MIB-626, an Oral Formulation of a Microcrystalline Unique Polymorph of β-Nicotinamide Mononucleotide, Increases Circulating Nicotinamide Adenine Dinucleotide and its Metabolome in Middle-Aged and Older Adults

Abstract

Background

Nicotinamide adenine dinucleotide (NAD) precursors, nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) extend healthspan and ameliorate some age-related conditions in model organisms. However, early-phase trials of NAD precursors have yielded varying results and their pharmacokinetics remain incompletely understood. Here, we report the pharmacokinetics and pharmacodynamics of MIB-626, a microcrystalline unique polymorph βNMN formulation.

Methods

In this double-blind, placebo-controlled study, 32 overweight or obese adults, 55–80 years, were block-randomized, stratified by sex, to 1 000-mg MIB-626 once daily, twice daily, or placebo for 14 days. NMN, NAD, and NAD metabolome were measured using liquid chromatography–tandem mass spectrometry.

Results

Participant characteristics were similar across groups. MIB-626 was well tolerated and frequency of adverse events was similar across groups. Blood NMN concentrations on Day 14 in MIB-626-treated groups were significantly higher compared to placebo (1.7-times and 3.7-times increase above baseline in 1 000 mg once-daily and twice-daily groups in mean AUC_last, respectively). MIB-626 treatment was associated with substantial dose-related increases in blood NAD levels. Blood levels of NAD metabolites were higher in NMN-treated participants on Days 8 and 14 than at baseline. Changes in NMN or NAD levels were not related to sex, body mass index, or age. Very little unmodified NMN was excreted in the urine.

Conclusion

MIB-626 1 000 mg once-daily or twice-daily regimens were safe and associated with substantial dose-related increases in blood NAD levels and its metabolome. These foundational data that were obtained using a pharmaceutical-grade βNMN, standardized sample collection, and validated liquid chromatography–tandem mass spectrometry assays, should facilitate design of efficacy trials in disease conditions.

Dietary supplement, Geroscience, MIB-626, Nicotinamide adenine dinucleotide, Nicotinamide mononucleotide

The “geroscience hypothesis” posits that age-related diseases can be prevented or delayed by targeting common mechanisms of aging (1); among these mechanisms, the important role of sirtuins and nicotinamide adenine dinucleotide (NAD) in regulating the aging process has been long recognized (2–4). NAD plays an important role in ATP synthesis and serves as a cofactor for sirtuins and other enzymes involved in DNA repair, mitochondrial function, chromosomal integrity, metabolism, and epigenetic and posttranslational regulation of gene expression (3,4).

NAD levels decrease throughout life span (5,6); the age-related decline in NAD levels has been linked to age-related diseases. In mice, raising NAD levels increases their life span and healthspan (7–11); improves glucose and lipid metabolism, and mitochondrial energetics; attenuates the development of diet-induced obesity, diabetes, and hepatic steatosis; protects the heart from ischemic damage; and promotes liver regeneration (12). Raising intracellular NAD also has been reported to improve mitochondrial energetics and aerobic capacity (13,14). Therefore, administration of NAD precursors, such as niacinamide, nicotinamide mononucleotide (NMN), and nicotinamide riboside (NR) to increase NAD levels is being explored as a therapeutic strategy to prevent and treat age-related diseases. Unsurprisingly, sales of over-the-counter (OTC) NAD boosters have witnessed substantial growth.

Although preclinical data look promising, clinical trials of NAD precursors have yielded varying results (15–22) likely due to heterogeneity of study populations, dose regimens, and relatively small sample size and short intervention durations. The doses used in some initial studies were relatively low. NMN and NR sold as OTC dietary supplements have suffered from variable manufacturing quality. NAD, NMN, and NR are susceptible to rapid degradation, and assays for the measurement of NAD, NMN, and their metabolites have been challenging. Only limited information is available on the pharmacokinetics (PK) and pharmacodynamics (PD) of βNMN and NR in humans.

We report here the PK and PD of MIB-626, a tablet microcrystalline formulation of a cGMP NAD precursor, βNMN, in overweight or obese, medically stable, middle-aged, and older adults. The trial evaluated the safety and tolerability and the ability of varying doses of oral MIB-626, to raise intracellular NAD and its metabolome, and biomarkers of metabolic function. MIB-626 is a pharmaceutical GMP preparation of NMN with a unique crystalline structure (polymorph) that has been milled to a desired particle size range to optimize absorption. Unlike niacin, NMN does not activate the GPR109A receptor and does not cause flushing. We utilized a validated protocol of sample collection in 4% trichloroacetic acid to ensure preanalytical stability. We also used validated liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays for the measurement of NMN, NAD, and NAD metabolome.

Method

This single-center, randomized, double-blind, placebo-controlled, multiple ascending dose study was approved by the Institutional Review Board of Massachusetts General Brigham Healthcare System. All participants provided written informed consent.

Study Participants

Participants were community-dwelling, overweight or obese (body mass index [BMI] 28–40 kg/m²), middle-aged and older men, and postmenopausal women, 55–80 years, without significant medical problems. We excluded participants using a supplement containing nicotinamide, niacin, NMN, or NR; smokers; or those with aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >1.5 times the upper limit of normal, hematocrit <37%, creatinine >1.5 mg/dL, QTc >450 ms; or diabetes mellitus.

Intervention and Comparator

Participants were given the specified dose of the MIB-626 tablets each containing 500 mg of microcrystalline NMN or matching placebo tablets, ingesting either two 500 mg MIB-626 or 2 placebo tablets once daily or twice daily for 14 days. The dose selection was guided by PK data from our single ascending dose study that revealed that oral 250, 500, and 750 mg of MIB-626 did not consistently raise NAD levels. Participants were followed up for 28 days after the last dose.

Participant Allocation and Dose Escalation

Thirty-two eligible participants, 16 in each of the 2 cohorts (8 men and 8 women) were randomized in a 3:1 ratio, stratified by sex, using concealed block randomization with a block size of 4 to receive either the specified regimen of MIB-626 or placebo for 14 days. The first cohort received either 1 000-mg MIB-626 or placebo once daily. Dose escalation was sequential and proceeded based on predefined dose-limiting toxicity (DLT) and stopping criteria. DLT dose was defined as the dose at which no more than 2 MIB-626-treated participants within a given dose cohort exhibited grade 2 or worse AE caused by the investigational product, >60 ms prolongation of QTc from baseline, or an absolute QTc >500 ms. Enrollment to 1 000 mg twice-daily cohort commenced after the Safety Review Committee had reviewed the safety data at the lower dose and approved dose escalation to 1 000 mg twice daily.

Study Procedures

On Days 1 and 14, baseline blood samples were drawn after an overnight fast for NMN, NAD, and NAD metabolome predose; study product was administered at time 0; and blood was drawn for blood counts and chemistries at 4 hours, and for NMN, NAD, and NAD metabolome at 2, 4, 6, and 24 hours after administration of study product. A 4-hour urine collection was made.

Blood samples were collected for PK assessment of NMN levels and PD assessment (NAD and NAD metabolome including nicotinamide [NAM], 1-methyl nicotinamide [1-methyl NAM], and N-methyl-2-pyridone-5-carboxamide [2-PY]-NAM, and NR in plasma) predose (−15 minutes) and at 0, 2, 4, and 24 hours on Day 1; 0 and 1 hour on Day 8; and predose (−15 minutes) and at 1, 2, 4, 6, and 24 hours on Day 14. Urine samples were collected for NMN, and major metabolites NAM and 2-PY-NAM on Days 1 and Day 14 at 0 and 24 hours. The participants returned on Day 8 for blood counts and chemistries and for measurement of NMN, NAD, and NAD metabolome.

Measurement of NMN, NAD, and NAD Metabolome in Blood and Urine

We measured NAD and NAD metabolome in the whole blood versus peripheral blood mononuclear cells (PBMCs) to avoid issues such as accelerated metabolite degradation by lysed PBMCs and variability in normalization of PBMC abundance in whole blood. NAD levels in whole blood are a good marker of NAD and its metabolome in PBMCs (22). To ensure preanalytical stability of the analytes, whole blood was collected in 4% trichloroacetic acid in a ratio of 1–6 v/v blood to TCA and centrifuged, and the supernatant was stored at −80°C.

Detailed methods for measurements of NMN, NAD, and NAD metabolome in blood and urine are given in Supplementary Appendix. Briefly, blood NMN and NAD concentrations were measured in a GLP laboratory using validated LC-MS/MS methods. Linear range for NMN was 0.2–20.0 μg/mL and for NAD 5.0–500.0 μg/mL. Interassay coefficients of variation for NMN assay were 6.0%, 3.1%, and 3.0% in low (0.36 μg/mL), medium (7.4 μg/mL), and high (14 μg/mL) quality control pools, respectively, and for the NAD assay 8.7%, 6.7%, and 4.6% at concentrations of 19.2, 197, and 392 μg/mL, respectively.

PK and PD Analyses

The PK and PD analyses were performed using noncompartmental methods with Phoenix Win-Nonlin software version 8.0 or Statistical Analysis Software (SAS Institute Inc., Cary, NC; version 9.4). One participant was excluded from the parameter calculation for NMN due to insufficient data. For each participant, PK parameters of C_max, T_max, and AUC_last were calculated, based on baseline-corrected blood concentrations of each analyte. Terminal elimination half-life t_1/2 could not be calculated as terminal phase of concentration versus time profile did not have 3 or more timepoints excluding C_max. The relation between dose and PK parameters (C_max [maximum concentration], T_max [time at which C_max was achieved], AUC_last [baseline-corrected area under concentration curve on Day 14]) was evaluated using a Kruskal–Wallis nonparametric test. Relation between NMN PK parameters and dose was assessed using analysis of variance. Geometric least squares mean ratio estimates and 90% confidence intervals (CIs) extracted from this model were employed to assess the magnitude of relation. Association between PK parameters and sex, age, and BMI was analyzed using linear regression models. Relations between intervention arms and change in safety outcomes were analyzed using mixed-effects regression with dose level, visit, and visit-by-arm interaction included in the model. Unstructured covariance matrix was assumed; however, if the model did not converge, a compound symmetry structure was utilized. Outcomes were expressed as changes from baseline and analyses were adjusted for baseline value. Point estimates and 95% CIs were employed to assess the magnitude of association.

Results

Ninety-one participants were screened for eligibility, 32 (16 men and 16 women) met eligibility criteria and were randomized; 8 to the placebo group, 12–1 000 mg once-daily group, and 12 to 1 000 mg twice-daily group (CONSORT diagram, Supplementary Figure 1). One participant was discontinued due to an adverse event. Thirty-one participants completed the study and were included in the analyses.

Baseline Characteristics of the Participants

Baseline characteristics of participants in the study arms were similar (Supplementary Table 1). Participants included 16 men and 16 women (n = 16) with mean ± SD age 63.9 ± 6.1 years, body weight 82.0 ± 9.3 kg, and BMI 29.1 ± 2.9 kg*m⁻².

Adverse Events and Safety Laboratory Data

There were no serious adverse events, and frequency of adverse events was similar across groups (Supplementary Table 2). One participant in 1 000 mg twice-daily dose was discontinued between Days 8 and 14 due to diarrhea. Changes from baseline in clinical laboratory analytes in MIB-626-treated groups did not differ from placebo (Supplementary Figure 2). Two participants—one randomized to the 1 000 mg daily dose and one randomized to placebo—experienced mild AST and ALT elevations on Day 14; AST and ALT levels in both participants returned toward baseline after discontinuation of study product. There was no clinically significant change from baseline in vital signs or QTc interval in any group.

Circulating NMN Concentrations

The 1 000 mg twice-daily regimen was associated with a significantly greater increase in NMN levels compared to the 1 000 mg once-daily regimen (geometric mean ratios [90% CI] were 1.98 [1.12, 3.48] and 3.32 [1.14, 9.67] for C_max and AUC_last, respectively; Figure 1; Table 1). There was a statistically significant difference between MIB-626-treated groups and placebo in blood NMN concentrations on Day 14 (p = .007 and p = .037 for C_max and AUC_last, respectively; Table 1). About 1 000 mg once-daily regimen was associated with an average 2.7- and 1.7-times increase above baseline and twice-daily treatment resulted in 4.5- and 3.7-times increase in mean C_max and AUC_last, respectively, compared to placebo (Figure 1 and Table 1). NMN C_max and AUC_last were higher in participants treated with 1 000 mg twice-daily regimen than in those treated with once-daily regimen. No significant sex differences in blood NMN exposures (C_max and AUC_last) were observed across treatment groups. The NMN AUC_last values were not significantly associated with BMI and age.

Table 1.

Pharmacokinetics Parameters for NMN, NAD, and Metabolites

Variable	Parameter (unit)	Placebo (N = 8)	MIB-626—1 000 mg Once Daily (N = 12)	MIB-626—1 000 mg Twice Daily (N = 12)	p
NMN	Cmax (µg/mL)	0.0326 (128)	0.0882 (53.9)*	0.148 (59.2)*	.0066
	Tmax (h)*	0.40 (0.00, 23.78)	4.00 (0.00, 24.00)*	1.07 (0.00, 4.00)*	.0643
	AUClast (h*µg/mL)	0.663 (84.2)‡	1.12 (85.8)*	2.48 (70.7)†	.0366
NAD	Cmax (µg/mL)	1.36 (74.9)	23.0 (35.1)*	40.4 (40.1)*	<.0001
	Tmax (h)*	2.05 (0.00, 23.92)	5.88 (0.00, 24.40)*	2.00 (0.00, 24.23)*	.7029
	AUClast (h*µg/mL)	14.1 (124.9)	459 (31.0)*	867 (43.6)*	<.0001
NAM	Cmax (ng/mL)	10.2 (123)	65.2 (55.5)*	140 (80.2)*	.0002
	Tmax (h)*	0.98 (0.00, 6.02)	2.00 (1.00, 5.98)*	4.07 (0.00, 24.37)*	.0436
	AUClast (h*ng/mL)	63.7 (133)§	787 (59.5)*	1 810 (98.8)*	.0004
1-Methyl NAM	Cmax (ng/mL)	7.57 (88.4)	146 (41.8)*	276 (38.7)*	<.0001
	Tmax (h)*	2.00 (0.00, 5.87)	5.87 (3.92, 6.03)*	4.23 (2.00, 24.37)*	.0031
	AUClast (h*ng/mL)	54.8 (205)‖	2 300 (40.1)*	4 430 (31.5)*	<.0001
2-PY-NAM	Cmax (ng/mL)	103 (101.2)	2 150 (27.8)*	4 230 (32.2)	<.0001
	Tmax (h)*	0.48 (0.00, 23.97)	5.87 (3.92, 6.00)*	5.83 (2.00, 23.85)	.1148
	AUClast (h*ng/mL)	1 670 (103)‡	42 500 (33.5)*	82 300 (32.1)	<.0001
NR	Cmax (ng/mL)	0.406 (104)	1.30 (44.3)*	1.48 (96.6)*	.0083
	Tmax (h)*	0.00 (0.00, 23.97)	2.10 (0.00, 5.98)*	5.80 (0.00, 24.12)*	.0482
	AUClast (h*ng/mL)	3.59 (97.5)‡	10.5 (59.8)*	18.6 (112)*	.0504

Notes: NMN = nicotinamide mononucleotide; NAD = nicotinamide adenine dinucleotide; NAM = nicotinamide; 1-methyl NAM = 1-methylnicotinamide; NR = nicotinamide riboside; 2-PY = N-methyl-2-pyridone-5-carboxamide. Mean (coefficient of variation) presented for C_max and AUC_last. Median (min, max) presented for T_max. p values for Kruskal–Wallis test; parameters are calculated using baseline-corrected concentrations, where baseline is −15 minutes sample on Day 1.

*n = 11.

^†n = 10.

^‡n = 5.

^§n = 6.

^‖n = 7.

Figure 1.

Circulating concentrations of NAD and NMN, and NAD metabolome on Days 1 and 14 by treatment arms. A, B: Mean circulating concentrations of NAD (A) and NMN (B) on Days 1 and 14 with the corresponding 95% confidence intervals are shown. C–F: Circulating NAD metabolome (2-PY, NAM, 1-methyl nicotinamide, and NR) on Days 1 and 14 by treatment arms. Mean circulating concentrations of 2-PY, NAM, 1-methyl nicotinamide, and NR on Days 1 and 14 with corresponding 95% confidence intervals are shown. NMN = nicotinamide mononucleotide; NAD = nicotinamide adenine dinucleotide; NAM = nicotinamide; 1-methyl NAM = 1-methyl nicotinamide; NR = nicotinamide riboside; 2-PY = N-methyl-2-pyridone-5-carboxamide.

NAD Levels

MIB-626 treatment was associated with a dose-related increase in blood NAD levels from baseline to Day 14 (Figure 1). On Day 1, blood NAD levels showed only a modest increase from baseline to 24 hours in NMN-treated participants. NAD levels were substantially higher on Day 14 than at baseline or Day 1 in participants treated with 1 000 mg once-daily and 1 000 mg twice-daily regimens compared with the placebo group. NAD levels on Day 8 were higher than on Day 1 and were similar to the levels on Day 14 in both NMN dose groups. NAD C_max and AUC_last on Day 14 were significantly different between the 2 NMN-treated and placebo-treated groups (p < .001 and p < .001, respectively; Table 1).

Increases in NAD levels from baseline to Day 14 were related to NMN dose and increase in NMN levels from baseline to Day 14 (p < .001, R² = 0.57). The AUC_last NAD values were not significantly associated with sex, BMI, and age (all p > .05).

Circulating Levels of NAD Metabolome

Among the circulating metabolites of NAD, 2-PY was the most abundant (Figure 1). Circulating concentrations of all 4 metabolites (2-PY, NAM, 1-methyl NAM, and NR) increased from baseline to 24 hours on Day 1. Metabolite concentrations were higher on Days 8 and 14 than on Day 1 and baseline in both MIB-626-treated groups, but did not differ on Days 8 and 14. C_max and AUC_last for the 4 metabolites were not significantly associated with sex, BMI, or age.

Urinary Metabolites

Urinary NMN concentrations standardized to creatinine levels were similar among treatment groups with no apparent sex differences; thus, very little oral NMN is eliminated unchanged in the urine (Table 2). On Days 8 and 14, mean urinary NAM and 2-PY-NAM concentrations were higher in NMN-treated groups compared to the placebo group.

Table 2.

Urinary Concentrations of Major NAD Metabolites Standardized to Urinary Creatinine by Treatment Arm

Day	Scheduled Time (h)	Placebo	MIB-626—1 000 mg Once Daily	MIB-626—1 000 mg Twice Daily
	NMN	(N = 8)	(N = 12)	(N = 12)
1	0	333 (59.1)	357 (51.1)	232 (71.9)
	24	262 (44.7)	380 (35.5)	367 (74.3)
8	0	290 (42.8)	343 (61.9)	290 (36.6)*
14	0	299 (57.6)	396 (59.7)*	301 (55.7)*
	24	333 (47.2)†	384 (62.5)	308 (41.6)*
	Nicotinamide	(N = 8)	(N = 12)	(N = 12)
1	0	132 (48.9)	221 (65.9)	242 (42.5)
	24	126 (50.0)	248 (50.8)	576 (65.4)
8	0	155 (26.6)	321 (61.1)	690 (48.5)*
14	0	152 (45.8)	390 (46.9)*	543 (61.7)*
	24	174 (56.8)†	290 (49.3)	703 (43.8)*
	2-PY	(N = 8)	(N = 12)	(N = 12)
1	0	18 500 (71.6)	16 300 (39.1)	15 400 (30.0)
	24	14 200 (41.8)	103 000 (32.8)	183 000 (49.4)
8	0	14 900 (44.9)	114 000 (28.0)	264 000 (43.9)*
14	0	17 800 (61.0)	131 000 (43.2)*	255 000 (54.6)*
	24	16 700 (64.2)†	137 000 (37.9)	273 000 (40.0)*

Notes: NMN = nicotinamide mononucleotide; NAD = nicotinamide adenine dinucleotide; 2-PY = N-methyl-2-pyridone-5-carboxamide. Values are expressed as ng/mg creatinine. Mean (coefficient of variation) presented by time point.

*n = 11.

^†n = 7.

Metabolic Markers

Change in fasting glucose levels did not differ among groups at any visit (all p > .05; Supplementary Table 3). Serum total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, uric acid, and free fatty acids did not differ significantly among groups at any time point (all p > .05).

Discussion

Oral administration of 1 000-mg MIB-626 once daily or 1 000-mg twice daily was safe and well tolerated and associated with substantial dose-related augmentation of blood NAD levels. Increases in blood NAD concentrations were associated with substantial increases in the circulating levels of its major metabolites 2-PY, NAM, and 1-methyl nicotinamide, indicating that both dose regimens were effective in augmenting NAD and its metabolome. As doses less than 1 000 mg in a phase 1a study did not consistently raise NAD levels in healthy volunteers, subsequent efficacy trials should use regimens of 1 000-mg MIB-626 daily or higher.

These findings are important in the context of the resveratrol experience. In spite of promising data in preclinical studies (23), clinical trials of resveratrol failed to find evidence of efficacy in any disease state. Subsequent PK studies found only trace amounts of unmodified resveratrol in plasma (24) and no human study provided evidence of an increase in NAD levels. Therefore, carefully performed PK studies are necessary before proceeding to efficacy trials, and it is important to demonstrate that the administered dose raises the blood levels of the key PD target NAD.

The study also provided important information about the time course of NAD increase and systemic metabolism of NMN. NMN and NAD concentrations increased gradually after administration of the first dose. Blood NMN and NAD levels were similar on Days 8 and 14 and the NMN and NAD concentrations did not vary during the 24-hour period on Day 14. Very little NMN was excreted in an unchanged form in the urine and 2-PY was its most abundant circulating and urinary metabolite. We did not find a major effect of sex, BMI, and age although the age and BMI range of participants was narrow and sample size was small.

The drug was safe and well tolerated. There were no serious adverse events and mild adverse events were similar among groups. One participant assigned to 1 000 mg daily dose and one assigned to placebo showed mild AST and ALT elevation that returned to normal.

Short-term treatment with MIB-626 did not significantly affect metabolic variables such as serum glucose, insulin, HOMA-IR, lipids, and uric acid. The trial was neither large enough nor long enough to detect differences in glucose metabolism and insulin sensitivity. These data are consistent with other reports (15,17,18) that also have not found significant effects of NMN or NR on glucose, insulin, lipids, and uric acid during short-term treatment. A randomized trial of 250-mg MIB-626 once daily for 10 weeks did not find significant changes in glucose and insulin levels but reported improvements in insulin-stimulated glucose disposal and muscle insulin signaling (15). Other studies have shown improvements in inflammation markers (21). Some short-term trials have reported promising results in preventing acute kidney injury in patients undergoing cardiac surgery (25) and in patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (26). Uncontrolled trials have reported attenuation of disease severity in SARS-CoV-2 infection by a cocktail of molecules that included NR (27).

These findings should be viewed in the context of trial’s strengths and some limitations. We used a pharmaceutical-grade formulation of microcrystalline βNMN manufactured by a cGMP process. Blood samples were collected in 4% TCA to ensure preanalytical stability. NMN, NAD, and NAD metabolites were measured using validated LC-MS/MS assays. The PK data revealed robust dose proportionality of increments in NAD levels with NMN dose and changes in NMN levels. This early-phase trial was neither designed nor powered for evaluation of efficacy outcomes.

Conclusion

Oral administration of 1 000-mg MIB-626 once daily or twice daily was safe and associated with dose-related increases in blood NAD levels and its metabolome. These PK and PD data obtained using a pharmaceutical-grade formulation of microcrystalline βNMN, standardized procedures for sample collection, and validated LC-MS/MS assays for NMN and NAD should facilitate the design of randomized trials to determine the efficacy of NAD augmentation in disease conditions.

Funding

This project was funded by a research grant from Metro International Biotech.

Conflict of Interest

S.B. reports receiving research grants from National Institute on Aging, National Institute of Nursing Research, National Institute of Child Health and Human Development - National Center for Medical Rehabilitation Research, Alivegen, AbbVie, and Metro International Biotech and consultation fees from OPKO. These grants are managed by the Brigham and Women’s Hospital. S.L. and D.L. are employees of Metro International Biotech.

Author Contributions

The decision to publish and the selection of the journal was made by the corresponding author, who served as the Principal Investigator of this project. S.L., a consultant to Metro International Biotech, assisted in the pharmacokinetic analyses. D.L., an employee of Metro International Biotech, contributed to the development of the bio-analytical methods for the NAD metabolome, wrote the sections related to the composition of the formulation, and participated in the review of the manuscript.