Abstract
Background:
This study evaluated the potential of activating the fuel-sensing enzymes Adenine monophosphate (AMP)-activated protein kinase and the deacetylase sirtuin1, to promote weight loss. We tested the efficacy of a fixed dose combination of the amino acid leucine and 2 well-characterized agents with established safety profiles to modulate energy metabolism and facilitate weight loss.
Study Question:
Will a combination of L-leucine with low-dose metformin and sildenafil produce a novel synergistic interaction that reduces body weight?
Study Design:
We conducted a 24-week randomized controlled trial evaluating the effect on weight loss of leucine 1.1 g and sildenafil 1.0 mg or 4.0 mg, with and without metformin 500 mg (Leu/Sil 1.0, Leu/Sil 4.0, Leu/Met/Sil 1.0, and Leu/Met/Sil 4.0 twice/day). We enrolled 267 participants who were 18–65 years of age without diabetes and with the body mass index (BMI) of 30–45 kg/m2.
Measures and Outcomes:
The primary endpoint was percentage weight change after 24 weeks. Adverse events were evaluated. The primary analysis was performed using the perprotocol population analysis of covariance estimation. Subgroup analyses of patients residing above certain threshold limits at baseline and in populations at increased risk of obesity were assessed post-hoc as exploratory end points.
Results:
Placebo-adjusted mean bodyweight reductions in the Leu/Met/Sil 1.0, Leu/Met/Sil 4.0, and Leu/Sil 4.0 groups were −1.99%, −1.69%, and −1.67% (P = 0.015, 0.035, and 0.036, respectively). The most common adverse events were gastrointestinal-related and occurred in the metformin-treated groups consistent with metformin treatment. In African Americans, Leu/Met/Sil 1.0 produced 5.4% mean weight loss. In participants with BMI <40 kg/m2 treated with Leu/Met/Sil 1.0, the weight loss increased to 2.84%, particularly in participants with baseline insulin ≥12mU/L (3.5%).
Conclusions:
Leu/Met/Sil 1.0 and 4.0 and Leu/Sil 4.0 reduced body weight, but Leu/Met/Sil 1.0 was associated with robust weight loss in African Americans, and individuals with BMI 30–39.9 kg/m2, especially participants with hyperinsulinemia.
Keywords: obesity, weight loss, African Americans, insulin resistance
INTRODUCTION
Human adiposity is influenced by complex interactions between genetic, biologic, behavioral, and environmental factors that act in concert to preserve body weight over long periods of time.1 Reducing food intake or increasing physical activity are recommended as the first approach for obesity management. However, the signals emanating from weight loss are received and integrated by neural circuits as an energy gap between appetite and metabolic requirements. The counter-regulatory cascade of adaptive mechanisms prompted by weight loss is persistent, saturated with redundancies, and primed to restore depleted energy reserves.2 Therefore, most people find it difficult to adhere to a lifestyle change regimen and it cannot be sustained.3-5 Interventions that influence changes in body weight must disrupt the balance between energy intake and expenditure, substrate utilization, and the storage of excess calories.1 The ability to sustain lifestyle changes over time presents a challenge for most individuals. Even small improvements induced by the addition of pharmacotherapy to lifestyle changes could provide the impetus to adhere to a diet and exercise regimen.
Combination therapy that targets multiple pathways involved in energy metabolism has the potential to accentuate an overall effect and do so at low doses which would less likely precipitate adverse events. Adenine monophosphate–activated protein kinase (AMPK) is a fuel-sensing enzyme whose activation maintains cellular energy stores by switching on catabolic pathways such as oxidative metabolism and mitochondrial biogenesis, whereas switching off anabolic pathways not essential for survival such as triglyceride synthesis and cell proliferation. AMPK acts in coordination with another fuel-sensing enzyme, the NAD+-dependent type III deacetylase, sirtuin 1 (SIRT1). AMPK enhances SIRT1 activity by increasing cellular NAD+ levels. Reciprocally, SIRT1 regulates the activation of AMPK through the acetylation of liver kinase B1.6,7 The activation of AMPK and SIRT1 not only increases energy expenditure but also diminishes oxidative and endoplasmic reticulum stress and low-grade inflammation in various tissues.6
The branched chain amino acid leucine and its metabolites have been shown to increase SIRT1 activity by lowering the Km for NAD+.8,9 Thus, at low concentrations of NAD+ (characteristic of an energy-replete state), leucine stimulates SIRT1 activity and serves as a partial mimetic of an energy-depleted state. Metformin increases hepatic insulin sensitivity and rodent models suggest that its action may be mediated by AMPK.10 Nitric oxide (NO) activates SIRT1 which in turn deacetylates and activates endothelial NO synthase to produce NO in a positive feedback loop (Figure 1).11-14
FIGURE 1.
Schema of mechanism of action for the leucine, metformin, and sildenafil combination therapy to treat obesity. NAD+/NADH, nicotinamide adenine dinucleotide; AMP/ATP, adenine monophosphate/adenine triphosphate; Leu, leucine; Met, metformin; Sil, sildenafil; SIRT1, sirtuin 1; LKB1, liver kinase B1; NAMPT, nicotinamide phosphoribosyltransferase; ; eNOS, endothelial nitric oxide synthase.
To enhance AMPK and SIRT1 action in tandem, we tested a combination of leucine, metformin, and the endothelial NO synthase activator sildenafil (NS-0200) shown to act in synergy to increase fat oxidation.15 We found that the combination reduced body weight, triglycerides, and hemoglobin A1c in subjects with obesity.16 NS-0200 also reduced hepatic fat in patients with nonalcoholic fatty liver disease.17 This synergy permitted low doses of each compound which minimized adverse events, without reducing the antidiabetic effects of metformin. The objective of the present study was to evaluate the effect of NS-0200 on weight loss in the absence of any lifestyle intervention in subjects with obesity, and explore the possibility of obtaining similar results with 2 fixed-dose combinations of leucine and sildenafil (excluding metformin).
METHODS
Study design and participants
We completed a 24-week randomized, double-blind, placebo-controlled, multicenter, parallel-group, phase 2 trial. The study was conducted at 15 clinical sites across the United States and in accordance with the ethical principles set forth in the Declaration of Helsinki. The protocol and informed consent document were approved by the institutional review board of each clinical site. Eligible participants were adults between 18 and 65 years of age with the body mass index (BMI) between 30 kg/m2 and 45 kg/m2. Self-reported body weight should not have fluctuated by more than 5%, and health should have been stable in the 90 days preceding screening. Participants who had diseases associated with metabolism or were taking medications associated with weight changes or had clinically significant laboratory tests were excluded. A complete list of inclusion and exclusion criteria is provided in the Supplemental Digital Content 1 (see, http://links.lww.com/AJT/A80). All participants provided written informed consent; the trial was registered on ClincalTrials.gov (registration number NCT03364335).
Randomization and masking
Subjects were assigned a randomization number in a strict chronological order. Eligible subjects were randomized to 1 of 5 treatment groups: (A) placebo, (B) 1.1 g leucine + 1.0 mg sildenafil (NS-0300, Leu/Sil 1.0), (C) 1.1 g leucine + 4.0 mg sildenafil (NS-0300, Leu/Sil 4.0), (D) 1.1 g leucine + 1.0 mg sildenafil + 500 mg metformin (NS-0200, Leu/Met/Sil 1.0), or (E) 1.1 g leucine + 4.0 mg sildenafil + 500 mg metformin (NS-0200, Leu/Met/Sil 4.0). Since the dosing was twice daily, each participant ingested twice the amounts listed for each compound. Participants, investigators, and sponsors were masked to the study treatment assignments. The formulation and packaging of medications is provided in the SDCII.
Procedures
The study consisted of a 1-week screening period and 24 weeks of treatment. The study design is presented in Figure 2. Subjects reported to the clinic at screening, baseline, and every 4 weeks through week 24. To maintain engagement and enhance subject retention, participants were contacted by telephone 2 weeks after each clinic visit. Body weight and vital signs were measured, adverse events reviewed, and medications recorded at all visits. Waist circumference was measured at all visits except the screening visit. Laboratory parameters were assessed at screening, baseline, week 12, and at the end of the study. In addition, at the screening and end-of-study visit, a complete medical history, physical examination, 12-lead electrocardiogram, and urinalysis assessment were completed. Subjects reported for all the visits in the morning after a 10-hour overnight fast (except water). At the visits following randomization, the medication dose was ingested in the clinic after completion of study procedures.
FIGURE 2.
Study visits over the screening and 24-week treatment periods.
Outcomes
The primary endpoint was the relative percentage change in body weight from baseline to week 24. Prespecified secondary endpoints were percentage of participants with weight loss of 5% or more or 10% or more of baseline, absolute change in weight, and waist circumference; changes in glucose metabolism (hemoglobin A1c and fasting glucose), to; cardiovascular risk factors (blood pressure and fasting lipids), and biomarker of inflammation (C-reactive protein), to; and other comorbidities including hypertension, hyperlipidemia, diabetes, and systemic inflammation. Furthermore, subgroup analyses of patients residing above certain threshold limits at baseline for each of the comorbidities were assessed post-hoc as exploratory end points. Safety was evaluated throughout the study by examination of adverse events, concomitant medications, clinical laboratory evaluations, vital signs, physical examinations, and 12-lead electrocardiograms.
Statistical analysis
Using a 2-sided, 2-sample comparison (t test) of means at the alpha = 0.05 level of significance, sample sizes of 23 subjects per treatment provided approximately 90% power to detect a mean treatment difference of 3 kg body weight (~3.0–3.3% weight loss over 2 weeks), assuming a common standard deviation of 25%. However, detection of significant changes in obesity comorbidities required a sample size of 40 subjects per treatment group to detect a 6 mm Hg change in blood pressure. The assumptions of effect size and standard deviation are based on our previous clinical trial of NS-0200 and represent the anticipated treatment effect of fixed-dose combinations of leucine and sildenafil, with or without metformin, compared with placebo.16 A sufficient number of individuals were screened to enroll at least 250 subjects, with a maximum of 70% female (~50/per treatment arm). Assuming a 20% dropout rate, approximately 200 subjects (~40/per treatment arm) were expected to complete treatment through week 24 (end of study). The intent-to-treat (ITT) population included all randomized subjects regardless of adherence to the protocol. To estimate the pharmacologic activity, the per-protocol (PP) population was used. The PP population included all ITT subjects who had adequate exposure to the study medication (>85%) during the 24-week period and completed the end of study procedures with valid measurements and had no major protocol deviations. In addition, descriptive statistics were used to summarize baseline values for all analyzed endpoints. These summaries are presented for the ITT population using the last observation carried forward approach.
A mixed-model of analysis of covariance was used to analyze percentage change in body weight from baseline to week 24 in the PP and ITT populations. The analysis of covariance included treatment group and time as the main effects with baseline weight as the covariate. The least squares means (LSMs) and SEs for the changes from baseline to week 24 were derived from the model for each treatment. Each of the fixed-dose combinations of leucine and sildenafil with/without metformin treatment groups (treatment B, C, D and E) were compared with the placebo group (treatment A), and the LSMs for the treatment difference (treatment B, C, D, or E minus treatment A), the SEs, and the P-values were computed. There was no adjustment for multiple comparisons because of the exploratory nature of this early-phase study. The significance was set at α = 0.05.
All secondary efficacy endpoints were analyzed with mixed-effects models similar to the primary endpoint analyses. For endpoints that required logarithm-transformation to ensure data normality, the change from baseline was calculated as the log-transformed post-baseline data minus the log-transformed baseline data. These changes from baseline data were examined as the dependent variable and the log of the baseline covariate was included as a covariate. The resulting estimates were exponentially transformed back to the original scale to yield geometric LSM ratios and the SEs of the geometric mean ratios (calculated as the geometric LSM ratio multiplied by the SE of the LSM of the log-transformed data). For the exploratory analysis in the PP group, the means and SE of the mean for the changes from baseline to week 24 were determined and similarly analyzed using a mixed-effect model. Analyses were conducted using SAS 9.2 or above (SAS Institute Inc., Cary NC).
RESULTS
Of the 267 participants randomized, 212 completed the trial, and 202 met the a priori compliance criteria (PP group). The results for the PP and ITT groups were similar. However, the PP group had measured values for the endpoints throughout the study and was designated a priori for the primary efficacy assessment. The most common reason for treatment discontinuation was withdrawal of consent which occurred similarly among the groups. The analysis population and disposition of participants are presented in Table 1. The baseline characteristics analyzed using the ITT population is summarized in Table 2.
Table 1.
The analysis population and participant disposition over the course of the study.
| Treatment arms | ||||||
|---|---|---|---|---|---|---|
| Analysis population |
Placebo treatment A* (N = 53) n (%) |
NS-0300 treatment B* (N = 54) n (%) |
NS-0300 treatment C* (N = 54) n (%) |
NS-0200 treatment D* (N = 52) n (%) |
NS-0200 treatment E* (N = 54) n (%) |
Enrolled population (N = 267) n (%) |
| Randomized† | 53 (100) | 54 (100) | 54 (100) | 52 (100) | 54 (100) | 267 (100) |
| ITT‡ | 52 (98.1) | 51 (94.4) | 53 (98.1) | 50 (96.2) | 52 (96.3) | 258 (96.6) |
| Per-protocol§ | 42 (79.2) | 40 (74.1) | 42 (77.8) | 37 (71.2) | 41 (75.9) | 202 (75.7) |
| Participant disposition | ||||||
| Completed study | 45 (84.9) | 40 (74.1) | 43 (79.6) | 41 (78.8) | 43 (79.6) | 212 (79.4) |
| Treatment discontinuation | 8 (15.1) | 14 (25.9) | 11 (20.4) | 11 (21.2) | 11 (20.4) | 55 (20.6) |
| Withdrawal of consent | 3 (5.7) | 2 (3.7) | 2 (3.7) | 4 (7.7) | 3 (5.6) | 14 (5.2) |
| Adverse event | 1 (1.9) | 4 (7.4) | 2 (3.7) | 2 (3.8) | 1 (1.9) | 10 (3.7) |
| Investigator decision | 1 (1.9) | 0 | 0 | 0 | 0 | 1 (0.4) |
| Protocol violation | 0 | 1 (1.9) | 1 (1.9) | 0 | 1 (1.9) | 3 (1.1) |
| Lost to follow-up | 2 (3.8) | 3 (5.6) | 4 (7.4) | 5 (9.6) | 5 (9.3) | 19 (7.1) |
| Pregnancy | 0 | 1 (1.9) | 0 | 0 | 0 | 1 (0.4) |
| Others | 1 (1.9) | 3 (5.6) | 2 (3.7) | 0 | 1 (1.9) | 7 (2.6) |
Treatment: A = placebo; B = leucine 1.1 g + sildenafil 1 mg; C = leucine 1.1 g + sildenafil 4 mg; D = leucine 1.1 g + sildenafil 1 mg + metformin 500 mg; E = leucine 1.1 g + sildenafil 4 mg + metformin 500 mg.
The randomized population consists of all enrolled subjects randomized at baseline/visit 2 (day 1).
The ITT population includes all randomized subjects who received at least 1 dose of study medication and had at least 1 follow-up primary efficacy assessment performed.
The PP population includes all ITT subjects who received 80% of the randomized study medication during the 24-week period, have completed all study visits, and have adequately complied with the protocol as assessed by the investigator and sponsor before database lock.
Table 2.
Demographic and baseline characteristics by treatment.
| Intent-to-treat population (N = 258) | ||||||
|---|---|---|---|---|---|---|
| Demographic and baseline characteristics |
Placebo treatment A* (N = 52) |
NS-0300 treatment B* (N = 51) |
NS-0300 treatment C* (N = 53) |
NS-0200 treatment D* (N = 50) |
NS-0200 treatment E* (N = 52) |
All (N = 258) |
| Sex, n (%) | ||||||
| Male | 14 (26.9) | 22 (43.1) | 21 (39.6) | 11 (22.0) | 17 (32.7) | 85 (32.9) |
| Female | 38 (73.1) | 29 (56.9) | 32 (60.4) | 39 (78.0) | 35 (67.3) | 173 (67.1) |
| Age (yr)† | ||||||
| Mean (SD) | 40.8 (11.04) | 41.3 (11.57) | 39.2 (11.10) | 42.1 (12.58) | 42.1 (10.44) | 41.1 (11.32) |
| Race, n (%) | ||||||
| White | 40 (76.9) | 42 (82.4) | 39 (73.6) | 37 (74.0) | 40 (76.9) | 198 (76.7) |
| Black or African American | 9 (17.3) | 9 (17.6) | 13 (24.5) | 11 (22.0) | 11 (21.2) | 53 (20.5) |
| Asian | 0 | 0 | 1 (1.9) | 2 (4.0) | 1 (1.9) | 4 (1.6) |
| American Indian or Alaska Native | 1 (1.9) | 0 | 0 | 0 | 0 | 1 (0.4) |
| Unknown | 2 (3.8) | 0 | 0 | 0 | 0 | 2 (0.8) |
| Ethnicity, n (%) | ||||||
| Hispanic or Latino | 8 (15.4) | 12 (23.5) | 13 (24.5) | 12 (24.0) | 15 (28.8) | 60 (23.3) |
| Not Hispanic or Latino | 44 (84.6) | 39 (76.5) | 40 (75.5) | 38 (76.0) | 37 (71.2) | 198 (76.7) |
| Weight (kg) | ||||||
| Mean (SD) | 101.67 (13.856) | 105.05 (15.568) | 107.16 (17.962) | 100.18 (16.991) | 104.50 (17.405) | 103.75 (16.489) |
| BMI (kg/m2) | ||||||
| Mean (SD) | 35.773 (3.3067) | 37.179 (4.2849) | 37.900 (4.1995) | 36.425 (4.1266) | 37.152 (4.2327) | 36.892 (4.0801) |
| Fasting plasma glucose (mg/dL) | ||||||
| Mean (SD) | 95.5 (12.30) | 97.9 (8.97) | 95.2 (8.00) | 97.2 (11.87) | 95.5 (10.27) | 96.2 (10.37) |
| HbA1c (%) | ||||||
| Mean (SD) | 5.32 (0.355) | 5.45(0.365) | 5.36 (0.368) | 5.45 (0.399) | 5.45 (0.281) | 5.41 (0.357) |
Treatment: A = placebo; B = leucine 1.1 g + sildenafil 1 mg; C = leucine 1.1 g + sildenafil 4 mg; D = leucine 1.1 g + sildenafil 1 mg + metformin 500 mg; E = leucine 1.1 g + sildenafil 4 mg + metformin 500 mg.
N: number of subjects in the ITT population; n: number of subjects in the category; %: percentage calculated using the N as the denominator.
The primary endpoint was met for 3 treatment groups. At week 24, the placebo group showed a mean percentage body weight increase. The placebo-adjusted mean reduction in percentage body weight was greatest in Leu/Met/Sil 1.0, followed by Leu/Met/Sil 4.0, and then Leu/Sil 4.0. The percentage body weight increased at week 24 in Leu/Sil 1.0 (Table 3). In the analysis of the secondary outcomes, the changes in absolute body weight were consistent with the changes in percent body weight (Table 4). There were numerical improvements in all of the secondary outcomes consistent with the significant weight loss in the 3 treatment groups compared with placebo. However, they did not attain statistical significance except for total and LDL cholesterol and C-reactive protein. Placebo-adjusted total cholesterol (9.0 mg/dL, SE: 4.16, P = 0.03) and LDL cholesterol (6.8 mg/dL, SE: 3.20, P = 0.03) reduced in Leu/Met/Sil 4.0. C-reactive protein fell in Leu/Met/Sil 1.0 (geometric LSM ratio:0.71, SE: 0.09, P = 0.005), and also fell in Leu/Sil 1.0 (geometric LSM ratio: 0.77, SE: 0.09, P = 0.03).
Table 3.
Primary endpoint—percent body weight change from baseline to week 24 in the per protocol and ITT populations.
| ANCOVA* analysis for primary endpoint per protocol population (N = 202) | |||||
|---|---|---|---|---|---|
| Parameter statistics | Placebo treatment A† | NS-0300 treatment B† | NS-0300 treatment C† | NS-0200 treatment D† | NS-0200 treatment E† |
| n | 42 | 40 | 42 | 37 | 41 |
| LS mean (SE) | 1.14 (0.56) | 0.65 (0.37) | −0.54 (0.56) | −0.86 (0.60) | −0.56 (0.57) |
| LS mean placebo-adjusted (SE | −0.49 (0.80) | −1.67 (0.79) | −1.99 (0.82) | −1.69 (0.80 | |
| P-value | 0.5461 | 0.0364 | 0.0156 | 0.0349 | |
| ANCOVA* analysis for primary endpoint intent to treat population (N = 258) | |||||
| Parameter statistics | Placebo treatment A† | NS-0300 treatment B† | NS-0300 treatment C† | NS-0200 treatment D† | NS-0200 treatment E† |
| n | 52 | 51 | 53 | 50 | 52 |
| LS mean (SE) | 1.17 (0.48) | 0.55 (0.49 | −0.33 (0.48) | −0.65 (0.50) | −0.25 (0.48) |
| LS mean placebo-adjusted (SE) | −0.62 (0.69) | −1.50 (0.69) | −1.82 (0.69) | −1.42 (0.69) | |
| P-value | 0.3722 | 0.0298 | 0.0090 | 0.0389 | |
Analysis of covariance (ANCOVA) model: with treatment group and time as the main effects and baseline body weight as the covariate. Treatment A is used as the reference group.
Treatment: A = placebo; B = leucine 1.1 g + sildenafil 1 mg; C = leucine 1.1 g + sildenafil 4 mg; D = leucine 1.1 g + sildenafil 1 mg + metformin 500 mg; E = leucine 1.1 g + sildenafil 4 mg + metformin 500 mg.
LS, least squares; N, number of subjects in the PP population; n, number of subjects in the category.
Table 4.
Absolute weight change from baseline to week 24 in the per protocol and intent to treat populations.
| ANCOVA* analysis—PP population (N = 202) | |||||
|---|---|---|---|---|---|
| Parameter statistics | Placebo treatment A† | NS-0300 treatment B† | NS-0300 treatment B† | NS-0200 treatment D† | NS-0200 treatment E† |
| n | 42 | 40 | 42 | 37 | 41 |
| LS mean (SE) | 1.15 (0.57) | 0.64 (0.58) | −0.53 (0.57) | −0.77 (0.61) | −0.47 (0.57) |
| LS mean placebo-adjusted (SE) | −0.51 (0.81) | −1.68 (0.80) | −1.92 (0.83) | −1.61 (0.81) | |
| P-value | 0.5348 | 0.0379 | 0.0215 | 0.0464 | |
| ANCOVA* analysis—Intent to treat population (N = 258) | |||||
| Parameter statistics | Placebo treatment A† | NS-0300 treatment B† | NS-0300 treatment B† | NS-0200 treatment D† | NS-0200 treatment E† |
| n | 52 | 51 | 53 | 50 | 52 |
| LS mean (SE) | 1.13 (0.49) | 0.60 (0.50) | −0.34 (0.49) | −0.56 (0.50) | −0.18 (0.49) |
| LS mean placebo-adjusted (SE) | −0.53 (0.70) | −1.47 (0.70) | −1.69 (0.70) | −1.31 (0.70) | |
| P-value | 0.4514 | 0.0366 | 0.0174 | 0.0626 | |
Analysis of covariance (ANCOVA) model: with the treatment group and time as the main effects and baseline body weight as the covariate. Treatment A is used as the reference group.
Treatment: A = placebo; B = leucine 1.1 g + sildenafil 1 mg; C = leucine 1.1 g + sildenafil 4 mg; D = leucine 1.1 g + sildenafil 1 mg + metformin 500 mg; E = leucine 1.1 g + sildenafil 4 mg + metformin 500 mg.
LS, least squares; N, number of subjects in the PP population; n, number of subjects in the category.
Exploratory analysis in the PP subgroups yielded interesting results in the Leu/Met/Sil 1.0 treatment group. African-Americans exhibited markedly greater placebo-adjusted weight loss than the other participants who received Leu/Met/Sil 1.0 treatment (5.4%, SE: 1.29, P < 0.01). In addition, in the Leu/Met/Sil 1.0 treatment group, participants with BMI between 35 kg/m2 and 40 kg/m2 (obesity: class 2) lost 4.1% (P < 0.0001) of placebo-adjusted body weight and in those with BMI between 30 kg/m2 and 40 kg/m2 (obesity: classes 1 and 2), the reduction was 2.84% (P < 0.001,Figure 3A-C). Furthermore, in a subgroup analysis excluding participants with BMI >40 kg/m2 (obesity: class 3), those with insulin resistance or baseline fasting insulin concentration ≥12 mU/L,18 demonstrated greater placebo-adjusted weight loss (3.5%) than participants with <12 mU/L (1.99%, P < 0.001, Figure 4A-C). The enhanced effect with the exclusion of participants with BMI >40 kg/m2 suggests that this subgroup was less responsive to the treatment.
FIGURE 3.
(A–C)
Placebo; 
Leu/Sil 1.0; 
2Leu/Sil 4.0; 
Leu/Met/Sil 1.0; 
Leu/Met/Sil 4.0. Placebo-adjusted percentage change in body weight among (A) African American participants, (B) participants with class II obesity, and (C) participants with class I and class II obesity. Values are mean ± SEM. Leu, leucine; Met, metformin; Sil, sildenafil. ****P <0.0001.
FIGURE 4.
(A–C) Placebo-adjusted percentage change in body weight among (A) all participants with BMI <40 kg/m2, (B) participants with BMI <40 kg/m2 and fasting insulin ≤12 mU/L, and (C) participants with BMI <40 kg/m2 and fasting insulin ≥12 mU/L. Values are mean ± SEM.
Table 5 shows a summary of all treatment-emergent adverse events (TEAEs). Gastrointestinal events, primarily diarrhea, were the most common. TEAEs observed in the treatment groups and were mostly of mild (54.9% of subjects) or moderate (31% of subjects) intensity. There were 7 TEAEs that were rated as severe, 1 in Leu/Sil 1.0 (tooth abscess), 1 in Leu/Sil 4.0 (pneumonia mycoplasmal), 3 in Leu/Met/Sil 1.0 pyrexia, rhabdomyolysis, and metastatic malignant melanoma), and 2 in Leu/Met/Sil 4.0 (cholelithiasis and adnexal torsion). Three subjects reported 4 serious TEAEs including tooth abscess and hypokalemia in 1 subject (Leu/Sil 1.0), anal cancer (Leu/Sil 1.0) in 1 subject, and metastatic malignant melanoma (Leu/Met/Sil 1.0) in 1 subject; however, the serious TEAEs were deemed not related to the study treatment.
Table 5.
Summary of treatment-emergent adverse events (TEAEs), data are reported as n (%).
| ITT population (N = 258) | ||||||
|---|---|---|---|---|---|---|
| Category | Placebo treatment A* N = 52 |
NS-0300 treatment B* N = 51 |
NS-0300 treatment C* N = 53 |
NS-0200 treatment D* N = 50 |
NS-0200 treatment E* N = 52 |
B, C, D, and E pooled (N = 206) |
| All TEAEs† | ||||||
| No. of subjects, n(%) | 29 (55.8) | 36 (70.6) | 28 (52.8) | 39 (78.0) | 40 (76.9) | 143 (69.4) |
| No. of events | 69 | 83 | 51 | 96 | 78 | 308 |
| All serious TEAEs | ||||||
| No. of subjects, n(%) | 0 | 2 (3.9) | 0 | 1 (2.0) | 0 | 3 (1.5) |
| No. of events | 0 | 3 | 0 | 1 | 0 | 4 |
| All drug-related TEAEs | ||||||
| No. of subjects, n(%) | 16 (30.8) | 14 (27.5) | 11 (20.8) | 22 (44.0) | 21 (40.4) | 68 (33.0) |
| No. of events | 26 | 17 | 14 | 39 | 31 | 101 |
| All TEAEs leading to study discontinuation | ||||||
| No. of subjects, n (%) | 1 (1.9) | 3 (5.9) | 1 (1.9) | 2 (4.0) | 0 | 6 (2.9) |
| No. of events | 1 | 3 | 1 | 2 | 0 | 6 |
| Frequent ≥5% TEAEs | ||||||
| Abdominal pain | 1 (1.9) | 0 | 1 (1.9) | 3 (6.0) | 1 (1.9) | 5 (2.4) |
| Anthralgia | 0 | 4 (7.8) | 0 | 0 | 0 | 4 (1.9) |
| Back pain | 0 | 0 | 1 (1.9) | 3 (6.0) | 1 (1.9) | 5 (2.4) |
| C-reactive protein increase | 1(1.9) | 0 | 0 | 0 | 3 (5.8) | 3 (1.5) |
| Constipation | 3 (5.8) | 2 (3.9) | 1 (1.9) | 1 (2.0) | 1 (1.9) | 5 (2.4) |
| Diarrhea | 1 (1.9) | 2 (3.9) | 4 (7.5) | 21 (42.0) | 12 (23.1) | 39 (18.9) |
| Headache | 4 (7.7) | 6 (11.8) | 0 | 4 (8.0) | 5 (9.6) | 15 (7.3) |
| Hypertension | 3 (5.8) | 0 | 0 | 0 | 0 | 0 |
| Increased appetite | 2 (3.8) | 4 (7.8) | 2 (3.8) | 2 (4.0) | 3 (5.8) | 11 (5.3) |
| Influenza | 0 | 1 (2.0) | 2 (3.8) | 3 (6.0) | 0 | 6 (2.9) |
| Nasopharyngitis | 1 (1.9) | 5 (9.8) | 0 | 1 (2.0) | 2 (3.8) | 8 (3.9) |
| Nausea | 1 (1.9) | 4 (7.8) | 1 (1.9) | 4 (8.0) | 6 (11.5) | 15 (7.3) |
| Pharyngitis | 0 | 3 (5.9) | 0 | 0 | 0 | 3 (1.5) |
| Sinusitis | 1 (1.9) | 0 | 0 | 0 | 4 (7.7) | 4 (1.9) |
| Upper respiratory tract infection | 5 (9.6) | 6 (11.8) | 2 (3.8) | 4 (8.0) | 4 (7.7) | 16 (7.8) |
| Viral infection | 2 (3.8) | 3 (5.9) | 3 (5.7) | 2 (4.0) | 1 (1.9) | 9 (4.4) |
N: number of subjects in the ITT population; n: number of subjects in the category; %:percentage calculated using the N as the denominator. MedDRA Dictionary Version 19. Subjects experiencing multiple episodes of a given adverse events are counted once at the maximum intensity category and once in each preferred term.
Treatment-emergent adverse event (TEAE) is defined as an adverse event occurring on (or) after the first dose of randomized study medication, (or) existing before the time of and worsening after the time of the first dose of randomized study medication.
Treatment: A = placebo; B = leucine 1.1 g+ sildenafil 1 mg; C = leucine 1.1 g + sildenafil 4 mg; D = leucine 1.1 g + sildenafil 1 mg + metformin 500 mg; E = leucine 1.1 g + sildenafil 4 mg + metformin 500 mg.
Seven TEAEs reported by 7 participants, including hypertension (placebo), diarrhea and metastatic malignant melanoma, (Leu/Met/Sil 1.0), and abdominal pain, increased appetite, anal cancer, and rash (Leu/Sil 1.0) led to discontinuation of the study. In the analysis of the TEAEs with a frequency of >5% in any group, gastrointestinal events were the most common and occurred largely in the groups receiving metformin. Mild and moderate gastrointestinal disorders were reported by 50% and 38% of participants in the Leu/Met/Sil 1.0 and 4.0 groups, respectively. The increase in gastrointestinal disorder-related TEAEs was consistent with metformin treatment.19
DISCUSSION
This study was designed to evaluate the effect of 1.1 g leucine and 1 mg or 4 mg sildenafil combinations (NS-0300) and the NS-0300 combinations with 500 mg metformin (NS-0200) on body weight, compared with placebo. The study represents the second evaluation of the concomitant administration of leucine, sildenafil, and metformin and the initial evaluation of the concomitant administration of leucine and sildenafil without metformin. All of the combinations except 1.1 g leucine and 1 mg sildenafil achieved weight losses over 24 weeks that were significantly greater than placebo. The NS-0200 formulation containing leucine (1.1 g), metformin (500 mg), and sildenafil (1 mg) was most effective, and substantiates the results of our previous study demonstrating a 2.4 kg weight loss over 16 weeks.16
The placebo-adjusted 24-week weight loss of 1.99%, 1.69%, and 1.67% in the Leu/Met/Sil 1.0, Leu/Met/Sil 4.0, and Leu/Sil 4.0 treatments, respectively, is less than the 52-week weight loss achieved with lifestyle modification by the clinically approved antiobesity agents orlistat (approximately 6%), lorcaserin (approximately 6%), phentermine–topiramate (approximately 8%–10%), and naltrexone–bupropion (approximately 5%).20 These trials of approved antiobesity medications included a lifestyle intervention. As was demonstrated in a previous study investigating the effect of leucine, the lifestyle intervention may be expected to augment the overall effect.21 The enhanced effect of Leu/Met/Sil 1.0 compared with Leu/Met/Sil 4.0 is indicative of the effects of synergistic combinations where the optimal ratio may not be the highest dose of each drug.22
The prevalence of traditional cardiovascular risk factors such as hypertension, diabetes, obesity, and atherosclerotic cardiovascular risk underlies the earlier onset of cardiovascular disease among African Americans compared with Whites. Across the age spectrum, obesity rates are higher in African Americans compared with Whites.23 Leu/Met/Sil 1.0 treatment without lifestyle modification given to African Americans produced reductions in percent body weight at 24 weeks that were comparable with that produced by clinically approved antiobesity medications in 52 weeks. Although the trials are different and the participants are different, the finding is of interest, especially because African Americans are more impervious to weight loss than Whites.24 Therefore, the effect of Leu/Met/Sil 1.0 in African Americans and participants with Class I and II obesity, particularly those with hyperinsulinemia, bears investigation in trials exceeding 24 weeks that include participation in a lifestyle intervention.
Both fixed-dose combinations of Leu/Sil (NS-0300) and Leu/Met/Sil (NS-0200) were safe and well tolerated. There were no deaths or serious TEAEs that were considered related to the treatments. The most commonly reported TEAEs (incidence >5%) among participants receiving NS-0300 were upper respiratory tract infection, diarrhea, headache, and increased appetite. The most commonly reported TEAEs among participants receiving NS-0200 were primarily gastrointestinal conditions, such as diarrhea and nausea. In general, the TEAEs were reported as mild to moderate in intensity. This observed increase in gastrointestinal side effects in participants receiving NS-0200 was consistent with the well-known effects of metformin which was included in that formulation.
A limitation of the study is that the fluctuation in bodyweight 3 months before randomization was self-reported and not controlled. In addition, the study is limited by its design to primarily test the effects of NS-0200 and NS-0300 on weight loss at 24 weeks. The duration of the trial precludes conclusions regarding the sustainability of the reported weight loss. Furthermore, obesity treatment guidelines recommend that for individuals with BMI 30 kg/m2, or BMI 27 kg/m2 and at least 1 obesity-related comorbidity, who are motivated to lose weight, pharmacotherapy is an optional addition to lifestyle-change efforts. However, our study did not require participants to restrict intake or include nutrition counseling, and the reported weight loss is without any lifestyle changes. The subgroup analyses were exploratory. Therefore, by definition, the study was not powered to detect differences between the groups. Nevertheless, these analyses were conducted based on data to show that obesity and its comorbidities are more likely to afflict African Americans and individuals with insulin resistance.23,25
CONCLUSIONS
The primary efficacy endpoint of NS-0300 and NS-0200 treatment-related reduction in percentage body weight at week 24 versus placebo was achieved in this study for 1 dose of NS-0300 (4 mg sildenafil) and both doses of NS-0200. Although these results were achieved without an accompanying diet and exercise program, they were small and lack clinical significance. Exploratory analyses revealed more robust effects of NS-0200 (Leu/Met/Sil 1.0) in participants with BMI <40 kg/m2 particularly in participants with hyperinsulinemia. African-Americans, a racial group known to be prone to developing obesity, experienced weight loss with NS-0200 (Leu/Met/Sil 1.0) treatment comparable with a clinically approved antiobesity agent. These post-hoc exploratory analyses provide data to support the hypothesis that Leu/Met/Sil 1.0 may have therapeutic value in the treatment of obesity in specific populations. Further clinical evaluation is warranted to determine the strength of these observations, durability of the therapeutic effect, and the responses in ethnic populations.
Supplementary Material
Acknowledgments
Supported by NuSirt Biopharma and in part by U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH), which funds the Louisiana Clinical and Translational Science Center.
Footnotes
F. L. Greenway, O. Kolterman, and G. A. Fleming are advisors for the NuSirt Biopharma. M. B. Zemel is an employee of NuSirt Biopharma. The remaining authors have no conflicts of interest to declare.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.americantherapeutics.com).
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