RESUMEN
Despite advances in treatment, a significant proportion of patients with chronic lymphocytic leukemia (CLL) will relapse with drug-resistant disease. The imipridones, ONC-201 and ONC-212, are effective against a range of different cancers, including acute myeloid leukemia (AML) and tumors of the brain, breast, and prostate. These drugs induce cell death through activation of the mitochondrial protease, caseinolytic protease (CIpP), and the unfolded protein response (UPR). Here we demonstrate that the novel imipridone analog, TR-57, has efficacy as a single agent and synergises with venetoclax against CLL cells under in vitro conditions that mimic the tumor microenvironment. Changes in protein expression suggest TR-57 activates the UPR, inhibits the AKT and ERK1/2 pathways and induces pro-apoptotic changes in the expression of proteins of the BCL-2 family. The study suggests that TR-57, as a single agent and in combination with venetoclax, may represent an effective treatment option for CLL.
Asunto(s)
Apoptosis , Compuestos Bicíclicos Heterocíclicos con Puentes , Sinergismo Farmacológico , Leucemia Linfocítica Crónica de Células B , Sulfonamidas , Humanos , Sulfonamidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Respuesta de Proteína Desplegada/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de Señal/efectos de los fármacosRESUMEN
To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 microM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 microM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 microM) and SE-3 (200 microM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 microM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 microM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Edulcorantes/farmacología , Papilas Gustativas/efectos de los fármacos , Gusto/efectos de los fármacos , Regulación Alostérica , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Pirimidinas/química , Pirimidinas/farmacología , Receptores Acoplados a Proteínas G/genética , Sacarosa/administración & dosificación , Sacarosa/análogos & derivados , Sacarosa/química , Sacarosa/farmacología , Edulcorantes/administración & dosificación , Edulcorantes/química , Papilas Gustativas/metabolismo , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
The mitochondrial ClpP protease is responsible for mitochondrial protein quality control through specific degradation of proteins involved in several metabolic processes. ClpP overexpression is also required in many cancer cells to eliminate reactive oxygen species (ROS)-damaged proteins and to sustain oncogenesis. Targeting ClpP to dysregulate its function using small-molecule agonists is a recent strategy in cancer therapy. Here, we synthesized imipridone-derived compounds and related chemicals, which we characterized using biochemical, biophysical, and cellular studies. Using X-ray crystallography, we found that these compounds have enhanced binding affinities due to their greater shape and charge complementarity with the surface hydrophobic pockets of ClpP. N-terminome profiling of cancer cells upon treatment with one of these compounds revealed the global proteomic changes that arise and identified the structural motifs preferred for protein cleavage by compound-activated ClpP. Together, our studies provide the structural and molecular basis by which dysregulated ClpP affects cancer cell viability and proliferation.
Asunto(s)
Mitocondrias , Proteómica , Mitocondrias/metabolismo , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , ProteolisisRESUMEN
We recently described the identification of a new class of small-molecule activators of the mitochondrial protease ClpP. These compounds synthesized by Madera Therapeutics showed increased potency of cancer growth inhibition over the related compound ONC201. In this study, we describe chemical optimization and characterization of the next generation of highly potent and selective small-molecule ClpP activators (TR compounds) and demonstrate their efficacy against breast cancer models in vitro and in vivo. We selected one compound (TR-107) with excellent potency, specificity, and drug-like properties for further evaluation. TR-107 showed ClpP-dependent growth inhibition in the low nanomolar range that was equipotent to paclitaxel in triple-negative breast cancer (TNBC) cell models. TR-107 also reduced specific mitochondrial proteins, including OXPHOS and TCA cycle components, in a time-, dose-, and ClpP-dependent manner. Seahorse XF analysis and glucose deprivation experiments confirmed the inactivation of OXPHOS and increased dependence on glycolysis following TR-107 exposure. The pharmacokinetic properties of TR-107 were compared with other known ClpP activators including ONC201 and ONC212. TR-107 displayed excellent exposure and serum t1/2 after oral administration. Using human TNBC MDA-MB-231 xenografts, the antitumor response to TR-107 was investigated. Oral administration of TR-107 resulted in a reduction in tumor volume and extension of survival in the treated compared with vehicle control mice. ClpP activation in vivo was validated by immunoblotting for TFAM and other mitochondrial proteins. In summary, we describe the identification of highly potent new ClpP agonists with improved efficacy against TNBC, through targeted inactivation of OXPHOS and disruption of mitochondrial metabolism.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Animales , Endopeptidasa Clp/química , Endopeptidasa Clp/metabolismo , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Péptido Hidrolasas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Despite advances in therapy, a significant proportion of patients with chronic lymphocytic leukemia (CLL) relapse with drug resistant disease. Novel treatment approaches are required, particularly for high risk disease. The imipridones represent a new class of cancer therapy that has been investigated in pre-clinical and clinical trials against a range of different cancers. We investigated the effects of the imipridone, ONC-212, against CLL cells cultured under conditions that mimic aspects of the tumour microenvironment and a TP53ko CLL cell line (OSU-CLL-TP53ko). ONC-212 induced dose-dependent apoptosis, cell cycle arrest and reduced the migration of CLL cells in vitro, including cells from patients with TP53 lesions and OSU-CLL-TP53ko cells. The effects of ONC-212 were associated with protein changes consistent with activation of the mitochondrial protease, CIpP, and the integrated stress response. We also observed inhibition of pathways downstream of the B-cell receptor (BCR) (AKT and MAPK-ERK1/2) and a pro-apoptotic shift in the balance of proteins of the BCL2 family of proteins (BCL2, MCL1, BCLxL, BAX and NOXA). In conclusion, the study suggests ONC-212 may represent an effective treatment for high risk CLL disease by inhibiting multiple facets of the BCR signaling pathway and the pro-survival effects of the BCL2-family proteins.
RESUMEN
In humans, bitter taste is mediated by 25 TAS2Rs. Many compounds, including certain active pharmaceutical ingredients, excipients, and nutraceuticals, impart their bitter taste (or in part) through TAS2R8 activation. However, effective TAS2R8 blockers that can either suppress or reduce the bitterness of these compounds have not been described. We are hereby reporting a series of novel 3-(pyrazol-4-yl) imidazolidine-2,4-diones as potent and selective TAS2R8 antagonists. In human sensory tests, S6821 and S7958, two of the most potent analogues from the series, demonstrated efficacy in blocking TAS2R8-mediated bitterness and were selected for development. Following data evaluation by expert panels of a number of national and multinational regulatory bodies, including the US, the EU, and Japan, S6821 and S7958 were approved as safe under conditions of intended use as bitter taste blockers.
Asunto(s)
Hidantoínas/farmacología , Pirazoles/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Gusto/efectos de los fármacos , Animales , Café/química , Descubrimiento de Drogas , Estabilidad de Medicamentos , Humanos , Hidantoínas/síntesis química , Hidantoínas/toxicidad , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/toxicidad , Ratas , Relación Estructura-ActividadRESUMEN
A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src-family kinases (SFKs). These type II inhibitors were optimized using a cellular Lck-dependent proliferation assay and are capable of inhibiting Lck at single-digit nanomolar concentrations. This scaffold is likely to serve a valuable template for developing potent inhibitors of a number of SFKs.
Asunto(s)
Bencimidazoles/farmacología , Descubrimiento de Drogas , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Animales , Bencimidazoles/síntesis química , Bencimidazoles/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be â¼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with â¼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a â¼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.
Asunto(s)
Antineoplásicos/farmacología , Endopeptidasa Clp/antagonistas & inhibidores , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía de Afinidad , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Activación Enzimática , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Imidazoles , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Piridinas , PirimidinasRESUMEN
A series of nonsteroidal glucocorticoid receptor (GR) ligands based on a 6-indole-1,2,3,4-tetrahydroquinoline scaffold are reported. Structure-activity relationship (SAR) of the pendent indole group identified compound 20 exhibiting good GR binding affinity (K(i)=1.5nM) and 100- to 1000-fold selectivity over MR, PR, and AR while showing activity in an E-selectin repression assay.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Indoles/farmacología , Quinolinas/farmacología , Receptores de Glucocorticoides/efectos de los fármacos , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Sitios de Unión , Evaluación Preclínica de Medicamentos , Indoles/síntesis química , Indoles/química , Ligandos , Estructura Molecular , Quinolinas/síntesis química , Quinolinas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Modification on a lead series of [1,4]oxazino[3,2-g]quinolin-7-ones at the 2-position led to selective androgen receptor modulators with improved in vivo activity. The most potent analog (-)-33a exhibited full maintenance of levator ani muscle at 3mg/kg and reduced activity on ventral prostate weight in a 2-week orally-dosed and orchidectomized rat maintenance assay.
Asunto(s)
Anabolizantes/farmacología , Oxazinas/farmacología , Próstata/efectos de los fármacos , Quinolonas/farmacología , Receptores Androgénicos , Administración Oral , Anabolizantes/síntesis química , Antagonistas de Receptores Androgénicos , Andrógenos , Animales , Masculino , Modelos Químicos , Orquiectomía , Tamaño de los Órganos/efectos de los fármacos , Oxazinas/síntesis química , Próstata/anatomía & histología , Quinolonas/síntesis química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Testosterona/farmacologíaRESUMEN
A series of 4-amino-6-benzimidazole-pyrimidines was designed to target lymphocyte-specific tyrosine kinase (Lck), a member of the Src kinase family. Highly efficient parallel syntheses were devised to prepare analogues for SAR studies. A number of these 4-amino-6-benzimidazole-pyrimidines exhibited single-digit nanomolar IC(50)s against Lck in biochemical and cellular assays. These 4-amino-6-benzimidazole-pyrimidines represent a new class of tyrosine kinase inhibitors.
Asunto(s)
Bencimidazoles/antagonistas & inhibidores , Química Farmacéutica/métodos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Pirimidinas/antagonistas & inhibidores , Enfermedades Autoinmunes/tratamiento farmacológico , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/química , Solubilidad , Relación Estructura-Actividad , Familia-src Quinasas/metabolismoRESUMEN
The lead optimization of a novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. The chemical instability of the dihydro-benzo[a]carbazole lead 2 was successfully addressed in the design and evaluation of compounds which also demonstrated improved potency compared to 2. Members of the scaffold have been identified which are full agonists that demonstrate cellular functional potency <50 nM. Analog 21 demonstrates equivalent efficacy in the human megakaryocyte differentiation (CFU-mega) assay compared to Eltrombopag.
Asunto(s)
Derivados del Benceno/síntesis química , Derivados del Benceno/farmacología , Carbazoles/síntesis química , Carbazoles/farmacología , Receptores de Trombopoyetina/agonistas , Trombopoyetina , Derivados del Benceno/química , Carbazoles/química , Técnicas Químicas Combinatorias , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Megacariocitos/metabolismo , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Receptores de Trombopoyetina/química , Relación Estructura-Actividad , Trombopoyetina/química , Trombopoyetina/metabolismoRESUMEN
A novel series of benzo[a]carbazole-based small molecule agonists of the thrombopoietin (Tpo) receptor is reported. Starting from a 3.4 microM high throughput screen hit, members of this series have been identified which are full agonists with functional potency <50 nM and oral bioavailability in mice.
Asunto(s)
Derivados del Benceno/síntesis química , Derivados del Benceno/farmacología , Carbazoles/síntesis química , Carbazoles/farmacología , Receptores de Trombopoyetina/agonistas , Trombopoyetina , Administración Oral , Animales , Derivados del Benceno/química , Carbazoles/química , Técnicas Químicas Combinatorias , Diseño de Fármacos , Humanos , Ratones , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Receptores de Trombopoyetina/química , Relación Estructura-Actividad , Trombopoyetina/química , Trombopoyetina/metabolismoRESUMEN
A toxicological evaluation of N-(1-((4-amino-2,2-dioxido-1H-benzo[c][1,2,6]thiadiazin-5-yl)oxy)-2-methylpropan-2-yl)-2,6-dimethylisonicotinamide (S2218; CAS 1622458-34-7), a flavour with modifying properties, was completed for the purpose of assessing its safety for use in food and beverage applications. S2218 exhibited minimal oxidative metabolism in vitro, and in rat pharmacokinetic studies, the compound was poorly orally bioavailable and rapidly eliminated. S2218 was not found to be mutagenic in an in vitro bacterial reverse mutation assay, and was found to be neither clastogenic nor aneugenic in an in vitro mammalian cell micronucleus assay. In subchronic oral toxicity studies in male and female rats, the NOAEL was 140 mg/kg bw/day (highest dose tested) for S2218 sulfate salt (S8069) when administered as a food ad-mix for 13 consecutive weeks. Furthermore, S2218 sulfate salt demonstrated a lack of maternal toxicity, as well as adverse effects on fetal morphology at the highest dose tested, providing a NOAEL of 1000 mg/kg bw/day for both maternal toxicity and embryo/fetal development when administered orally during gestation to pregnant rats.
RESUMEN
Toxicological evaluations of two N-alkyl benzamide umami flavour compounds, N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide (S807, CAS 745047-51-2) and (R)-N-(1-methoxy-4-methylpentan-2-yl)-3,4-dimethylbenzamide (S9229, CAS 851669-60-8), were completed for the purpose of assessing their safety for use in food and beverage applications. Both S807 and S9229 undergo rapid oxidative metabolism by both rat and human liver microsomes in vitro. In pharmacokinetic studies in rats, the systemic exposure to S9229 on oral administration is very low at all doses (% F < 1%), while that of S807 demonstrated a non-linear dose dependence. In metabolism studies in rats, hydroxylation of the C-4 aryl methyl group was found to be the dominant metabolic pathway for S9229. The dominant metabolic pathway for S807 in the rat involved oxidative scission of the methylenedioxy moiety to produce the corresponding 3,4-dihydroxybenamide which is further converted by Phase II metabolic enzymes to the 3- and 4-O-methyl ethers as well as their corresponding glucuronides. Both S807 and S9229 were not found to be mutagenic or clastogenic in vitro, and did not induce micronuclei in polychromatic erythrocytes in vivo. In a subchronic oral toxicity study in rats, the no-observed-effect-level (NOEL) for S807 was 20 mg/kg bw/day when administered in the diet for 13 weeks. The no-observed-adverse-effect-level (NOAEL) for S9229 in rats was 100 mg/kg bw/day (highest dose tested) when administered in the diet for 28 consecutive days.
RESUMEN
A toxicological evaluation of two novel bitter modifying flavour compounds, 3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)imidazolidine-2,4-dione (S6821, CAS 1119831-25-2) and 3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5,5-dimethylimidazolidine-2,4-dione (S7958, CAS 1217341-48-4), were completed for the purpose of assessing their safety for use in food and beverage applications. S6821 undergoes oxidative metabolism in vitro, and in rat pharmacokinetic studies both S6821 and S7958 are rapidly converted to the corresponding O-sulfate and O-glucuronide conjugates. S6821 was not found to be mutagenic or clastogenic in vitro, and did not induce micronuclei in bone marrow polychromatic erythrocytes in vivo. S7958, a close structural analog of S6821, was also found to be non-mutagenic in vitro. In short term and subchronic oral toxicity studies in rats, the no-observed-adverse-effect-level (NOAEL) for both S7958 and S6821 was 100 mg/kg bw/day (highest dose tested) when administered as a food ad-mix for either 28 or 90 consecutive days, respectively. Furthermore, S6821 demonstrated a lack of maternal toxicity, as well as adverse effects on fetal morphology at the highest dose tested, providing a NOAEL of 1000 mg/kg bw/day for both maternal toxicity and embryo/fetal development when administered orally during gestation to pregnant rats.
RESUMEN
A toxicological evaluation of a umami flavour compound, 2-(((3-(2,3-dimethoxyphenyl)-1H-1,2,4-triazol-5-yl)thio)methyl)pyridine (S3643; CAS 902136-79-2), was completed for the purpose of assessing its safety for use in food and beverage applications. S3643 undergoes extensive oxidative metabolism in vitro with rat microsomes producing the S3643-sulfoxide and 4'-hydroxy-S3643 as the major metabolites. In incubations with human microsomes, the O-demethyl-S3643 and S3643-sulfoxide were produced as the major metabolites. In pharmacokinetic studies in rats, the S3643-sulfoxide represents the dominant biotransformation product. S3643 was not found to be mutagenic or clastogenic in vitro, and did not induce micronuclei in CHO-WBL cells. In subchronic oral toxicity studies in rats, the no-observed-adverse-effect-level (NOAEL) for S3643 was 100 mg/kg bw/day (highest dose tested) when administered in the diet for 90 consecutive days.
RESUMEN
A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of alpha-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.
Asunto(s)
Inhibidores de Caspasas , Hepatopatías/tratamiento farmacológico , Ácidos Pentanoicos/síntesis química , Adulto , Alanina Transaminasa/sangre , Animales , Apoptosis/efectos de los fármacos , Aspartato Aminotransferasas/sangre , Disponibilidad Biológica , Caspasa 3 , Colestasis/tratamiento farmacológico , Colestasis/patología , Ensayos Clínicos Fase I como Asunto , Semivida , Hepatitis C Crónica/tratamiento farmacológico , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Humanos , Células Jurkat , Hígado/efectos de los fármacos , Hígado/patología , Hepatopatías/enzimología , Hepatopatías/etiología , Ratones , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
A toxicological evaluation of 4-amino-5-(3-(isopropylamino)-2,2-dimethyl-3-oxopropoxy)-2-methylquinoline-3-carboxylic acid(S9632; CAS 1359963-68-0), a flavour with modifying properties,was completed for the purpose of assessing its safety for use in food and beverage applications. No Phase I biotransformations of S9632 were observed in rat or human microsomes in vitro, and in rat pharmacokinetic studies, the compound was poorly orally bioavailable and rapidly eliminated. S9632 was not found to be mutagenic or clastogenic in vitro, and did not induce micronuclei or indicate interactions with the mitotic spindle in an in vivo mouse micronucleus assay at oral doses up to 2000 mg/kg. In subchronic oral toxicity studies in rats, the NOEL was 100 mg/kg/day (highest dose tested) for S9632 when administered as a food ad-mix for 90 consecutive days. Furthermore, S9632 demonstrated a lack of maternal toxicity, as well as adverse effects on fetal morphology at the highest dose tested, providing a NOEL of 1000 mg/kg/day for both maternal toxicity and embryo/fetal development when administered orally during gestation to pregnant rats.