Absorption, Distribution and Excretion
Six male and six female Sprague-Dawley Crl:CDBR rats were administered a single dose of 15 mg/kg body weight via gavage or intravenous injection. The rats were housed individually in metabolic cages, and urine and feces were collected intermittently over 72 hours post-administration. Three additional rats were given a single oral dose of 120 mg/kg body weight of 14 C-neosweet. All rats were sacrificed after 72 hours, and their carcasses were preserved for analysis. Radiolabeling was measured in all samples, and metabolites in urine and feces were also measured. Within 48 hours following oral administration, over 90% of the radiolabeled material was recovered in urine and feces. Within 72 hours following oral administration of (14)C-neostatin at doses of 15 or 120 mg/kg body weight, 8.5–10.8% and 84.5–87.2% of the radiolabeled material, respectively, were excreted in urine and feces. Following intravenous administration of (14)C-neostatin at doses of 15 mg/kg body weight, approximately 35% and 59% of the radiolabeled material, respectively, were excreted in urine and feces. Less than 0.3% of the radiolabeled material was recovered from animal carcasses within 72 hours following oral or intravenous administration. Unconverted neostatin was detected only in the urine of female rats 0–6 hours after intravenous administration, representing 3.7% of the administered dose. No altered neostatin was detected in the feces of any animal, regardless of dose or route of administration. Sprague-Dawley Crl:CDBRVAF Plus rats were administered a single oral gavage of 15 mg/kg body weight of (14)C-neostatin and randomly assigned to four groups. In the first group of rats (three males and three females), blood was collected at intervals within 24 hours after administration, and cellular and plasma components were separated and analyzed for radiolabeling. In the second group of rats (two males and two females), urine, feces, and exhaled gases were collected in glass metabolic cages for 72 hours after administration. The cadavers were dissolved to analyze residual radiolabeling, and urine and feces were mixed to analyze metabolites and total radiolabeling. In the third group (two males and two females), blood was collected for analysis after anesthesia at 0.5 hours or 2 hours after administration. In the fourth group (two male rats), bile ducts and gastric tubes were inserted after anesthesia. Radiolabeled neotame was administered through the gastric tube, and bile was collected at intervals within 48 hours after administration. Urine and feces were collected from 0-24 hours and 24-48 hours after administration, and radiolabeling was measured. After oral administration of (14)C-neotame, the concentration of radiolabeled substances in the plasma of female rats peaked at 30 minutes after administration, and in male rats at 1 hour after administration, and then rapidly declined. The major metabolite identified in plasma, urine, feces, and bile was deesterified neotame. Excretion of (14)C-neotame was monitored over 72 hours. 8–10% of the radiolabeled substance was recovered from urine, 90–92% from feces, and 0.01–0.03% from exhaled breath. After 72 hours, 0.11–0.13% of the radiolabeled substance remained in the carcass. Urinary excretion in males was almost complete within 12 hours, while in females it continued for more than 24 hours. Most fecal excretion occurred between 6 and 24 hours after administration. Urinary excretion in males of Group 4 was similar to that in other groups, with approximately 5–9% of the administered dose excreted in urine. Bile excretion accounted for approximately 5.7% of the administered dose, while fecal excretion accounted for approximately 85%. Very little radiolabeled substance remained in the carcass. In a study investigating the distribution and elimination of neotame radioactive material using whole-body autoradiography, eight pregnant and eight non-pregnant Sprague-Dawley rats were administered a single dose of 15 mg/kg body weight of 14C-neotame via gavage. Rats were sacrificed at different time points within 24 hours of administration and their cadavers were disposed of according to previous study methods. The tissue distribution of the radiolabeled material was similar in both pregnant and non-pregnant rats. At 0.5 and 2 hours post-administration, the concentration of the radiolabeled material in the placenta was low, similar to that in other peripheral tissues and circulating blood. No radiolabeled material was detected in the fetus at any time point. Shortly after administration, the concentration of the radiolabeled material reached its peak, initially present in the gastric contents, gastrointestinal tract, liver, kidneys, and bladder, while concentrations were lower in other parts of the body. At subsequent time points, the radiolabeled material was observed to be excreted through excretory organs. No accumulation of the radiolabeled material in tissues was observed, and concentrations were extremely low after 24 hours. There was no significant difference in the tissue distribution time curves between pregnant and non-pregnant rats. In a study investigating the distribution of neotame in rat tissues, 21 male Lister-Hooded rats were administered a single oral dose of 15 mg/kg body weight of 14C-neotame via gavage. One male and one female rat were sacrificed at 0.5, 2, 6, 12, and 24 hours post-administration. The rats were fixed, rapidly frozen, and the cadavers were sectioned in six sagittal planes for analysis using autoradiography. Qualitative assessment of radiomarkers in male and female rats showed that the highest levels of radiomarkers were found in rats sacrificed at the earliest post-administration time. Radiomarker levels decreased rapidly over time. At 0.5 and 2 hours post-administration, most radiomarkers were present in the stomach, digestive tract, liver, kidneys, and bladder, with smaller amounts distributed in other parts of the body. Radiomarker levels in the central nervous system were extremely low, and no binding to pigmented skin or eyes was observed. These levels are consistent with the circulation of radiomarkers in the blood. At subsequent time points (6, 12, and 24 hours), radiomarkers were observed excreting through excretory organs. Twenty-four hours after administration, only trace amounts remained in the animals, and no signs of tissue accumulation were observed. For more complete data on the absorption, distribution, and excretion of neotame (11 species in total), please visit the HSDB record page. Metabolites/Metabolites…Neotame is carbon-14 labeled at position 1 of the dimethyl butyl side chain and carbon-13 labeled on both terminal methyl groups of the same side chain. …Volunteers ingested a single dose of the labeled test substance in water at a dose of approximately 0.25 mg/kg, equivalent to the amount of neotame required for 1 liter of beverage. Neotame is rapidly absorbed but not completely, and is rapidly excreted. An average of 98% of the administered radioactive material is recovered in urine and feces, with the majority excreted within 72 hours after administration. The average plasma concentration of neotame peaks at 0.4 hours and declines with a half-life of 0.6 hours. The major metabolite of neotame is desesterified neotame, formed by the hydrolysis of the methyl ester group. The mean plasma concentration of this metabolite peaked at 1 hour, approximately 2.5 times that of neotame, and decreased with a half-life of 1.5 hours. Deesterified neotame accounted for approximately 80% of the excreted dose. Two other metabolites were present at concentrations exceeding 1% of the dose. One metabolite (approximately 4.9% of the dose) was present in feces and identified as N-(3,3-dimethylbutyl)-L-aspartic acid. The other metabolite was present in urine and identified by LC/MS/MS, NMR, and the original synthesis as a carnitine ester of 3,3-dimethylbutyric acid. The presence of all neotame metabolites at concentrations of 1% or higher of the dose was confirmed in the animal models used in the safety studies, thus confirming the safety of these metabolites. As part of the safety testing, we conducted studies to evaluate the absorption, distribution, pharmacokinetics, metabolism, and excretion of neotame in experimental rats and dogs. For this purpose, we labeled the neotame dimethylbutyl side chain with carbon-14 at position 1 and administered it to animals at doses of 15 or 120 mg/kg body weight. In rats and dogs, neotame is rapidly but incompletely absorbed after oral administration and is rapidly excreted without any signs of accumulation. In rats, the absorbed carbon-14 is primarily distributed in the gastrointestinal tract and metabolic and excretory organs (liver, kidneys, and bladder). Neotame is almost undetectable in (stable) plasma or…/excrement after oral administration. This is likely due to higher plasma esterase activity. The major metabolite of neotame is desesterified neotame, formed by the hydrolysis of the methyl ester group. In rats, the mean plasma concentration of this metabolite peaks at 0.5 hours and decreases with a half-life of 1 hour. In dogs with lower plasma esterase activity, neotame was detected in both plasma and excrement after oral administration. The mean plasma concentration of neotame peaks at 0.5 hours and decreases with a half-life of 0.4 hours. In rats and dogs, desesterified neotame accounts for approximately 70-80% of the excreted dose after oral administration. Other metabolites detected included N-(3,3-dimethylbutyl)-L-aspartic acid (approximately 2% of the administered dose in rats and dogs) and a β-glucuronide conjugate of 3,3-dimethylbutyric acid (approximately 5% of the administered dose in rats and dogs). Additionally, carnitine esters of 3,3-dimethylbutyric acid were detected in the urine of female rats. Following oral administration, approximately 20-30% of the administered dose is absorbed and rapidly converted to the major metabolite N-(N-(3,3-dimethylbutyl)-L-α-aspartic acid)-L-phenylalanine (deesterified neotame) and several minor metabolites. Neotame and its metabolites are rapidly excreted in urine and feces. …The primary metabolic pathway is the deesterification of neotame to N-[N-(3,3-dimethylbutyl)-L-α-aspartic acid]-L-phenylalanine and methanol. Secondary metabolites include N-(3,3-dimethylbutyl)-L-aspartic acid (generated from neotame via peptide or amide hydrolysis); 3,3-dimethylbutyric acid (also known as 3,3-dimethylbutanoic acid); carnitine conjugates of 3,3-dimethylbutyric acid; and glucuronide conjugates of 3,3-dimethylbutyric acid. Six male and six female Sprague-Dawley Crl:CDBR rats were administered a single dose of 15 mg/kg body weight via gavage or intravenous injection to each group. Rats were housed individually in metabolic cages, and urine and feces were collected periodically within 72 hours post-administration. Three additional rats were given a single oral dose of 120 mg/kg body weight. All rats were sacrificed after 72 hours, and their carcasses were preserved for analysis. Radiolabeled substances were measured in all samples, and metabolites in urine and feces were also measured. …48 hours later, the major metabolite found in urine was deesterified neotame, regardless of route of administration or dose. Following oral administration, low concentrations of N-(3,3-dimethylbutyl)-L-aspartic acid (NC-00754) were detected (approximately 10% of the level of deesterified neotame). The parent compound was found only in the urine of female rats following intravenous administration (3.7% of the administered dose); it was not detected in the urine of other groups. In addition, low concentrations of glucuronide metabolites were detected in urine (0.4–0.5% of the administered dose), regardless of dose or route of administration. Two minor metabolites were identified, each at levels below 1.6% of the administered dose. In feces, deesterified neotame was the major metabolite (approximately 70–80% of the dose after oral administration). Low levels of N-(3,3-dimethylbutyl)-aspartic acid (NC-00754) were detected, at 0.8–2.5% of the administered dose. The study also found low concentrations of unidentified metabolites, accounting for 0.7–1.2% of the administered dose. For more complete metabolite/metabolite data on neotame (9 metabolites in total), please visit the HSDB record page.

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Biological Half-Life
Healthy men (mean age ± standard deviation, 28 ± 6 years) were given a single dose of 0.10, 0.25, or 0.50 mg/kg body weight of neotame solution after an overnight fast (7, 6, and 6 men per dose group, respectively). A total of 18 men completed the study. Clinical evaluation and laboratory testing were performed immediately before and approximately 48 hours after administration. …Neotame is rapidly eliminated, with a half-life of 0.61 to 0.75 hours. The rapid disappearance of neotame in urine (undetectable after 8 hours) confirms its short half-life. …The plasma half-life of deesterified neotame was calculated to be approximately 2 hours.

[1]. Effects of the Artificial Sweetener Neotame on the Gut Microbiome and Fecal Metabolites in Mice. Molecules. 2018 Feb 9;23(2):367.

[2]. Stability of Aspartame and Neotame in Pasteurized and In-Bottle Sterilized Flavoured Milk. Food Chem. 2016 Apr 1;196:533-8.

Neotame is a dipeptide composed of N-(3,3-dimethylbutyl)-L-aspartic acid and L-phenylpropionate methyl ester units linked by peptide bonds. It is both an environmental pollutant and an exogenous substance and sweetener. Mechanism of Action The sweet taste receptor is a heterodimer composed of two G protein-coupled receptors, T1R2 and T1R3. Previous experimental studies using chimeric and mutant sweet taste receptors have shown that at least three potential binding sites exist in this heterodimer receptor. The receptor's activity against the artificial sweeteners aspartame and neotame depends on residues in the N-terminal domain of human T1R2. Conversely, the receptor's activity against the sweetener sodium cyclohexylsulfamate and the sweetness inhibitor lactitol depends on residues in the transmembrane domain of human T1R3. Furthermore, the receptor activity against the sweet taste protein brazzein depends on the cysteine-rich domain of human T1R3.
The sweet taste protein brazzein (a recombinant protein with the same sequence as the natural protein but lacking the N-terminal pyroglutamic acid residue (Asp2 is the N-terminal residue in the numbering system)) activates the human sweet taste receptor, a heterodimeric G protein-coupled receptor composed of taste receptor type 1 2 (T1R2) and taste receptor type 1 3 (T1R3) subunits. To elucidate the key amino acids responsible for this interaction, we mutated residues in brazzein and its two receptor subunits. The effects of brazzein mutations were examined in a human taste assay panel and in vitro experiments (involving the recombinantly expressed receptor subunits in human embryonic kidney cells); the effects of receptor mutations were examined in vitro. We mutated residues at three putative interaction sites on the surface of brazzein: site 1 (Loop43), site 2 (N-terminus and C-terminus, as well as adjacent Glu36, Loop33), and site 3 (Loop9-19). The basic residues at site 1 and the acidic residues at site 2 were crucial for positive responses in each assay. The Y39A mutation (site 1) significantly reduced the positive response. The large side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, and the presence of the native disulfide bond in Loop9-19 (site 3) were also necessary for a positive response. Results from receptor mutagenesis and chimeras indicated that brazzein interacts with both T1R2 and T1R3, and that the Venus flytrap module of T1R2 is crucial for the agonistic effect of brazzein. With one exception, mutations in all receptor residues at the hypothetical interaction sites predicted by the wedge model failed to lead to the expected reduction in glycosidase activity. The exception was hT1R2 (human sweet taste receptor T1R2 subunit): R217A/hT1R3 (human sweet taste receptor T1R3 subunit), which exhibited a slight selective reduction in glycosidase activity due to an amino acid substitution in the 2-leaf region at the junction of the two subunits. However, because this mutation enhances the positive cooperativity of binding to several ligands thought to bind to both T1R subunits (glycosidase, mononitrate, and sucralose), but has no effect on ligands that bind to a single subunit (neotame and sodium cyclohexylsulfamate), we believe that this site is involved in inter-subunit interactions rather than directly binding to glycosidase. Our results support the existence of multi-point interactions between glycosidases and sweet receptors, and that the mechanism is not based on the proposed wedge model.

C20H30N2O5

378.4626

378.215

165450-17-9

(R)-Neotame-d3

9810996

White to off-white solid powder

1.1±0.1 g/cm3

535.8±60.0 °C at 760 mmHg

80.9-83.4ºC

277.9±32.9 °C

0.0±1.5 mmHg at 25°C

1.530

4.73

3

6

12

27

495

2

CC(C)(C)CCN[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)OC

HLIAVLHNDJUHFG-HOTGVXAUSA-N

InChI=1S/C20H30N2O5/c1-20(2,3)10-11-21-15(13-17(23)24)18(25)22-16(19(26)27-4)12-14-8-6-5-7-9-14/h5-9,15-16,21H,10-13H2,1-4H3,(H,22,25)(H,23,24)/t15-,16-/m0/s1

(3S)-3-(3,3-dimethylbutylamino)-4-[[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino]-4-oxobutanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~264.23 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.61 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.61 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.61 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)