Since it exerted potent effects in vitro, we determined whether FBA-TPQ could also show anti-tumor effects in vivo. Nude mice bearing Panc-1 xenograft tumors were treated with vehicle or FBA-TPQ at 5 or 10 mg/kg/day, 5 days per week. Compared with control group, treatments of 5 and 10 mg/kg/day resulted in 77.8 (P < 0.01) and 90.1% (P < 0.01) inhibition of tumor growth, respectively (Figure 3a,b), with significant tumor regression or complete remission being seen in both treated groups. No significant host toxicity (using body weight as a surrogate marker) was observed at any of the doses (Figure 3c), suggesting that the FBA-TPQ can be safely given as a novel therapeutic agent.

Previous studies have shown that FBA-TPQ has nanomolar/low micromolar IC₅₀ values against prostate, breast, and ovarian cancer cell lines [13,14,15]. The compound inhibited cell growth, decreased cell proliferation, induced apoptosis, and arrested cell cycle distribution. In addition, normal cells were not as sensitive to the compound. In vivo study demonstrated that FBA-TPQ has potent activity against breast and ovarian cancer xenograft tumors [14,15]. Mechanistic studies revealed that FBA-TPQ down-regulated MDM2, E2F1, Bcl-2, PI3K proteins, up-regulated Fas, Bax, p53/p-p53, ATM/p-ATM, γH2AX, and led to increased cleavage of caspases-3, -8 and -9 both in vitro and in vivo [13,14,15]. These findings suggested that FBA-TPQ has potent anti-cancer activity due to inhibition of oncogenes, activation of the DNA damage response and PI3K-Akt pathways. In summary, our preclinical efficacy and safety data indicate that FBA-TPQ can be a candidate anticancer drug for future development. In subsequent studies, we developed analytical methods and determined pharmacological properties of the compound, including initial pharmacokinetic and metabolic studies.

The chromatographic conditions, such as the mobile phase composition, elution gradients, and column temperature, were optimized during the development of the RRLC methods. Acetonitrile was chosen over methanol for its ability to reduce back-pressure and achieve better separation. BA-TPQ (Figure 1b) was chosen as the internal standard due to its similarity to FBA-TPQ in terms of extractability and chromatographic behavior [11,12,22,23].

To determine the extent to which FBA-TPQ is bound by plasma proteins, pooled plasma samples were examined for their capacity to bind the compound at concentrations of 0.5 μM and 5 μM. FBA-TPQ was considerably bound to proteins in mouse plasma, with 72.32% ± 2.53%; and 77.78% ± 1.17% of the compound being bound to plasma proteins at concentrations of 0.5 μM and 5.0 μM, respectively. Extensive binding of a candidate compound to plasma proteins can impair its biodistribution and may therefore impact its therapeutic efficacy. However, compounds that are highly bound to proteins display longer half-lives, durations of action, and higher volumes of distribution. In the present study, FBA-TPQ exhibited considerable protein binding, which may have limited the in vitro degradation of the compound, as described below.

As a preliminary study of the metabolism of FBA-TPQ, the compound was incubated with S9 fractions containing Phase I and Phase II metabolic enzymes and their co-factors for 15, 30, 45, or 60 min. These studies indicated that FBA-TPQ undergoes significant metabolism in the presence of both Phase I and Phase II enzymatic components, with approximately 14.3% of the compound being metabolized by Phase I and 1.8% of the compound being metabolized by Phase II components during the 60 min incubation. No specific peaks that seemed to correspond to unique metabolites were identified using the current RRLC method.

To assess the disposition of FBA-TPQ in vivo, pharmacokinetic studies in BALB/c were conducted. The results of the mean plasma concentration-time curves of FBA-TPQ, and tissue concentration-time curves of FBA-TPQ, after intravenous or intraperitoneal injection in BALB/c mice are presented in Figure 5. Two-compartmental model (WinNonlin 6.0 Model 7, Pharsight Corporation: Mountain View, CA, USA, 2009) was used to analyze the data obtained following intravenous (iv) and intraperitoneal (ip) administration of the compound. The pharmacokinetic parameters are presented in Table 4.

After a single bolus intravenous dose of FBA-TPQ (5 mg/kg), there were relatively high concentrations of the compound in the plasma for the first 30 minutes, with a rapid decline thereafter, as indicated by its half-life of 0.248 h, and clearance rate of 8.46 (L/hr/kg) in BALB/c mice (Table 4). The iminoquinone was no longer detectable in mouse plasma 4 h after the injection. The highest overall concentrations in the tissues examined were in the order kidneys > lungs > heart > spleen > plasma ≈ liver > brain.

Following intraperitoneal administration, the concentration of the compound in plasma decreased rapidly from 542.5 ng/mL at 5 min to 40.25 ng/mL at 2 h, to undetectable levels 4 h after administration. The highest overall tissue concentrations after intraperitoneal injection were in the order: kidneys > spleen > lungs > liver > heart > brain. The intraperitoneal bioavailability of FBA-TPQ in BALB/c mice was estimated to be 28.91%, based on the AUC values.

The pharmacokinetic profiles for the tissues obtained in our study suggest that there is high drug accumulation in the lungs, kidneys, and spleen of the mice, and that it reaches low concentrations in the brain. Furthermore, pharmacokinetic modeling confirmed that these tissues exhibited the highest drug exposure (AUC), longest half-life, and lowest clearance values, indicating that these organs may be potential sites of FBA-TPQ toxicity.

Our study also investigated the pharmacokinetic profile of FBA-TPQ following both intravenous (5 mg/kg) and intraperitoneal (10 mg/kg) administration. Based on the AUC values, the bioavailability of FBA-TPQ following intraperitoneal injection was relatively low, presumably because FBA-TPQ underwent extensive metabolism after ip injection and/or distributed in tissues quickly. Ultimately, it would be optimal if the compound could be administered orally, because patients greatly prefer oral agents. Studies are currently underway to develop a method for oral delivery of the compound.

FBA-TPQ and BA-TPQ (Figure 1a,b) were kindly provided by Dr. Sadanandan Velu (University of Alabama at Birmingham). Acetonitrile and methanol were purchased from Fisher Scientific (Atlanta, GA, USA). All other chemicals were purchased from Sigma Chemical Company (St. Louis, MO, USA). Heparinized samples of non-Swiss albino mouse plasma were obtained from Lampire Biological Laboratories (Pipersville, PA, USA). Hepatic S9 fractions (20 mg/mL) from male ICR/CD-1 mice were purchased from Celsis In Vitro Technologies (Chicago, IL, USA). All chemicals and solvents used for sample preparation and the rapid resolution liquid chromatography analysis were of analytical grade. Cell culture supplies and media, phosphate-buffered saline (PBS), fetal bovine serum (FBS), sodium pyruvate, non-essential amino acids, and penicillin-streptomycin were obtained from Invitrogen (Carlsbad, CA, USA).

Human pancreatic cancer cells were obtained from the American Type Culture Collection (Rockville, MD). All cell culture media contained 10% FBS and 1% penicillin/streptomycin. HPAC cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 2 µg/mL insulin, 5 µg/mL transferrin, 40 ng/mL hydrocortisone, 10 ng/mL epidermal growth factor and 5% fetal bovine serum. Panc-1 cells were cultured with RPMI 1640 containing 1 mM HEPES buffer, 25 µg/mL gentamicin, 1.5 g/L sodium bicarbonate, and 0.25 µg/mL amphotericin B. Mia PaCa-2 and IMR-90 cells were grown in DMEM media.

The effect of the test compound on human pancreatic cancer cell viability, expressed as the percentage of cell survival, was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The MTT assay is colorimetric assay for measuring the activity of enzymes that reduce MTT to formazan dyes, giving a purple color. Cells were grown in 96-well plates at 4–5 × 10³ cells per well and exposed to different concentrations of the test compound (0, 0.1, 0.25, 0.5, 0.75, 1, 5 and 10 µM). After incubation for different times, 10 µL of the MTT solution (5 mg/mL; Sigma; St. Louis, MO, USA) were added into each well. The plates were incubated for 2–4 h at 37 °C. The supernatant was then removed and the formazan crystals were dissolved with 100 µL of DMSO. The absorbance at 570 nm was recorded using an OPTImax microplate reader (Molecular Devices; Sunnyvale, CA, USA). The cell survival percentages were calculated by dividing the mean OD of compound-containing wells by that of DMSO-containing control wells. Three separate experiments were accomplished to determine the IC₅₀ values.

Following a similar protocol as above, cells in early and late stages of apoptosis were detected using an Annexin V-FITC apoptosis detection kit from BioVision (Mountain View, CA, USA), according to the manufacturer’s protocol. For apoptosis experiments, 2–3 × 10⁵ cells were exposed to the test compound (0, 0.1, 0.5 and 1.0 µM) and incubated for 48 h prior to analysis. Cells were collected and washed with serum-free media. Cells were then re-suspended in 500 µL of Annexin V binding buffer followed by addition of 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI). The samples were incubated in the dark for 5 min at room temperature and analyzed with a Becton Dickinson FACSCalibur instrument (Ex = 488 nm; Em = 530 nm). Cells that were positive for Annexin V-FITC alone (early apoptosis) and Annexin V-FITC and PI (late apoptosis) were counted. The apoptotic index was calculated against the untreated control cells.

To determine the effect of FBA-TPQ on the cell cycle, cells (2–3 × 10⁵) were exposed to the test compounds (0, 0.1, 0.5 or 1 µM) and incubated for 24 h prior to analysis. Cells were trypsinized, washed with PBS, and fixed in 1.5 mL of 95% ethanol and 0.5 mL of 0.9% saline solution at 4 °C overnight, followed by incubation with RNAse and staining with propidium iodide (Sigma). The DNA content was determined by flow cytometry. The cell cycle distribution was evaluated by comparing with that of the control cells.

An Agilent 1200 Series Rapid Resolution Liquid Chromatography system (Agilent Technologies, Waldbronn, Germany) consisted of an on-line degaser, a binary pump, a high performance SL autosampler, a thermostated column compartment, and a photodiode array UV-vis detector. Determination of FBA-TPQ was achieved using a Zorbax XDB-C₁₈ (1.8 μm, 50 × 4.6 mm) analytical column with a LiChroCART 100 RP-18 guard column. The column eluate was monitored by a diode array detector at 354 nm. The temperature was maintained at 35 °C. The system was set to use a linear gradient at a flow rate of 0.9 mL/min, increasing the acetonitrile-water ratio from 10:90 at 0 min to 38:62 at 3.5 min, keeping this ratio until 5 min, and then increasing to 85:15 in 0.1 min, maintaining this ratio for 1 min, and then returning to 10: 90 in 0.4 min, for a total running time of 8 min.

A 1 mM stock solution of FBA-TPQ was prepared in methanol and stored at −80 °C. A series of standard working solutions with FBA-TPQ concentrations of 0.1, 0.25, 0.5, 1.0, 5.0, 10.0, 50.0 and 100.0 μM were obtained by further dilution of the stock solution with methanol. The standard serum samples for calibration were prepared at concentrations of 0.1, 0.25, 0.5, 1.0, 5.0, and 10.0 μM in control (drug-free) pooled mouse plasma.

Calibration curves were prepared by spiking 20 μL of the appropriate standard solution and 20 μL BA-TPQ (IS) into 160 μL of blank mouse plasma. The 40 µM BA-TPQ solution was prepared by diluting a stock solution of 100 µM BA-TPQ with methanol. Quality control samples of low, medium and high concentrations were prepared to assess the accuracy and precision of the bioanalytical method. For the calibration of the tissue samples, a plasma sample was supplemented with tissue homogenates in PBS.

Acetonitrile (600 μL) and BA-TPQ (IS, 20 μL) were added to 200 μL of plasma to precipitate proteins. The mixture was vigorously vortexed for 1 min and centrifuged at 14,000 × g for 10 min. Then, the supernatant was transferred to a glass tube and evaporated to dryness at 40 °C under a stream of nitrogen in a TurboVap^® LV Concentration Workstation (Caliper Lifesciences, Hopkinton, MA, USA). The resulting supernatant was transferred to a new glass tube and evaporated to dryness. Finally, samples were reconstituted in 200 μL mobile phase, and 10 μL was injected into the RRLC system for analysis.

To confirm its accuracy, selectivity, reproducibility, and specificity, the method was validated as follows: Evaluation of the method selectivity was performed by analyzing the blank plasma from six different preparations to test for interference at the retention times of the analyte. The linearity of the method was determined by plotting the peak area ratios of the analyte to the internal standard (IS) against the concentrations of FBA-TPQ in mouse plasma and various tissues. This was done in duplicate on three consecutive validation days. The precision and accuracy of the method were assessed by the determination of QC samples at three concentration levels, in six replicates, on three validation days. For the intra- and inter-day precision, we required that there was less than 15% variation, and also required the accuracy to be within ±15%, which is within the limits proposed in the US FDA guidelines [24]. Limit of detection (LOD) was the lowest concentration of an analyte that the bioanalytical procedure can reliably differentiate from background noise. The LLOQ, defined as the lowest concentration on the calibration curve at which both precision and accuracy were less than or equal to 20%, was evaluated by analyzing samples prepared in six replicates on three consecutive validation days [24]. The recovery of FBA-TPQ was determined by comparing the mean peak areas of the pretreated QC samples at three concentration levels (six samples each) to those of spike-after-extraction samples.

The stability of FBA-TPQ was evaluated at two concentrations (0.5 and 5.0 µM). The compound was incubated under the following conditions: 37 °C for 0, 1, 2, 4, and 8 h; 4 °C for 0, 1, 4, 8, and 24 h; and −80 °C for 0, 1, 2, 4, and 12 weeks. At the designated time points, the samples were extracted and analyzed using the established RRLC analytical method. The stability of FBA-TPQ was assessed by comparing the initial concentration (time 0) with the final concentration following incubation of the compound at the noted temperatures, and expressing this difference as a percentage of the initial concentration.

The extent of binding to mouse plasma proteins by FBA-TPQ was determined using a micro-ultrafiltration system as previously described [25]. FBA-TPQ was dissolved in methanol and added to pooled mouse plasma to yield final concentrations of 0.5 µM and 5.0 µM. Control solutions were prepared in methanol to account for non-specific binding of FBA-TPQ to the filter membranes. Duplicate preparations of each concentration were incubated at 37 °C for 1 h, before being placed into the sample reservoirs of Amicon Centrifree^® ultrafiltration tubes (30 kDa exclusion; Millipore).

A preliminary study of the metabolism of FBA-TPQ was done using murine hepatic S9 fractions containing Phase I and II metabolic enzymes. The reaction mixture contained 10 μM FBA-TPQ (in methanol), 1 mg/mL S9 and 100 mM Tris buffer (pH 7.4) (experimental controls did not contain the hepatic S9 fractions.) Metabolic reactions were initiated by adding NADPH-regenerating systems to the reaction mixtures, and samples were incubated in a water bath at 37 °C. Duplicate aliquots of the mixtures were taken at 0, 15, 30, 45, and 60 min, and the samples were processed and analyzed. The percent of FBA-TPQ that was metabolized by Phase I and Phase II enzymes was calculated by comparing the initial concentration of the iminoquinone to the measured concentration following incubation.

Experimental data are expressed as means and standard deviations, and the significance of differences was analyzed by ANOVA or Student’s t-test, as appropriate.