Materials and methods
The batch of S807 used for the in vitro/in vivo metabolism and pharmacokinetic studies, (Batch no 061001T01L, purity 99.9%), was synthesized at Derivados Quimicos Fine Chemicals,
The studies conducted in Murcia, Spain, utilized the procedures outlined in US Patent Nos 7,476,399 B2, 8,8124121 B2, and 8,895050 B2 (Tachdjian et al., 2009, 2012, 2014a) The S807 batch was employed for in vitro and in vivo genotoxicity assessments, as well as for 21-day range-finding toxicity and 90-day subchronic toxicity studies (Lot no [insert lot number]).
The compound 4KL0071A, with a purity greater than 99.95%, was synthesized at Albany Molecular Research, Inc in Albany, NY, utilizing a consistent synthetic method This specific batch, identified as S9229, was employed in various studies, including in vitro and in vivo metabolism, pharmacokinetics, genotoxicity assessments, and 28-day toxicity evaluations.
The compound with ID 50764226, exhibiting a chemical purity greater than 98.8% and an optical purity exceeding 99.8%, was synthesized at Senomyx in San Diego, CA This synthesis followed the procedures outlined in the relevant US Patents for S807 The specific batch of S9229 utilized for the in vitro metabolism study conducted by Senomyx is identified by its Batch ID.
58490390, chemical purity >97%) was also synthesized at Senomyx using the same procedure
All genetic toxicology studies were conducted in compliance with the FDA Good Laboratory Practices (GLP) regulations 21 CFR Part 58 (FDA, 2006) and OECD guidelines (OECD, 1998)
The experimental design adhered to the OECD Guidelines for the Testing of Chemicals - 471, 473, and 474 (OECD, 1997abc) Additionally, the 28-day and 90-day toxicology studies in rats were conducted in accordance with the United States Food and Drug Administration (FDA) Guidelines (FDA, 2010) on Toxicological Principles for the Safety of Food Ingredients and the FDA Good Laboratory Practice (GLP) Regulations, 21 CFR Part 58.
PharmOptima in Portage, MI conducted in vitro microsomal metabolism studies on S9229 using male and female rat liver microsomes (Lot no 1010122 and 0710104) as well as mixed gender human microsomes.
In vitro microsomal metabolism studies, along with pharmacokinetic and in vivo metabolism studies for compounds S807 and S9229, were performed at Senomyx in San Diego, CA These studies utilized male and female rat liver microsomes (Lot no 1410271 and 1310205) and mixed gender human microsomes The initial compound was obtained from XenoTech in Lenexa, KS.
The analytical methods for in vitro metabolism, pharmacokinetics, and in vivo metabolism studies, sourced from XenoTech in Lenexa, KS, are detailed in the Supplementary Data section available online.
Genotoxicity studies for S807 were performed both in vitro and in vivo at Nucro-Technics in Scarborough, Ontario, Canada The reverse bacterial mutation assay utilized strains of S typhimurium and E coli, along with rat liver S9 supernatant obtained from Sprague-Dawley rats treated with Aroclor™ 1254, sourced from Molecular Toxicology Inc Additionally, the in vitro chromosome aberration test employed the Chinese hamster ovary cell line WBL from the University of Guelph, while rat liver S9 for this test was derived from Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone, also provided by Molecular Toxicology Inc.
Genotoxicity studies for S9229 were performed both in vitro and in vivo at BioReliance in Rockville, MD The S typhimurium tester strains originated from Dr Bruce Ames’ Master cultures, while the E coli strains were sourced from the National Collection of Industrial and Marine Bacteria in Aberdeen, Scotland Tester strains TA100, TA1535, and TA1537 were acquired from Molecular Toxicology Inc in Boone, NC Peripheral blood lymphocytes for the preliminary toxicity test and chromosome aberration assay were collected from a healthy, non-smoking 31-year-old female donor with no recent history of radiotherapy, viral infections, or drug administration The rat liver S9 used in the study was derived from the supernatant fraction of liver homogenate from Sprague-Dawley rats treated with Aroclor™.
1254) used in the reverse bacterial mutation and chromosome aberration assays was obtained from Molecular Toxicology Inc., Boone, NC
The 21-day range-finder and 90-day subchronic toxicity studies on S807 were performed at Covance Laboratories Inc in Vienna, VA, while the 28-day toxicity study on S9229 took place at MPI Research in Mattawan, WI Detailed descriptions of the study designs are provided in the respective sections below, and comprehensive data tables for the genotoxicity, as well as the 21-day, 28-day, and 90-day toxicity studies on S807 and S9229, are available in the Supplementary Data section published online.
Results and study designs
Absorption, distribution, metabolism, excretion
The in vitro metabolism of S807 and S9229 was investigated using rat and human liver microsomes Additionally, pharmacokinetic studies and in vivo metabolism assessments of both compounds were conducted in male and female Sprague-Dawley rats.
3.1.1 In vitro metabolism of S807 and S9229 by rat and human liver microsomes
The study examined the oxidative metabolism of S807 and S9229 using Sprague-Dawley rat and human liver microsomes to compare metabolic profiles and evaluate the rat's suitability for toxicology research Two potential oxidative metabolites of S9229, formed by mono-hydroxylation of the 3,4-dimethylphenyl group, were synthesized along with their O-demethylated counterparts Additionally, a reference standard for the metabolite resulting from the demethylenation of the 1,3-benzodioxole moiety of S807 was also created.
Solutions of S807 (10 μM) or S9229 (10 μM) were incubated with pooled liver microsomes from both rat and human (0.5 mg/mL) in the presence of NADPH at 37 °C for varying durations of 10, 20, or 60 minutes The samples were then quenched with acetonitrile, while control samples included time zero and 60-minute incubates without NADPH.
Buspirone and loperamide were concurrently tested alongside the test compounds to validate the functionality of the microsomes Following centrifugation, the precipitated microsomes were separated from the supernatant, which contained the parent compound and its metabolites The supernatants underwent analysis using LC-QTOF/MS (Agilent iFunnel 6550A MS QTOF, positive mode) with an Agilent 1290 Infinity Binary pump and autosampler, utilizing a Waters CSH C18 column (50 x 2.1 mm, 1.7 μm) and a gradient system of 0.1% formic acid/water (v/v) and acetonitrile to assess the metabolism of S807 and S9229 Further details on the experimental and analytical methods are available in the Supplementary Data section.
Both S807 and S9229 were metabolized significantly faster by rat microsomes compared to human microsomes, with only 0.47% of S807 remaining in rats versus 65% in humans after 60 minutes Six potential Phase I metabolites were identified, with the major metabolite, M251A-1, accounting for approximately 34.4% in rats and 15.2% in humans of the initial S807 peak area The identity of M251A-1 was confirmed through LC-MS/MS comparison with a synthetic sample Additionally, minor metabolites resulting from mono-hydroxylation of the 4-heptamine side chain were observed, including M279A-1 and M279B-1, as well as M267A-1 and M267B-1 from the demethylenated metabolite M251A-1 The specific hydroxylation positions in these metabolites remain undetermined.
1) observed in the rat microsomal incubations was an olefin which results from the loss of water from the hydroxylated metabolite(s) M267A-1 and/or M267B-1 No dihydroxylated metabolites were observed in either the rat or human microsomal incubations
In the study of S9229, approximately 0.33% in rats and 22.6% in humans of the parent compound remained after 60 minutes of microsomal incubation Fourteen potential Phase I metabolites were identified, with significant transformations including hydroxylation of aryl methyl groups and the iso-butyl side chain, as well as demethylation of the side chain methyl ether The major metabolite, the C-4 hydroxymethyl compound (M279D-2), accounted for about 20.2% in rats and 21.2% in humans of the initial S9229 peak area at the 60-minute mark Other metabolites, such as the C-3 hydroxymethyl (M279E-2) and C-3,4 dihydroxymethyl (M295C-2), were also detected but at lower levels Additionally, a minor aryl aldehyde (M277B-2) was observed, resulting from the oxidation of M279D-2 or M279E-2 The demethylated analogs of the parent compound and the hydroxylated metabolites (M249A-2, M265A-2, and M265B-2) were also present in both species The identities of these metabolites were confirmed through direct comparison with synthetic samples using LC-MS/MS.
In both rat and human microsomal incubations of S9229, several minor metabolites were identified, including mono-hydroxylated compounds derived from the iso-butyl group of the parent compound (M279A-2, M279B-2, M279C-2) and two aryl methyl hydroxylated compounds (M295A-2 and M295B-2) Additionally, a metabolite (M277A-2) formed from the loss of water from the side chain of M295A-2 or M295B-2, along with another metabolite (M263A-2) resulting from the further oxidation of a hydroxylated metabolite of M249A-2, were also detected as minor components in the rat microsomal incubations.
The metabolic profiles of S807 and S9229 in rat and human liver microsomes were qualitatively similar, producing the same oxidative metabolites in both species This similarity confirms that rats are a suitable model for assessing the potential toxicity of S807 and S9229.
3.1.2 Pharmacokinetics and in vivo metabolism of S807 in rats
The pharmacokinetic (PK) parameters and oral bioavailability of S807 were assessed in male and female Sprague-Dawley rats following single intravenous or oral administration Plasma samples were analyzed for metabolites resulting from S807 incubations with rat liver microsomes For the intravenous administration, a total of 4 male and 4 female rats were bolus injected with S807 at a dosage of 1 mg/kg body weight, using a solution of 20% PEG400, 10% ethanol, 2% DMSO, and 68% sterile saline Blood samples were collected from an implanted jugular cannula at pre-dose and approximately 2 and 5 hours post-administration.
In a study involving Sprague-Dawley rats, both male and female subjects were administered a single oral dose of S807 at varying concentrations (20, 50, or 200 mg/kg bw) in a 1% methyl cellulose solution Blood samples were collected at multiple time points post-dose, including 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours, using implanted jugular cannulae The plasma samples were analyzed for S807 and its metabolites via LC-MS/MS, employing a Waters XSelect TM CSH C18 column and a gradient system with formic acid in water and acetonitrile Detection was performed using a Q-Trap mass spectrometer in positive ionization mode, focusing on specific mass transitions for the parent compound and internal standard The resulting plasma concentration-time data were processed using non-compartmental analysis with Phoenix WinNonlin version 6.2.
Mass transition pairs selected for metabolite exposure analysis were based on findings from a secondary LC-MS/MS method This method aimed to identify in vivo metabolites by measuring the precise mass of the parent ion and its fragment ions.
Pooled plasma samples from male and female rats, dosed orally at 200 mg/kg, were analyzed using LC-QTOF/MS (Agilent iFunnel 6550A MS QTOF in positive mode) The analysis was conducted with an Agilent 1290 Infinity Binary pump and an Agilent 1290 Infinity autosampler, utilizing a Waters CSH C18 column (50 x 2.1 mm, 1.7 μm) with a gradient system of 0.1% formic acid in water and acetonitrile Data processing was performed using Agilent MassHunter software.
Details of the analytical methods can be found in the Supplementary Data section Test article formulations prepared for this study were analyzed for concentration by HPLC-UV (240 nm)
The pharmacokinetic parameters for S807 are shown in Table 3
In a study on intravenous administration of S807, the terminal half-life in plasma was found to be 3.02 ± 0.24 hours for male rats and 3.02 ± 0.92 hours for female rats The mean plasma clearance (CL) averaged 30.7 mL/min/kg for males, representing 55.6% of hepatic blood flow, while females had a higher clearance rate of 47.2 mL/min/kg, accounting for 85.5% of hepatic blood flow Additionally, the volume of distribution at steady-state (Vss) was 4630 mL/kg for males and 5610 mL/kg for females, which corresponds to 6.93 and 8.40 times the total body water volume, respectively.
In a study on oral administration of S807, the plasma half-life values increased with higher doses, ranging from 1.14 ± 0.35 to 2.49 ± 0.07 hours in male rats and 1.07 ± 0.67 to 2.62 ± 0.20 hours in female rats This increase in half-life was accompanied by a significant rise in both Cmax and AUClast, which exceeded dose-proportionality across the oral dose range Notably, escalating the dose from 20 to 200 mg/kg bw led to a dramatic increase in systemic exposure, with AUClast rising 555-fold in males and 651-fold in females Additionally, oral bioavailability (%F) varied from 1.85-2.43% at the 20 mg/kg bw dose to 102.9-157.9% at the 200 mg/kg bw dose.
Although Cmax and AUClast values were very similar for male and female rats at both the 20 and
Genotoxicity and mutagenicity studies
Both S807 and S9229 were assessed for their genotoxic potential using a standard five-strain Ames test, chromosome aberration analysis, and an in vivo micronucleus test, as detailed in Table 8 All genetic toxicology studies adhered to the FDA Good Laboratory Practice (GLP) regulations outlined in 21 CFR Part 58.
(2006) and OECD guidelines (1998) The data tables for the genotoxicity studies can be found in the Supplemental Material
3.2.1 Bacterial reverse mutation test (5-strain Ames)
S807 and S9229 were tested for their ability to induce point mutations in various strains of S typhimurium (TA98, TA100, TA1535, TA1537) and E coli (WP2 uvrA), both with and without metabolic activation using rat liver S9 from Aroclor™ 1254-induced rats This evaluation adhered to the OECD Guideline for Testing of Chemicals No 471, specifically the Bacterial Reverse Mutation Test (OECD, 1997a).
The study investigated the concentrations of S807 and S9229, ranging from 21 to 5000 μg per plate and 50 to 5000 μg per plate, respectively Neither compound exhibited toxicity at any concentration, as indicated by normal background lawns and colony counts comparable to negative controls S9229 showed no precipitate, while S807 presented precipitation at higher concentrations, affecting colony counting and limiting analyzable plates to either 1670 or 5000 μg/plate Importantly, neither S807 nor S9229 increased revertant colonies in any test conditions, confirming the absence of mutagenic effects Positive controls validated the assay's sensitivity and the metabolic activity of liver preparations.
The study concluded that neither S807 nor S9229 exhibited mutagenic effects on S typhimurium strains TA98, TA100, TA1535, TA1537, or the E coli strain WP2 uvrA, regardless of the presence or absence of metabolic activation during the testing conditions.
3.2.2 In vitro chromosome aberration test
The study examined the ability of S807 and S9229 to cause structural and numerical chromosome aberrations in mammalian cells, with and without the addition of a rat liver fraction (S9) The experimental approach adhered to the OECD Guideline for the Testing of Chemicals – 473, which outlines the In Vitro Mammalian Chromosome Aberration Test.
In the case of S807, cultures of Chinese hamster ovary cells (CHO-WBL) were treated for 3 and
In a study assessing the effects of S807, cultures were exposed for 18 hours in a non-activated test system and for 3 hours with S9 from rats induced with phenobarbital and 5,6-benzoflavone The concentrations of S807 tested ranged from 21 to 5000 μg/mL, with solvent and positive controls (mitomycin C, -S9; cyclophosphamide, +S9) included Notably, precipitate was observed in cell cultures at concentrations of 62 μg/mL and above The exposure to S807 produced a "U" shaped curve of Relative Cell Growth (RCG), with the lowest RCG recorded at 560 μg/mL, ranging from 7-37% across all test conditions, rather than at the highest concentration of 5000 μg/mL.
In the absence of S9, concentrations of 0, 21, 190, 1670, and 5000 μg/mL in both 3 and 18 hour cultures exhibited a Relative Cytotoxicity Grade (RCG) of 19% or higher, along with a Relative Mitotic Index (RMI) of 28% or greater, making them suitable for chromosome analysis For the 3 hour exposure with S9, the concentrations included 0 and 21 μg/mL.
S807 was tested at concentrations of 560, 1670, and 5000 μg/mL, resulting in a Relative Cytotoxicity Growth (RCG) of ≥37% and a Relative Mitotic Index (RMI) of ≥100%, leading to its selection for chromosome analysis The study found no structural or numerical chromosome aberrations in S807 treated cultures, consistent with the concurrent solvent controls, while positive control agents successfully induced expected chromosome aberrations Consequently, it was concluded that S807 does not induce chromosomal aberrations in cultured CHO-WBL cells, adhering to regulatory guidelines.
In the study of S9229, human peripheral blood lymphocytes were exposed for 4 and 20 hours in a non-activated system, and for 4 hours with S9 from Aroclor™ 1254-induced rats A preliminary toxicity test determined the dose range for the cytogenetic test, revealing significant toxicity at doses of ≥789 µg/mL in both non-activated and S9-activated 4-hour groups, and at ≥263 µg/mL in the non-activated 20-hour group Consequently, the selected doses for the chromosome aberration assay were 158 to 800 µg/mL for the 4-hour exposure groups and 25 to 280 µg/mL for the 20-hour exposure group Additionally, solvent and positive control cultures (mitomycin C for -S9; cyclophosphamide for +S9) were included in the definitive assay.
Visible precipitate appeared in the treatment medium at dose levels of 322 µg/mL and above, while doses of 280 µg/mL and below remained soluble initially By the end of the treatment period, precipitate was noted at dose levels of 460 µg/mL or higher in both non-activated and S9-activated 4-hour exposure groups, with doses of 322 µg/mL and below still being soluble In the non-activated 20-hour exposure group, all doses were soluble at the conclusion of the treatment Doses for microscopic analysis were selected based on mitotic inhibition, specifically the lowest dose that achieved at least a 50% reduction in the mitotic index compared to the solvent control and two lower doses The analyzed dose levels included 225, 280, and 322 µg/mL for the non-activated and S9-activated 4-hour exposure groups, and 55, 110, and 158 µg/mL for the non-activated 20-hour exposure group.
Under the specified test conditions, S9229 treatment did not result in any structural or numerical chromosome aberrations beyond those observed in the solvent controls, with no significant differences (p > 0.05, Fisher’s Exact Test) In contrast, the positive control agents successfully induced chromosome aberrations as anticipated (p ≤ 0.01, Fisher’s Exact Test) Therefore, it was concluded that S9229 exposure did not cause chromosome aberrations in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes, both in the presence and absence of rat liver S9, in compliance with regulatory guidelines.
3.2.3 In vivo micronucleus assay in mice
S807 and S9229 were assessed for their potential clastogenic activity and impact on the mitotic apparatus by measuring the increase in micronucleated polychromatic erythrocytes (mnPCEs) in the bone marrow of CD-1 mice The evaluation adhered to the OECD Guideline for the Testing of Chemicals No 474, focusing on the Mammalian Erythrocyte Micronucleus Test Preliminary dose-range finding studies were conducted to evaluate the toxicity of the test substances and to establish the maximum tolerated dose (MTD) or maximum feasible dose (MFD) for the main assay.
For the dose range finding phase of the study with S807, three groups of Swiss Albino (CD-1) mice (3 animals/sex/group; Charles River, Canada) were treated with either 500, 1000, or
In a study involving S807, a total of 2000 mg/kg body weight (bw) was administered as a solution in DMSO (132 mg/mL) via intraperitoneal injection Additionally, a fourth group consisting of three male and three female mice received 800 mg/kg bw of S807, while a fifth group of three male mice was given 700 mg/kg bw of the same compound, using the same administration route Notably, fatalities were observed in all dose groups at or above the specified dosages.
In a study involving CD-1 mice, a maximum tolerated dose (MTD) of S807 was determined to be 700 mg/kg body weight (bw) when administered as a DMSO solution via intraperitoneal injection within 48 hours Following preliminary results, definitive dose levels of 175, 350, and 700 mg/kg bw were established, with 21 animals per group and a dose volume of 5.3 mL/kg bw As no significant differences in toxicity were noted between sexes, the main test was conducted exclusively with male mice In the definitive phase, DMSO at 5.8 mL/kg bw served as the negative control, while cyclophosphamide was utilized as the positive control at a dose of 40 mg/kg bw with a volume of 2 mL/kg bw.
Animals were observed for signs of toxicity during the course of these studies
In the definitive assay, seven animals from each group were euthanized at 24, 36, or 48 hours post-dosing, and femoral bone marrow was collected Bone marrow slides were prepared, fixed, and stained using May-Grunwald/Giemsa, allowing for the microscopic examination of polychromatic erythrocytes (PCEs) for micronuclei (mnPCEs) Additionally, the ratio of PCEs to total erythrocytes (TE) in the test article groups was compared to the vehicle control groups to assess the cytotoxicity of the test article.
In vivo toxicological studies
S807 underwent 21-day dose-range finding and 90-day subchronic toxicology studies in rats, adhering to FDA guidelines for food ingredient safety Additionally, S9229 was assessed in a 28-day subacute toxicology study in rats Summary data tables for these studies are available in the Supplemental Material.
3.3.1 21-day dose-range finding toxicity study on S807
This study aimed to assess the systemic toxicity of S807 in rats following 21 days of dietary administration, with the goal of determining appropriate doses for a subsequent 90-day subchronic toxicity study Male and female Crl:CD ® (SD)IGS BR rats (n = 5/sex/group) from Charles River Laboratories, Raleigh, NC, were divided into three treatment groups and received S807 through their diet at varying dose levels.
In the study, animals were divided into groups receiving 50, 100, or 200 mg/kg bw/day of the test substance, while a control group of five animals per sex received a vehicle diet The test substance was continuously administered through the diet during the treatment period, with dietary concentrations of S807 adjusted weekly based on body weight and food consumption to maintain consistent dosing At the end of the study, the animals were anesthetized with sodium pentobarbital, exsanguinated, and necropsied for analysis.
Survival, clinical observations, body weight, food consumption, hematology, clinical chemistry, organ weights, and macroscopic evaluations of all animals were used to assess potential toxicity
Liver samples from all animals were microscopically processed and examined, while kidney samples from both control and high-dose (200 mg/kg bw/day) groups underwent similar processing Additionally, macroscopic lesions were assessed in each animal.
All animals in the study survived until the scheduled euthanasia on Day 23, with no clinical observations linked to the test article Over the 21-day period, female subjects treated with 200 mg/kg bw/day exhibited a significant reduction in mean body weight gain of 38% (p ≤ 0.05) compared to controls Although mean absolute body weight and food consumption values did not show significant differences from control values, there were numerical decreases of 9.7% in body weight and 14.1% in food consumption by the end of the study Additionally, mean absolute body weights, body weight changes, and food consumption for males treated at 200 mg/kg bw/day, as well as for both males and females treated at 50 and 100 mg/kg bw/day, were not significantly different from their respective control values.
(TABLE 10) Clinical pathology alterations were limited to slightly lower erythrocyte counts and hemoglobin and hematocrit values in males, and slightly higher cholesterol values in males treated at 100 or
In a study comparing treatment groups to controls, cholesterol levels in females were significantly elevated at the highest dose of 200 mg/kg bw/day (p ≤ 0.05) Males at 100 and 200 mg/kg bw/day showed slightly higher globulin values, resulting in lower A/G ratios (p ≤ 0.05), although these changes did not exhibit a dose-response effect and lacked statistical significance Hemoglobin and hematocrit levels in males at the two higher doses were below control values, but the lack of significance was attributed to small group sizes and high variability Urinalysis parameters showed no meaningful changes, and while several clinical pathology values differed from controls, they were deemed incidental and unrelated to the test article administration.
In females treated with 100 and 200 mg/kg bw/day, relative liver weights as a percentage of body weight significantly increased (p ≤ 0.05), attributed to decreased body weights and slightly elevated liver weights, although the liver/brain weight ratio increase was not statistically significant Additionally, one male at the 200 mg/kg bw/day dosage exhibited decreased relative testicular weight due to bilateral testicular atrophy and hypospermia observed microscopically, which is not deemed related to the test article.
No macroscopic findings were linked to the administration of the test article; however, histomorphologic changes were noted in the livers of male and female subjects at doses of 50, 100, and 200 mg/kg bw/day These changes included vacuolization of centrilobular hepatocytes in males and scattered random hepatocytes in females, with multiple clear vacuoles indicating potential intracytoplasmic lipid accumulation This was corroborated by increased plasma cholesterol levels in the 100 and 200 mg/kg bw/day groups While the diagnosis was based on hematoxylin and eosin-stained liver sections, the possibility of cytoplasmic vacuolation due to phospholipidosis could not be excluded The severity of the observed changes was dose-dependent, yet no hepatocellular necrosis or increase in liver enzymes was detected in clinical evaluations Other microscopic findings in the liver or kidney were deemed incidental and unrelated to the test article administration.
In conclusion, S807 administered in the diet of rats for 21 days was considered palatable at 50,
In a study involving various animal subjects, doses of 100 and 200 mg/kg bw/day were administered, with female subjects receiving 200 mg/kg bw/day due to concerns over non-palatability and changes in body weight gains Notably, increased relative liver weights were observed in females treated at both 100 and 200 mg/kg bw/day, alongside histomorphological changes in the livers of both male and female subjects across all treatment groups Consequently, a No Observed Effect Level (NOEL) for S807 could not be determined.
3.3.2 13-week subchronic toxicity study on S807
This study aimed to assess the subchronic toxicity of S807 in rats over a 13-week period The test substance was incorporated into the diet of four groups, each consisting of twenty male and twenty female Crl:CD ® (SD)IGS BR rats, with dose levels of 0 (control), 2, 10, or 20 mg/kg body weight per day S807 was administered continuously through the diet, and dietary concentrations were adjusted weekly based on body weight and food consumption to maintain consistent dosing throughout the treatment duration.
The study assessed potential toxicity through various evaluations, including survival rates, clinical observations, body weight gain, food consumption, hematology, clinical chemistry, urinalysis, organ weights, macroscopic examinations, and histopathologic evaluations Daily cageside observations were conducted for each rat, with detailed clinical assessments performed weekly and prior to treatment initiation, focusing on changes in skin, fur, eyes, mucous membranes, and autonomic activity Weekly evaluations also recorded changes in posture, reactivity to handling, and any unusual behaviors such as clonic or tonic movements and stereotypies Gait changes were assessed by allowing the rats to walk freely Expanded clinical observations for neurotoxic effects were conducted on 10 rats of each sex per group, both before treatment and during Week 13, including Hand-Held and Open Field Observations to evaluate sensory reactivity to stimuli.
Ophthalmoscopic examinations were performed before treatment and at Week 13 for control and high-dose groups Hematology, coagulation, clinical chemistry, and urinalysis samples were collected from all animals at scheduled sacrifice, with urine collected overnight prior to blood collection Body weights were recorded before treatment, on the first day, Day 4, and weekly thereafter, while food consumption was measured on Day 4 and weekly After a minimum of 90 days of treatment, rats were anesthetized, exsanguinated, and necropsied, with 12 specified organ weights recorded at scheduled sacrifice.
A total of 44 protocol-specified tissues from each animal in the control and high-dose groups, as well as any animal that died or was sacrificed unexpectedly, were embedded in paraffin and examined microscopically after staining with hematoxylin and eosin At the end of the study, the livers of all rats were divided into left and right lobes; one lobe was flash frozen in OCT for Oil Red O staining to assess fat deposits, while the other lobe was preserved for histopathological testing Additionally, portions of the mandibular and mesenteric lymph nodes, Peyer’s patches, spleen, and thymus were preserved in OCT and stored at -60 to -80°C for potential future immunohistochemical analysis.
The study reported no deaths related to the test article, although one control female was found deceased on Day 72, with the cause of death undetermined upon microscopic examination The mean weekly body weights and changes for both male and female subjects receiving 2, 10, or 20 mg/kg bw/day were not significantly different from the control group Additionally, specific mean body weight changes were observed in males given 20 mg/kg bw/day during Week 6 and in those given 2 mg/kg bw/day during Week 11.
In a study, significant increases in body weight were observed in groups receiving 10 and 20 mg/kg bw/day compared to controls (p ≤ 0.05) By Week 12, males treated with 2, 10, and 20 mg/kg bw/day exhibited notably lower mean body weight changes (p ≤ 0.05), which were linked to significant alterations in food consumption However, due to the infrequent and inconsistent nature of these changes, along with the lack of uniform impact on body weights, they are not deemed adverse or directly associated with S807 treatment.
Discussion and conclusions
Toxicological evaluations were conducted on two N-alkyl benzamide umami flavor compounds, S807 and S9229, to assess their safety for food and beverage applications These compounds are part of a new series of N-alkyl benzamide agonists for the human umami receptor hTAS1R1/hTAS1R3, offering an umami flavor effect comparable to MSG at a concentration 1000 times lower.
The metabolic profile of S807 in rat and human liver microsomes was qualitatively similar, producing the same oxidative metabolites, indicating that rats are suitable for assessing S807's potential toxicity The primary metabolite identified in both species was catechol M251A-1, with oxidative demethylenation recognized as a key metabolic pathway for methylenedioxyphenyl derivatives, particularly for highly lipophilic compounds.
In a study on rats, S807 demonstrated significant non-linear pharmacokinetics during oral dosing, with bioavailability (%F) ranging from 1.85-2.43% at a dose of 20 mg/kg body weight to 102.9-157.9% at 200 mg/kg body weight Additionally, a notable gender difference was observed in the rate and extent of exposure at the 50 mg/kg body weight oral dose, which was not present at the 20 mg/kg body weight dose.
The data indicates that the significant rise in the bioavailability of S807 at higher doses, specifically 200 mg/kg bw, is likely attributed to the saturation of first-pass metabolism and/or tissue distribution Additionally, the observed gender difference at the 50 mg/kg bw dose implies that the clearance pathway for S807 reaches saturation at a lower concentration in males compared to females.
Research has shown that the metabolism of xenobiotics and the response to inducing agents differ between male and female rats, primarily due to variations in the profiles of CYP isozymes present in their livers (Kato and Yamazoe, 1992; Mugford and Kedderis, 1998).
In vivo studies reveal that the major metabolite formed during microsomal incubations is catechol M251A-1, which is quickly conjugated and oxidized, resulting in a total of thirteen Phase II metabolites At a dosage of 20 mg/kg bw, the dominant biotransformations are the mono-methyl ethers M265(A-B)-1 and glucuronide mono-methyl ether M441B-1, accounting for 41.70-44.17% and 29.26-35.74% of the total metabolite AUClast, respectively The 3-methoxy isomer M265B-1 is the primary O-methyl regioisomer produced from M251A-1 by catechol-O-methyltransferase (COMT) In vitro, both rat and human COMT enzymes preferentially catalyze the 3-O-methylation of various catechol substrates with anionic or cationic side chains, including 3,4-dihydroxybenzoic acid and catecholamines like dopamine The 3-O-methyl/4-O-methyl ratios for these substrates using rat COMT range from 5.5 to 19.8.
Catechol substrates with neutral or non-polar side chains, such as 4-ethyl catechol and N-acetyl dopamine, exhibit minimal preference for 3-O-methylation compared to 4-O-methylation, with reported ratios ranging from 0.46 to 2.0 when using rat COMT The varying meta/para ratios of these substituted catechols are linked to their distinct binding orientations within the active site of COMT.
COMT is expected to produce both mono-methyl ethers M265A-1 (4-O-methyl) and M265B-1 (3-O-methyl) from catechol M251A-1, which has a calculated logP value of 2.99, in roughly equal amounts due to its lack of preference for 3-O-methylation over 4-O-methylation In vitro studies with native porcine COMT and S-adenosyl-L-methionine confirmed this, yielding M265B-1 and M265A-1 in a ratio of 1.9 to 1 (Chi, 2016) However, following an oral dose of S807 in rats, M265B-1 concentrations were found to be 60- to 170-fold higher than those of M265A-1 throughout a 24-hour observation period Additionally, for the glucuronide mono-methyl ethers M441(A-B)-1, the 3-O-methyl regioisomer M441B-1 was present at significantly higher concentrations than the 4-O-methyl regioisomer M441A-1.
1, indicating that the lower concentration of M265A-1 relative to M265B-1 is not due to rapid conversion to the corresponding glucuronide M441A-1
The in vivo regioselectivity for 3-O-methylation of catechols has been linked to the selective demethylation of 4-O-methyl derivatives by microsomal enzymes, leading to an accumulation of the 3-O-methyl metabolite To investigate this phenomenon in catechol M251A-1, both mono-methyl regioisomers M265A-1 and M265B-1 were incubated with rat liver microsomes Additionally, a parallel experiment was conducted with an equimolar mixture of M265A-1 and M265B-1 to determine if one regioisomer influences the demethylation rate of the other The samples were analyzed for the loss of the parent compound and the production of the demethylated product M251A-1 and the side chain oxidation product M281(A-B)-1 at various time points.
20 and 60 minutes (see Table 13) The 4-O-methyl regioisomer M265A-1 was found to be metabolized by rat microsomes only slightly faster than its 3-O-methyl regioisomer M265B-1
After a 60-minute incubation period, 47.7% of M265A-1 remained, compared to 55.7% of M265B-1 The co-incubation experiment yielded similar findings, with both regioisomers generating catechol M251A-1 and hydroxylation products M281(A-B)-1 However, these products represented only a small fraction of the loss of the parent compound, assuming that the MS response factors for M265(A-B)-1 and its oxidation products M251A-1 and M281(A-B)-1 are comparable.
More M265A-1 products were produced than M265B-1, and both compounds showed a significant loss of the parent compound without NADPH, indicating metabolism via a CYP-independent pathway If the two O-methyl regioisomers are produced in similar quantities by rat COMT in vivo, the observed regioselectivity for 3-O-methylation may be due to the selective removal of the 4-O-methyl isomer M265A-1 through an alternative metabolic or elimination pathway.
An evaluation of the eight major metabolites of S807 reveals that at higher doses, the glucuronidation of catechol M251A-1 and its mono-methyl ethers M265(A-B)-1 becomes rate limiting, while the oxidation of the 4-heptamine moiety of S807 and M265(A-B)-1 emerges as a more significant metabolic pathway Additionally, the exposure data for M265B-1 indicates that the oxidative demethylenation of S807 to form M251A-1 may also be rate limiting at elevated doses, although this is counterbalanced by the saturation of the pathway that produces glucuronide M441B from mono-methyl ether M265B-1 Consequently, the saturation of the oxidative demethylenation pathway for S807 likely contributes to the substantial increase in S807 exposure with rising doses.
The metabolite profile of S9229 in both rat and human liver microsomes showed qualitative similarities, including hydroxylation of aryl methyl groups, the iso-butyl side chain, and demethylation of the side chain methyl ether, with the major metabolite being the C-4 hydroxymethyl compound M279D-2 Oral bioavailability of S9229 in rats was very low, ranging from 0.04% to 0.10%, and M279D-2 was identified as the dominant in vivo metabolite Additional metabolic pathways included oxidative demethylation and hydroxylation of various moieties of S9229 and its hydroxymethyl metabolites The findings suggest that rapid oxidative metabolism by CYP enzymes contributes to the poor oral bioavailability of the parent compound S9229.
Both S807 and S9229 were assessed for genotoxicity using a series of in vitro assays, including a bacterial reverse mutation assay and a chromosome aberration test, revealing that neither compound was mutagenic or clastogenic Further evaluation in an in vivo mouse micronucleus assay demonstrated that S807, administered intraperitoneally at doses up to 700 mg/kg, and S9229, given orally at doses up to 2000 mg/kg, were well tolerated and did not induce clastogenicity or aneugenicity in bone marrow erythrocytes Additionally, no significant reductions in the PCE/TE ratio were observed, indicating that neither compound was cytotoxic to bone marrow or inhibited erythropoiesis.
In a 21-day dose range-finding toxicity study of S807, significant findings included increased relative liver weights in female rats at doses of 100 and 200 mg/kg bw/day, along with histomorphological changes in the livers of both sexes indicative of intracytoplasmic lipid accumulation Consequently, the No Observed Effect Level (NOEL) was determined to be less than 50 mg/kg bw/day For the subsequent 13-week subchronic toxicology study, doses of 2, 10, and 20 mg/kg bw/day were selected to establish a NOEL for S807 while ensuring a safety margin for its use as a flavoring agent Due to observed cytoplasmic vacuolation in the livers at higher doses, liver samples from all animals were flash frozen and stained with Oil Red O for microscopic examination to assess potential lipid deposits and exclude phospholipidosis.