Vehicle development, pharmacokinetics and toxicity of the anti-invasive agent 4-fluoro-3’,4’,5’-trimethoxychalcone in rodents

Effective inhibitors of invasion and metastasis represent a serious unmet clinical need. We have recently identified 4-fluoro-3’,4’,5’-trimethoxychalcone or C16 as a potent anti-invasive molecule. In this paper, we report on the development of an optimized vehicle for oral administration of C16. We also explore its pharmacokinetic and toxicity profile in rodents as a prelude to a broad-scope evaluation as a pharmacological tool in animal models of disease. C16 showed suboptimal pharmacokinetics with limited oral bioavailability and whole blood stability. Rapid metabolism with elimination via glutathione conjugation was observed. An oral dosing routine using medicated gels was developed to overcome bioavailability issues and yielded sustained whole blood levels above the half maximal effective concentration (EC50) in a 7-day study. The compound proved well-tolerated in acute and chronic experiments at 300 mg/kg PO dosing. The medicated gel formulation is highly suitable for evaluation of C16 in animal models of disease.


A. Vehicle development
Quantification was performed by calculating the ratio of the peak area (counts) of C16 and the IS. Results are expressed as relative differences to the peak ratio at 0 h (%RE, Table A).    Peak ratios for stability assessment Peak ratios for C16/IS, relative to the first time point, were calculated for three wavelengths (Table D).  A). The samples were analyzed according to the method described above for the stability test of C16 in Medigel Sucralose (Table E).

b. Results
Individual plasma concentrations for C16 are shown in Table F and Table G. All data are expressed as ng/mL of the free drug. Samples that were below the limit of quantitation were not used in the calculation of averages. Plasma     Area under the curve, extrapolated to infinity; ND: Not determined; BLOQ: Below the limit of quantitation (1 ng/mL); a Not determined due to correlation coefficient (R 2 ) was less than 0.85; b Dose normalized by dividing the parameter by the nominal dose in mg/kg; c Bioavailability determined by dividing the individual dose normalized oral AUClast by the average dose normalized intravenous AUClast value.

a. Solution preparation
The dosing solution was prepared as a cassette to contain all three test articles in the same solutions using a vehicle consisting of 20% DMSO/10% (DMSO/Cremophor EL 1:1)/70% Milli-Q water.

b. Data
Individual blood concentrations and pharmacokinetic parameters are shown in Table H through Table J. All data are expressed as ng/mL of the free drug. Samples that were below the limit of quantification were not used in the

C. Stability and metabolism
For details on ADME experiments not included underneath, please consult ref. [

b. Results
The areas and percentages for the parent and metabolites have been calculated using MIM data where parent to parent transitions have been used, and assume that each metabolite has the same point of ionisation and the sensitivity of the metabolite has not been affected by the biotransformation. All remaining peaks in the sample were either in the control sample or not believed to be related to test compound. See Table M through Table P.    25  25  24  24  23  23  25  25  24  24  24  25  23  26  23  23  23  22  Irritability -

D.B. IP Administration
C16 was administered IP at an initial dose of 10 mg/kg to a group of 5 ICR derived male mice weighing 22±2 g. The animals were then observed for presence of acute toxic symptoms and autonomic effects during the first 30 min and after 1 h, 2 h, 6 h and 24 h (Table S-Table U). The next dose level was determined based on whether more than 50% animals died within 30 min after dosing. Gross necropsy was performed in all animals without tissue collection.

E.B. Toxicology
An overview of all details and raw data is presented in Table V-Table Z.  .7 Consumption was recorded at the same time each day (morning). a Relative overestimation gel consumption due to removal debris from bedding material and fecal matter. b Start undoped gels in 0, 100 and 300 mg/kg cohorts; c Start treatment with 0, 100 or 300 mg/kg C16 in medicated gels; d Animals were fasted during night between days 6 and 7. 5.4 6.7 6.1 5.7 6.1 6.1 Consumption was recorded at the same time each day (morning). a Start undoped gels in 0, 100 and 300 mg/kg cohorts; b Start treatment with 0, 100 or 300 mg/kg C16 in medicated gels; c Animals were fasted during night between days 6 and 7.

E.C. Whole blood C16 levels
The concentration of C16 in the individual hemolyzed mouse blood samples is presented in Table AA and Table BB. 1±27.8 BLOQ: below the limit of quantitation (0.5 ng/mL). Samples that were below the limit of quantification were not used in the calculation of averages.