of the early intervention study. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats.

Recently, measurement of density of sensory nerves in the skin and cornea as well as associated biosensitivity assays of these nerves have been promoted as possible surrogate markers for peripheral neuropathy (20,21). At the time of early intervention, thermal sensitivity and intraepidermal nerve fiber density of control and diabetic rats were 11.7 ± 0.6 s and 24.5 ± 1.0 profiles/mm vs. 20.0 ± 0.7 s and 20.2 ± 0.5 profiles/mm, respectively (P < 0.05 compared with control rats; n = 12). After the early intervention phase, thermal sensitivity and intraepidermal nerve fiber density were significantly impaired in untreated diabetic rats, with a greater loss of intraepidermal nerve fibers occurring with an additional 12 weeks of no treatment (Fig. 2A). Early intervention with valsartan partially improved/delayed the impairment in both thermal sensitivity and intraepidermal nerve fiber density, whereas early intervention with sacubitril/valsartan completely reversed the early decrease in thermal sensation and loss of sensory nerve fibers in the skin. With late intervention, valsartan significantly improved thermal sensitivity, whereas intraepidermal nerve fiber density was not improved (Fig. 2B). Late intervention with sacubitril/valsartan also significantly improved thermal sensitivity and partially improved intraepidermal nerve fiber density.

Figure 2

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on thermal nociception and intraepidermal nerve fiber (IENF) density in type 2 diabetic rats. Thermal sensitivity and intraepidermal nerve fiber density were examined after early (A) or late (B) intervention as described in the researchdesign andmethods. Data are presented as the mean ± SEM for thermal nociception in seconds and intraepidermal nerve fiber density in profiles per millimeter. At the time of early intervention, baseline thermal sensitivity and intraepidermal nerve fiber density of control and diabetic rats were 11.7 ± 0.6 and 24.5 ± 1.0 vs. 20.0 ± 0.7 and 20.2 ± 0.5, respectively (P < 0.05 compared with control rats; n = 12). Baseline values for the late intervention group were the thermal sensitivity and intraepidermal nerve fiber density for control and untreated diabetic rats at the end of the early intervention study. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

Figure 2

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on thermal nociception and intraepidermal nerve fiber (IENF) density in type 2 diabetic rats. Thermal sensitivity and intraepidermal nerve fiber density were examined after early (A) or late (B) intervention as described in the researchdesign andmethods. Data are presented as the mean ± SEM for thermal nociception in seconds and intraepidermal nerve fiber density in profiles per millimeter. At the time of early intervention, baseline thermal sensitivity and intraepidermal nerve fiber density of control and diabetic rats were 11.7 ± 0.6 and 24.5 ± 1.0 vs. 20.0 ± 0.7 and 20.2 ± 0.5, respectively (P < 0.05 compared with control rats; n = 12). Baseline values for the late intervention group were the thermal sensitivity and intraepidermal nerve fiber density for control and untreated diabetic rats at the end of the early intervention study. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

Data in Fig. 3, Supplementary Fig. 2, and Tables 1 and 2 demonstrate the effect of diabetes and treatment with valsartan or sacubitril/valsartan on subepithelial cornea nerve fiber length and cornea sensitivity as determined by a response to a hyperosmotic solution and Cochet-Bonnet filament esthesiometer. Subepithelial nerve fiber length of the cornea at time of treatment for the early intervention was 9.0 ± 0.3 and 5.7 ± 0.5 mm/mm2 for control and diabetic rats, respectively (P < 0.05 compared with control rats; n = 12). Corneal sensitivity as measured by responsiveness to a hyperosmotic solution at time of early intervention was 35.5 ± 5.5 and 68.9 ± 6.2 AUC for control and diabetic rats, respectively (P < 0.05 compared with control rats; n = 12). Applying an isotonic solution to the eye of control or diabetic rats elicits no response (data not shown) (17). Over the 12 weeks for the early intervention phase, both cornea nerve fiber length and sensitivity were further impaired (Fig. 3A). Treatment with valsartan provided no benefit, whereas early treatment with sacubitril/valsartan significantly improved/reversed diabetes-induced decrease in cornea nerve fiber length and sensitivity. Following the late intervention phase, cornea nerve fiber length remained significantly decreased compared with control rats but was not further reduced when compared with untreated rats following the early intervention (Fig. 3B). In contrast, cornea sensitivity was further impaired. Treating diabetic rats with valsartan trended to improve cornea nerve fiber length and sensitivity, but these outcome measures remained significantly impaired compared with control rats. Treating diabetic rats with sacubitril/valsartan significantly improved/reversed diabetes-induced impairment in both cornea nerve fiber length and sensitivity. Representative images of subepithelial cornea nerve fibers following the late intervention phase are provided in Supplementary Fig. 2. Cornea sensitivity was also measured by Cochet-Bonnet filament esthesiometer and demonstrated that both valsartan and sacubitril/valsartan treatment following early and late intervention improved cornea sensitivity (Tables 1 and 2). Considering that the rats are being physically handled during this evaluation, it may not be as sensitive as the examination using the hyperosmotic solution.

Figure 3

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on cornea nerve fiber length and cornea sensitivity in type 2 diabetic rats. Innervation of the subepithelial layer of the cornea and corneal reactivity was determined and quantified by determining response to a hyperosmotic solution after early (A) or late (B) intervention as described in the researchdesign andmethods. Data are presented as the mean ± SEM for innervation of the cornea in millimeters per square millimeter and for corneal sensitivity in AUC. Baseline subepithelial nerve density of the cornea at time of treatment for the early intervention for control and diabetic rats was 9.0 ± 0.3 and 5.7 ± 0.5 mm/mm2, respectively (P < 0.05 compared with control rats; n = 12). Baseline corneal sensitivity, as measured by responsiveness to a hyperosmotic solution, for control and diabetic rats at time of early intervention was 35.5 ± 5.5 and 68.9 ± 6.2 AUC, respectively (P < 0.05 compared with control rats; n = 12). Baseline values for the late intervention group were the subepithelial nerve density of the cornea and corneal sensitivity for control and untreated diabetic rats at the end of the early intervention study. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

Figure 3

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on cornea nerve fiber length and cornea sensitivity in type 2 diabetic rats. Innervation of the subepithelial layer of the cornea and corneal reactivity was determined and quantified by determining response to a hyperosmotic solution after early (A) or late (B) intervention as described in the researchdesign andmethods. Data are presented as the mean ± SEM for innervation of the cornea in millimeters per square millimeter and for corneal sensitivity in AUC. Baseline subepithelial nerve density of the cornea at time of treatment for the early intervention for control and diabetic rats was 9.0 ± 0.3 and 5.7 ± 0.5 mm/mm2, respectively (P < 0.05 compared with control rats; n = 12). Baseline corneal sensitivity, as measured by responsiveness to a hyperosmotic solution, for control and diabetic rats at time of early intervention was 35.5 ± 5.5 and 68.9 ± 6.2 AUC, respectively (P < 0.05 compared with control rats; n = 12). Baseline values for the late intervention group were the subepithelial nerve density of the cornea and corneal sensitivity for control and untreated diabetic rats at the end of the early intervention study. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

We have previously demonstrated that a decrease in vascular reactivity of epineurial arterioles, blood vessels that provide circulation to the sciatic nerve, is manifest before detection of impaired motor nerve conduction velocity (22). Therefore, we believe that if treatments are to be successful for diabetic peripheral neuropathy, they need also to provide protection to the neural vasculature. Data in Fig. 4A demonstrate that vascular relaxation to acetylcholine by diabetic rats was further impaired after an additional 12 weeks of no treatment (compare Fig. 4A to Supplementary Fig. 3 [top panel]). Early intervention in diabetic rats with valsartan prevented the progressive impairment, whereas early intervention in diabetic rats with sacubitril/valsartan reversed impaired vascular reactivity to acetylcholine. Late intervention with valsartan was unable to reverse vascular impairment of relaxation to acetylcholine. In contrast, late intervention with sacubitril/valsartan reversed the vascular dysfunction demonstrated by vasodilation to acetylcholine.

Figure 4

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on vascular relaxation by acetylcholine in epineurial arterioles of the sciatic nerve in type 2 diabetic rats. Relaxation by epineurial arterioles to acetylcholine was determined after early (A) or late (B) intervention as described in the researchdesign andmethods. Pressurized arterioles (40–50%) and incremental doses of acetylcholine were added to the bathing solution while recording steady-state vessel diameter. Data are presented as the mean of percent relaxation ± SEM. Baseline epineurial reactivity to acetylcholine at time of treatment for the early intervention for control and diabetic rats is presented in Supplementary Fig. 3 (top panel). Baseline values for epineurial reactivity to acetylcholine at the time of treatment for the late intervention for control and diabetic rats are the values of control and diabetic rats from the early intervention group. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

Figure 4

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on vascular relaxation by acetylcholine in epineurial arterioles of the sciatic nerve in type 2 diabetic rats. Relaxation by epineurial arterioles to acetylcholine was determined after early (A) or late (B) intervention as described in the researchdesign andmethods. Pressurized arterioles (40–50%) and incremental doses of acetylcholine were added to the bathing solution while recording steady-state vessel diameter. Data are presented as the mean of percent relaxation ± SEM. Baseline epineurial reactivity to acetylcholine at time of treatment for the early intervention for control and diabetic rats is presented in Supplementary Fig. 3 (top panel). Baseline values for epineurial reactivity to acetylcholine at the time of treatment for the late intervention for control and diabetic rats are the values of control and diabetic rats from the early intervention group. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

Calcitonin gene-related peptide is the most potent vasodilator of mammalian vessels (23). We have also shown that sensory nerves innervating epineurial arterioles of the sciatic nerve contain calcitonin gene-related peptide (24). In untreated diabetic rats, vascular relaxation to midrange doses of calcitonin gene-related peptide is decreased early in epineurial arterioles (Supplementary Fig. 3 [bottom panel]). Data in Fig. 5A and B demonstrate that early or late intervention in diabetic rats with valsartan did not significantly improve calcitonin gene-related peptide-mediated vascular relaxation. However, early or late intervention in diabetic rats with sacubitril/valsartan did significantly improve vascular relaxation to calcitonin gene-related peptide compared with untreated diabetic rats.

Figure 5

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on vascular relaxation by calcitonin gene-related peptide (CGRP) in epineurial arterioles of the sciatic nerve in type 2 diabetic rats. Relaxation by epineurial arterioles to calcitonin gene-related peptide was determined after early (A) or late (B) intervention as described in the researchdesign andmethods. Pressurized arterioles (40 mmHg and ranging from 60 to 100 µm luminal diameters) were constricted with phenylephrine (30–50%) and incremental doses of calcitonin gene-related peptide were added to the bathing solution while recording steady-state vessel diameter. Data are presented as the mean of percent relaxation ± SEM. Baseline epineurial reactivity to calcitonin gene-related peptide at time of treatment for the early intervention for control and diabetic rats is presented in Supplementary Fig. 3 (bottom panel). Baseline values for epineurial reactivity to calcitonin gene-related peptide at the time of treatment for the late intervention for control and diabetic rats are the values of control and diabetic rats from the early intervention group. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

Figure 5

Effect of valsartan (Val) or sacubitril (Sac)/valsartan on vascular relaxation by calcitonin gene-related peptide (CGRP) in epineurial arterioles of the sciatic nerve in type 2 diabetic rats. Relaxation by epineurial arterioles to calcitonin gene-related peptide was determined after early (A) or late (B) intervention as described in the researchdesign andmethods. Pressurized arterioles (40 mmHg and ranging from 60 to 100 µm luminal diameters) were constricted with phenylephrine (30–50%) and incremental doses of calcitonin gene-related peptide were added to the bathing solution while recording steady-state vessel diameter. Data are presented as the mean of percent relaxation ± SEM. Baseline epineurial reactivity to calcitonin gene-related peptide at time of treatment for the early intervention for control and diabetic rats is presented in Supplementary Fig. 3 (bottom panel). Baseline values for epineurial reactivity to calcitonin gene-related peptide at the time of treatment for the late intervention for control and diabetic rats are the values of control and diabetic rats from the early intervention group. The number of rats in the early and late intervention groups was the same as shown in Tables 1 and 2, respectively. *P < 0.05 compared with control rats; +P < 0.05 compared with untreated diabetic rats; ‡P < 0.05 compared with valsartan-treated rats.

Sacubitril/valsartan, a dual-action drug that blocks the angiotensin II receptor and neprilysin activity, has been approved by the U.S. Food and Drug Administration for the treatment of heart failure, and a recent meta-analysis of six randomized trials with ∼12,000 subjects showed that sacubitril/valsartan was associated with less drug risk than a placebo (2528).

In addition to the widely publicized effect of sacubitril/valsartan on heart failure, other organ systems and disease states may potentially benefit from inhibition of the renin-angiotensin system and neprilysin. In spontaneously hypertensive rats, treatment with sacubitril/valsartan improved endothelial dysfunction (29). Compared with irbesartan alone, dual inhibition of the renin-angiotensin system and neprilysin with irbesartan and thiorphan provided better protection against diabetic retinopathy in rats (30). In humans, neprilysin activity correlated with BMI and measures of insulin resistance with increasing levels in subjects with multiple cardiovascular risk factors (31). In a recent study, Jordan et al. (32) demonstrated that 8 weeks of treatment of obese subjects with hypertension with sacubitril/valsartan was associated with a significant increase in insulin sensitivity. It was postulated that sacubitril/valsartan treatment restored natriuretic peptide deficiency and reduced renin-angiotensin system activity, which are associated with impaired oxidative metabolism and type 2 diabetes. Plasma neprilysin levels have been shown to increase in high-fat–fed mice (31). In this study, we have demonstrated that neprilysin activity is increased in the liver of type 2 diabetic rats. High levels of fatty acids and glucose have been shown to increase neprilysin activity in human microvascular endothelial cells (33). We have demonstrated that neprilysin immunoreactivity and superoxide are increased in epineurial arterioles of diabetic rats, and this increase is associated with vascular dysfunction, which can be prevented with vasopeptidase inhibitor treatment (6,9). We have also demonstrated that endothelial cells of epineurial arterioles express C-type natriuretic peptide that has vasodilatory properties that are decreased by diabetes but protected with vasopeptidase inhibitor treatment (9).

Patients with chronic kidney disease are at increased risk of progression to end-stage renal disease and cardiovascular events and ultimately renal transplant (34). Vasopeptidase inhibitors had shown promise as a treatment for chronic kidney disease in animal models, but as previously discussed, clinical development of this class of drugs was discontinued (35). Sacubitril/valsartan is now being studied as a potential treatment in a population of patients with chronic kidney disease as well as in animal models (34,36). This literature demonstrates that widespread studies are ongoing examining the potential benefits of angiotensin receptor-neprilysin inhibition in renal and cardiovascular diseases. However, the potential benefit of this class of drug on neural complications associated with diabetes has not been reported.

The preclinical studies presented in this study clearly demonstrate that early and late intervention with sacubitril/valsartan improved multiple end points associated with peripheral neuropathy as well as improved vascular reactivity of epineurial arterioles, blood vessels that provide circulation to the sciatic nerve. The effect of sacubitril/valsartan was superior to valsartan alone, indicating that the combination of inhibition of the renin-angiotensin system and neprilysin is more efficacious. We have previously demonstrated that suppressing the renin-angiotensin system in diabetic rats through inhibiting ACE or blocking the angiotensin II receptor improved both vascular and neural function, in part by reducing oxidative stress in vascular tissue (5,7,13,37). We have also demonstrated that blocking neprilysin activity improves neural function (49). Malik et al. (38), in a randomized double-blind controlled trial, demonstrated that the ACE inhibitor trandolapril improved diabetic neuropathy in normotensive patients. In cardiovascular autonomic neuropathy, neuropeptides and their receptors play a key regulatory role (39). These neuropeptides include natriuretic peptides and calcitonin gene-related peptide, which are degraded by neprilysin (39,40). Dysregulation of the expression of these neuropeptides can negatively affect cardiac homeostasis (39). Calcitonin gene-related peptide also plays a significant role in peripheral nerve regeneration and Schwann cell proliferation (41,42). Because neprilysin expression is increased in diabetes, protecting calcitonin gene-related and natriuretic peptides, including C-type natriuretic peptide, from abnormal degradation by blocking neprilysin activity is perhaps one mechanism by which sacubitril/valsartan treatment is improving or reversing diabetic peripheral and autonomic neuropathy.

There are a number of other mechanisms that have been shown to cause damage of the nervous system in diabetic peripheral neuropathy including the polyol pathway, accumulation of advanced glycation end products, and activations of poly(ADP-ribose) polymerase, hexosamine pathway, and protein kinase C (43). Many of these pathways and mechanisms lead to dysregulation of the mitochondria and an increase in oxidative and nitrosative stress (14,43). Blocking the renin-angiotensin system has been shown to reduce oxidative stress in obesity and diabetes and improve insulin sensitivity (44).

In summary, the primary finding from these preclinical studies was that sacubitril/valsartan was able to slow progression of diabetes-induced vascular and neural complications as evidence of the results from the early intervention protocol and also stimulates restoration as demonstrated by outcome measures from the late intervention protocol in a rat model of type 2 diabetes. The mechanisms contributing to the beneficial effects observed with sacubitril/valsartan treatment likely include reducing oxidative stress and protecting neuro- and vasoactive peptides. These results provide rationale for the continued study of sacubitril/valsartan as a potential new treatment for diabetic peripheral neuropathy.

Duality of Interest. This material is based upon work supported by Novartis (LCZ696BUS10T). No other potential conflicts of interest relevant to this article were reported.

Author Contributions. E.P.D. performed studies, evaluated data, prepared figures, and reviewed the manuscript. L.J.C., H.S., and A.O. performed studies, evaluated data, and reviewed the manuscript. M.A.Y. performed studies, evaluated data, and wrote the manuscript. M.A.Y. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the 27th Annual Meeting of the Diabetic Neuropathy Study Group of the European Association for the Study of Diabetes (NEURODIAB), Coimbra, Portugal, 9–11 September 2017.

1.
Feldman
EL
,
Nave
KA
,
Jensen
TS
,
Bennett
DLH
.
New horizons in diabetic neuropathy: mechanisms, bioenergetics, and pain
.
Neuron
2017
;
93
:
1296
1313
[PubMed]
2.
Gonçalves
NP
,
Vægter
CB
,
Andersen
H
,
Østergaard
L
,
Calcutt
NA
,
Jensen
TS
.
Schwann cell interactions with axons and microvessels in diabetic neuropathy
.
Nat Rev Neurol
2017
;
13
:
135
147
[PubMed]
3.
Yorek
MA
.
Vascular impairment of epineurial arterioles of the sciatic nerve: implications for diabetic peripheral neuropathy
.
Rev Diabet Stud
2015
;
12
:
13
28
[PubMed]
4.
Davidson
EP
,
Coppey
LJ
,
Yorek
MA
.
Early loss of innervation of cornea epithelium in streptozotocin-induced type 1 diabetic rats: improvement with ilepatril treatment
.
Invest Ophthalmol Vis Sci
2012
;
53
:
8067
8074
[PubMed]
5.
Davidson
EP
,
Coppey
LJ
,
Holmes
A
,
Yorek
MA
.
Effect of inhibition of angiotensin converting enzyme and/or neutral endopeptidase on vascular and neural complications in high fat fed/low dose streptozotocin-diabetic rats
.
Eur J Pharmacol
2012
;
677
:
180
187
[PubMed]
6.
Davidson
EP
,
Coppey
LJ
,
Holmes
A
,
Dake
B
,
Yorek
MA
.
Effect of treatment of high fat fed/low dose streptozotocin-diabetic rats with Ilepatril on vascular and neural complications
.
Eur J Pharmacol
2011
;
668
:
497
506
[PubMed]
7.
Oltman
CL
,
Davidson
EP
,
Coppey
LJ
,
Kleinschmidt
TL
,
Dake
B
,
Yorek
MA
.
Role of the effect of inhibition of neutral endopeptidase on vascular and neural complications in streptozotocin-induced diabetic rats
.
Eur J Pharmacol
2011
;
650
:
556
562
[PubMed]
8.
Oltman
CL
,
Davidson
EP
,
Coppey
LJ
,
Kleinschmidt
TL
,
Yorek
MA
.
Treatment of Zucker diabetic fatty rats with AVE7688 improves vascular and neural dysfunction
.
Diabetes Obes Metab
2009
;
11
:
223
233
[PubMed]
9.
Davidson
EP
,
Kleinschmidt
TL
,
Oltman
CL
,
Lund
DD
,
Yorek
MA
.
Treatment of streptozotocin-induced diabetic rats with AVE7688, a vasopeptidase inhibitor: effect on vascular and neural disease
.
Diabetes
2007
;
56
:
355
362
[PubMed]
10.
Burnett
JC
 Jr
.
Vasopeptidase inhibition: a new concept in blood pressure management
.
J Hypertens Suppl
1999
;
17
:
S37
S43
[PubMed]
11.
Pickering
TG
.
Effects of stress and behavioral interventions in hypertension: the rise and fall of omapatrilat
.
J Clin Hypertens (Greenwich)
2002
;
4
:
371
373
[PubMed]
12.
McMurray
JJ
,
Packer
M
,
Desai
AS
, et al.;
PARADIGM-HF Investigators and Committees
.
Angiotensin-neprilysin inhibition versus enalapril in heart failure
.
N Engl J Med
2014
;
371
:
993
1004
[PubMed]
13.
Coppey
LJ
,
Davidson
EP
,
Rinehart
TW
, et al
.
ACE inhibitor or angiotensin II receptor antagonist attenuates diabetic neuropathy in streptozotocin-induced diabetic rats
.
Diabetes
2006
;
55
:
341
348
[PubMed]
14.
Yorek
MA
.
The potential role of angiotensin converting enzyme and vasopeptidase inhibitors in the treatment of diabetic neuropathy
.
Curr Drug Targets
2008
;
9
:
77
84
[PubMed]
15.
von Lueder
TG
,
Wang
BH
,
Kompa
AR
, et al
.
Angiotensin receptor neprilysin inhibitor LCZ696 attenuates cardiac remodeling and dysfunction after myocardial infarction by reducing cardiac fibrosis and hypertrophy
.
Circ Heart Fail
2015
;
8
:
71
78
[PubMed]
16.
Gu
J
,
Noe
A
,
Chandra
P
, et al
.
Pharmacokinetics and pharmacodynamics of LCZ696, a novel dual-acting angiotensin receptor-neprilysin inhibitor (ARNi)
.
J Clin Pharmacol
2010
;
50
:
401
414
[PubMed]
17.
Yorek
MS
,
Davidson
EP
,
Poolman
P
, et al
.
Corneal sensitivity to hyperosmolar eye drops: a novel behavioral assay to assess diabetic peripheral neuropathy
.
Invest Ophthalmol Vis Sci
2016
;
57
:
2412
2419
[PubMed]
18.
Yorek
MS
,
Obrosov
A
,
Shevalye
H
, et al
.
Effect of diet-induced obesity or type 1 or type 2 diabetes on corneal nerves and peripheral neuropathy in C57Bl/6J mice
.
J Peripher Nerv Syst
2015
;
20
:
24
31
[PubMed]
19.
Coppey
L
,
Davidson
E
,
Lu
B
,
Gerard
C
,
Yorek
M
.
Vasopeptidase inhibitor ilepatril (AVE7688) prevents obesity- and diabetes-induced neuropathy in C57Bl/6J mice
.
Neuropharmacology
2011
;
60
:
259
266
[PubMed]
20.
Tavakoli
M
,
Petropoulos
IN
,
Malik
RA
.
Assessing corneal nerve structure and function in diabetic neuropathy
.
Clin Exp Optom
2012
;
95
:
338
347
[PubMed]
21.
Chen
X
,
Graham
J
,
Dabbah
MA
, et al
.
Small nerve fiber quantification in the diagnosis of diabetic sensorimotor polyneuropathy: comparing corneal confocal microscopy with intraepidermal nerve fiber density
.
Diabetes Care
2015
;
38
:
1138
1144
[PubMed]
22.
Coppey
LJ
,
Davidson
EP
,
Dunlap
JA
,
Lund
DD
,
Yorek
MA
.
Slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic nerve
.
Int J Exp Diabetes Res
2000
;
1
:
131
143
[PubMed]
23.
Brain
SD
,
Grant
AD
.
Vascular actions of calcitonin gene-related peptide and adrenomedullin
.
Physiol Rev
2004
;
84
:
903
934
[PubMed]
24.
Yorek
MA
,
Coppey
LJ
,
Gellett
JS
,
Davidson
EP
.
Sensory nerve innervation of epineurial arterioles of the sciatic nerve containing calcitonin gene-related peptide: effect of streptozotocin-induced diabetes
.
Exp Diabesity Res
2004
;
5
:
187
193
[PubMed]
25.
Ansara
AJ
,
Kolanczyk
DM
,
Koehler
JM
.
Neprilysin inhibition with sacubitril/valsartan in the treatment of heart failure: mortality bang for your buck
.
J Clin Pharm Ther
2016
;
41
:
119
127
[PubMed]
26.
Malek
V
,
Gaikwad
AB
.
Neprilysin inhibitors: a new hope to halt the diabetic cardiovascular and renal complications
?
Biomed Pharmacother
2017
;
90
:
752
759
[PubMed]
27.
Chrysant
SG
.
Pharmacokinetic, pharmacodynamic, and antihypertensive effects of the neprilysin inhibitor LCZ-696: sacubitril/valsartan
.
J Am Soc Hypertens
2017
;
11
:
461
468
[PubMed]
28.
Li
B
,
Zhao
Y
,
Yin
B
, et al
.
Safety of the neprilysin/renin-angiotensin system inhibitor LCZ696
.
Oncotarget
2017
;
8
:
83323
83333
[PubMed]
29.
Seki
T
,
Goto
K
,
Kansui
Y
,
Ohtsubo
T
,
Matsumura
K
,
Kitazono
T
.
Angiotensin II receptor-neprilysin inhibitor sacubitril/valsartan improves endothelial dysfunction in spontaneously hypertensive rats
.
J Am Heart Assoc
2017
;
6
:
006617
[PubMed]
30.
Prasad
T
,
Roksnoer
LC
,
Zhu
P
, et al
.
Beneficial effects of combined AT1 receptor/neprilysin inhibition (ARNI) versus AT1 receptor blockade alone in the diabetic eye
.
Invest Ophthalmol Vis Sci
2016
;
57
:
6722
6730
[PubMed]
31.
Standeven
KF
,
Hess
K
,
Carter
AM
, et al
.
Neprilysin, obesity and the metabolic syndrome
.
Int J Obes
2011
;
35
:
1031
1040
[PubMed]
32.
Jordan
J
,
Stinkens
R
,
Jax
T
, et al
.
Improved insulin sensitivity with angiotensin receptor neprilysin inhibition in individuals with obesity and hypertension
.
Clin Pharmacol Ther
2017
;
101
:
254
263
[PubMed]
33.
Muangman
P
,
Spenny
ML
,
Tamura
RN
,
Gibran
NS
.
Fatty acids and glucose increase neutral endopeptidase activity in human microvascular endothelial cells
.
Shock
2003
;
19
:
508
512
[PubMed]
34.
Judge
P
,
Haynes
R
,
Landray
MJ
,
Baigent
C
.
Neprilysin inhibition in chronic kidney disease
.
Nephrol Dial Transplant
2015
;
30
:
738
743
[PubMed]
35.
Cao
Z
,
Burrell
LM
,
Tikkanen
I
,
Bonnet
F
,
Cooper
ME
,
Gilbert
RE
.
Vasopeptidase inhibition attenuates the progression of renal injury in subtotal nephrectomized rats
.
Kidney Int
2001
;
60
:
715
721
[PubMed]
36.
Ushijima
K
,
Ando
H
,
Arakawa
Y
, et al
.
Prevention against renal damage in rats with subtotal nephrectomy by sacubitril/valsartan (LCZ696), a dual-acting angiotensin receptor-neprilysin inhibitor
.
Pharmacol Res Perspect
2017
;
5
:
e00336
.
[PubMed]
37.
Holmes
A
,
Coppey
LJ
,
Davidson
EP
,
Yorek
MA
.
Rat models of diet-induced obesity and high fat/low dose streptozotocin type 2 diabetes: effect of reversal of high fat diet compared to treatment with enalapril or menhaden oil on glucose utilization and neuropathic endpoints
.
J Diabetes Res
2015
;
2015
:
307285
38.
Malik
RA
,
Williamson
S
,
Abbott
C
, et al
.
Effect of angiotensin-converting-enzyme (ACE) inhibitor trandolapril on human diabetic neuropathy: randomised double-blind controlled trial
.
Lancet
1998
;
352
:
1978
1981
[PubMed]
39.
Ejaz
A
,
LoGerfo
FW
,
Pradhan
L
.
Diabetic neuropathy and heart failure: role of neuropeptides
.
Expert Rev Mol Med
2011
;
13
:
e26
[PubMed]
40.
McDowell
G
,
Coutie
W
,
Shaw
C
,
Buchanan
KD
,
Struthers
AD
,
Nicholls
DP
.
The effect of the neutral endopeptidase inhibitor drug, candoxatril, on circulating levels of two of the most potent vasoactive peptides
.
Br J Clin Pharmacol
1997
;
43
:
329
332
[PubMed]
41.
Chung
AM
.
Calcitonin gene-related peptide (CGRP): role in peripheral nerve regeneration
.
Rev Neurosci
2018
;
29
:
369
376
42.
Cheng
L
,
Khan
M
,
Mudge
AW
.
Calcitonin gene-related peptide promotes Schwann cell proliferation
.
J Cell Biol
1995
;
129
:
789
796
[PubMed]
43.
Sifuentes-Franco
S
,
Pacheco-Moises
FP
,
Rodriguez-Carrizalez
AD
,
Miranda-Diaz
AG
.
The role of oxidative stress, mitochondrial function, and autophagy in diabetic polyneuropathy
.
J Diabetes Res
2017
;
2017
:
1673081
44.
Ramalingam
L
,
Menikdiwela
K
,
LeMieux
M
, et al
.
The renin angiotensin system, oxidative stress and mitochondrial function in obesity and insulin resistance
.
Biochim Biophys Acta
2017
;
1863
:
1106
1114
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