Gender effect on the pharmacokinetics of thymoquinone: Preclinical investigation and in silico modeling in male and female rats.

Thymoquinone is the most biologically active constituent of Nigella sativa (black seed). A monoterpene compound chemically known as 2-methyl-5-isopropyl-1, 4-quinone. In this study, the gender-dependent pharmacokinetic behavior of thymoquinone in rats was investigated. Thymoquinone was administered orally (20 mg/kg) and intravenously (5 mg/kg) to male and female rats and blood samples were collected at specific time points. Plasma concentration-time curves were plotted and pharmacokinetic parameters were determined using the non-compartmental analysis. In addition, simulations of steady state concentrations of thymoquinone in male and female rats were performed using GastroPlus PK software. After oral administration, the maximum plasma concentration (Cmax) of thymoquinone was 4.52 ±â€¯0.092 µg/ml in male rats and 5.22 ±â€¯0.154 µg/ml in female rats (p = 0.002). Similarly, after intravenous administration, the Cmax was 8.36 ±â€¯0.132 µg/ml in males and 9.51 ±â€¯0.158 µg/ml in females (p = 0.550). The area under the plasma concentration-time curve (AUC)0-∞ following oral dosing was 47.38 ±â€¯0.821 µg/ml·h in females and 43.63 ±â€¯0.953 µg/ml·h in males (p = 0.014). Pharmacokinetics and plasma concentration vs. time profiles for multiple oral doses of thymoquinone in rats were predicted using a simulation model to compare the simulation results with the experimental plasma pharmacokinetic data. The differences observed in thymoquinone pharmacokinetics between male and female rats after a single dose were not evident for the simulated steady-state parameters. The findings suggest that the gender difference does not seem to play a significant role in thymoquinone disposition at steady state.

Toxicological evaluation of the flavour ingredient 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).

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.

Toxicological evaluation of a novel umami flavour compound: 2-(((3-(2,3-Dimethoxyphenyl)-1H-1,2,4-triazol-5-yl)thio)methyl)pyridine.

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.

Toxicological evaluation and metabolism of two N-alkyl benzamide umami flavour compounds: N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide and (R)-N-(1-methoxy-4-methylpentan-2-yl)-3,4-dimethylbenzamide.

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.

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 and 3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5,5-dimethylimidazolidine-2,4-dione.

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.

Toxicological evaluation of a novel cooling compound: 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-(2-thienylmethyl)acetamide.

A toxicological evaluation of a novel cooling agent, 2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-(2-thienylmethyl) acetamide (S2227; CAS 1374760-95-8), was completed for the purpose of assessing its safety for use in food and beverage applications. S2227 undergoes rapid oxidative metabolism in vitro, and in rat and dog pharmacokinetic studies is rapidly converted to its component carboxylic acid and secondary amine. S2227 was not found to be mutagenic or clastogenic in vitro, and did not induce micronuclei in polychromatic erythrocytes in vivo. The secondary amine hydrolysis product, N-(2-thienylmethyl)-1H-pyrazol-3-amine (M179), was also evaluated for genotoxicity. In subchronic oral toxicity studies in rats, the no-observed-adverse-effect-level (NOAEL) for S2227 was 100 mg/kg/day (highest dose tested) when administered by oral gavage for 90 consecutive days. Furthermore, S2227 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/day for both maternal toxicity and embryo/fetal development when administered orally during gestation to pregnant rats.

A tetravalent bispecific TandAb (CD19/CD3), AFM11, efficiently recruits T cells for the potent lysis of CD19(+) tumor cells.

To harness the potent tumor-killing capacity of T cells for the treatment of CD19(+) malignancies, we constructed AFM11, a humanized tetravalent bispecific CD19/CD3 tandem diabody (TandAb) consisting solely of Fv domains. The molecule exhibits good manufacturability and stability properties. AFM11 has 2 binding sites for CD3 and 2 for CD19, an antigen that is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. Comparison of the binding and cytotoxicity of AFM11 with those of a tandem scFv bispecific T cell engager (BiTE) molecule targeting the same antigens revealed that AFM11 elicited more potent in vitro B cell lysis. Though possessing high affinity to CD3, the TandAb mediates serial-killing of CD19(+) cells with little dependence of potency or efficacy upon effector:target ratio, unlike the BiTE. The advantage of the TandAb over the BiTE was most pronounced at lower effector:target ratios. AFM11 mediated strictly target-dependent T cell activation evidenced by CD25 and CD69 induction, proliferation, and cytokine release, notwithstanding bivalent CD3 engagement. In a NOD/scid xenograft model, AFM11 induced dose-dependent growth inhibition of Raji tumors in vivo, and radiolabeled TandAb exhibited excellent localization to tumor but not to normal tissue. After intravenous administration in mice, half-life ranged from 18.4 to 22.9 h. In a human ex vivo B-cell chronic lymphocytic leukemia study, AFM11 exhibited substantial cytotoxic activity in an autologous setting. Thus, AFM11 may represent a promising therapeutic for treatment of CD19(+) malignancies with an advantageous safety risk profile and anticipated dosing regimen.