SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

BPR0C261 is a synthetic small molecule compound cytotoxic against human cancer cells and active prolonging the lifespan of leukemia mice. In the present study, we further investigated the mechanisms of its anticancer action and found that BPR0C261 inhibited microtubule polymerization through interacting with the colchicine binding sites on tubulins, disrupted microtubule arrangement and caused cell cycle arrest at G2/M phase in cancer cells. BPR0C261 also inhibited the clonogenic growths of cancer cells and showed cytotoxicity against human cervical cancer cells of multidrug-resistant phenotype. In addition, BPR0C261 concentration-dependently inhibited the proliferation and migration of HUVECs and disrupted the endothelial capillary-like tube formations in HUVEC and rat aorta ring cultures. Given orally, BPR0C261 inhibited angiogenesis in s.c. implanted Matrigel plugs in mice. Notably, its IC50 values against the endothelial cell growths were approximately 10-fold lower than those against the cancer cells. It was found orally absorbable in mice and showed a good oral bioavailability (43%) in dogs. BPR0C261 permeated through the human intestinal Caco-2 cell monolayer, suggesting oral availability in humans. Orally absorbed BPR0C261 distributed readily into the s.c. xenografted tumors in nude mice in which the tumor tissue levels of BPR0C261 were found oral dose-dependent. BPR0C261 showed in vivo activities against human colorectal, gastric, and nasopharyngeal tumors in nude mice. Most interestingly, the combination of BPR0C261 plus cisplatin synergistically prolonged the lifespans of mice inoculated with murine leukemia cells. Thus, BPR0C261 is a novel orally active tubulin-binding antitumor agent with antimitotic, apoptosis-inducing, and vasculature disrupting activities. (Cancer Sci 2011; 102: 182–191)

Microtubules are proven molecular targets on which anticancer drugs have been shown to have promising efficacies when used in cancer patients. There are clinically available antimitotic microtubule destabilizers such as Vinca alkaloids (vincristine, vinblastine, and vinorelbine),(1) and microtubule stabilizers (epothilone and taxanes).(2,3) Tubulin binding agents interact with the microtubule assembly kinetics, induce aberrations in cell cycle progression, and cause cell death in cancer cells.(4) Alterations in tubulins are associated with the resistance to Vinca alkaloids(5) and taxanes.(6,7) Continuous efforts to find new tubulin targeting agents with improved drug properties and antitumor activity spectrum have been active.(8,9) Tubulin colchicine site binders that inhibit microtubule polymerization are promising novel anticancer compounds, including combretastatin A4 phosphate.(8,10–12) A number of new tubulin targeting agents are currently in preclinical and clinical development, among which only a few are given in oral formulations. However, most of these tubulin-targeted compounds have, so far, shown limited objective responses in clinical trials.(9)

Angiogenesis is the formation of new blood vessels from existing vasculatures. It is tightly regulated during physiological processes and involves complex molecular and cellular regulations. Angiogenesis is, however, tipped toward uncontrolled processes during pathogenesis and therefore implicated as the underlying mechanism for a number of chronic diseases, including tumorigenesis.(13) The first anti-angiogenic anticancer agent, bevacizumab, is indicated for the treatment of colorectal cancer and active as a single drug and in combination with cytotoxic agents in preclinical and clinical studies.(14,15) Orally active sorafenib exerts dual antitumor and anti-angiogenic activities and was recently proven to be beneficial to patients.(16) Increasing research efforts have therefore been focused on searching particularly for orally active antitumor compounds with anti-angiogenic activity. The advantages of oral dosing include better patient compliance,(17) ease of use, and low cost, compared to that of parenteral injection.(17,18) However, the clinically available orally active anticancer drugs are limited in choice and activity spectra. The search for orally active anticancer drugs with low toxicity and attainable chemical properties for proper formulations has been extensive,(10,19,20) and has led to the discovery of orally active tubulin-targeting anticancer agents.(9)

D-24851 is a microtubule disrupting anticancer agent that exerts oral antitumor activities in prolonging the lifespan of leukemia mice and suppressing the growth of rat sarcomas.(21) Structurally analogous to D-24851, a few hundred novel N-heterocyclic indolyl glyoxylamides were synthesized and BPR0C261, N-(3-methyl-5-isothiazolyl)-2-1-[(3-methyl-5-isoxazolyl-)methyl]-1H-3-indolyl-2-oxoacetamide (Fig. 1) was identified with cytotoxic and apoptosis induction activities against a panel of human cancer cells.(22) It was also previously shown to have oral activity in prolonging the lifespans of inbred mice inoculated withe murine leukemia P388 cells. In the present study, we further examined whether antimitosis and anti-angiogenesis contribute to the underlying mechanisms of the antitumor action of BPR0C261, and we explored its in vivo anticancer activity against human solid tumors. Pharmacokinetic parameters and oral bioavailability of BPR0C261 in mice and dogs were determined. The potential therapeutic benefit of a combinational chemotherapy of BPR0C261 plus a DNA-intercalating cisplatin was also investigated.

image

Figure 1.  Chemical structure of BPR0C261, N-(3-methyl-5-isothiazolyl)-2–1[(3-methyl-5-isoxazolyl-) methyl]-1H-3-indolyl-2-oxoacetamide.

Download figure to PowerPoint

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cell cultures.  Human colorectal Colo205, cervical KB, gastric MKN-45 and NUGC-3, and nasopharyngeal TW-039 cancer cells were propagated in RPMI-1640 containing 10% FBS (Invitrogen Taiwan, Taipei, Taiwan). KB-derived drug resistant KB100,(23) KB-VIN10, and KB-7D(24) cell lines were cultured in the presence of 100 nM camptothecin, 10 nM vincristine, and 7 μM VP-16, respectively. Murine leukemia P388 cells were cultured in RPMI-1640 containing 10% FBS and 50 μM 2-mercaptoethanol.

Tubulin polymerization assay.  Tubulin polymerization turbidity assay was carried out as previously reported.(25) Bovine brain microtubule-associated protein-rich heterodimeric tubulins (Cytoskeleton, Denver, CO, USA) polymerized in a 100-μL solution mixture of PIPES (100 mM), EGTA (1 mM), and MgCl2 (1 mM, pH 6.6) containing BPR0C261 of various concentrations. The turbidity of the solution was measured by the optical absorbance at the wavelength of 350 nm using a PowerWave X reader from BioTek Instruments (Winooski, VT, USA). The area under the polymerization versus time curve normalized to that of the vehicle control was used to estimate the IC50 value that inhibits 50% of tubulin polymerization activity.

Colchicine binding assay.  Binding of [3H]-colchicine ([ring C, methoxy-3H]-colchicine; PerkinElmer, Waltham, MA, USA) to bovine brain microtubule-associated protein-rich heterodimeric tubulins was measured by column centrifugation as described.(25,26) Solutions of tubulins (2.5 μM) and [3H]-colchicine (0.1 Ci/mmol, 2.5 μM) containing BPR0C261 and cold colchicine at the indicated concentrations were incubated in the polymerization buffer at room temperature for 1 h. The reaction solutions were centrifuged through Sephadex G-50 columns at 50 μL per column. The eluates were analyzed for radioactivity in c.p.m. using a scintillation counter. The percentage of the bound compound was calculated using the equation (c.p.m. with compound − c.p.m. without tubulin)/(c.p.m. with DMSO − c.p.m. without tubulin) × 100%.

Tubulin immunostaining.  Modified from a previous report,(25) human gastric cancer NUGC-3 cells were treated with 1 μM BPR0C261 for 16 h. The cells were washed with PBS and fixed with 4% formaldehyde at room temperature for 25 min. The cells were incubated in the staining solution (1 μg/mL Hoechst 33342 in 0.1% Triton X-100 in PBS) at room temperature for 5 min. After being rinsed with PBS, the fixed cells were then blocked with 5 mg/mL BSA at room temperature for 30 min followed by an incubation with monoclonal anti-β-tubulin mouse IgG1 (1:200 dilution, Cat. No. T42026) purchased from Sigma (St. Louis, MO, USA) at 4°C overnight. The cells were incubated with goat anti-mouse IgG-FITC conjugates (1:300 dilution, Cat. No. F0257; Sigma) at room temperature for 1 h. The cellular images were visualized and photographs taken using a fluorescence confocal laser-scanning microscope (TCS SP2; Leica, Heidelberg, Germany).

Flow cytometry analysis.  Human gastric cancer NUGC-3 cells seeded at 1 × 106 cells/10-cm dish were incubated with BPR0C261 at 37°C for 24 h. The cell pellets were collected, permeabilized with a buffer (0.5% Triton X-100, 0.2 μg/mL Na2EDTA2H2O, and 1% BSA in PBS), and placed on ice for 15 min. The mixture was then combined with pre-cooled methanol and centrifuged at 300g for 5 min. The nuclear DNAs were stained with a sodium citrate (3.4 mM) solution containing propidium iodide (50 μg/mL) and RNase A (5000 units/mL) in the dark at 4°C for 30 min. DNA contents were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Bedford, MA, USA).

DNA fragmentation assay.  Oligonucleosomal fragments of the genomic DNA isolated from the BPR0C261-treated human gastric cancer MKN-45 cells were analyzed as previously described.(25) MKN-45 cells (3 × 105 cells/mL) were incubated with BPR0C261 at the indicated concentrations for 48 h. Both floating and adherent cells were collected and the DNA fragments were extracted with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1 = v/v/v), separated on a 2% agarose gel, and visualized by ethidium bromide staining.

Clonogenic growth assay.  The inhibition activity of BPR0C261 on the clonogenic growth of human cancer cells was evaluated as previously reported.(27) Cervical KB and colorectal Colo205 cancer cells were seeded at 150 and 1000 cells/well, respectively, and treated with BPR0C261 at 1, 10, and 100 nM for 24 h. The cells were then allowed for clonogenic proliferation in the absence of BPR0C261 for 8–10 days, followed by fixation and staining with 0.5% crystal violet in 6% glutaraldehyde in PBS. The numbers of the growing clusters containing at least 25 crystal violet stained cells were counted and the IC50 values were determined using GraphPad software (San Diego, CA, USA).

Tumor histoculture system.  Histocultures of human gastric MKN-45 tumors were established as previously described.(28) The histocultures were treated with BPR0C261 at the indicated concentrations for 96 h, followed by an incubation with the culture medium containing 40 μM BrdU for 48 h. The BPR0C261 treated tumor histocultures were formalin-fixed, paraffin-embedded, and sliced into 5-μm thick sections for BrdU immunohistochemical staining and TUNEL assay.

Immunohistochemistry of BrdU.  The proliferating BrdU-incorporated tumor cells in the histocultured tissue sections were immunostained and visualized as previously reported.(28) Tumor sections were incubated with BrdU mouse monoclonal antibody M744 (1:250 dilution) from Dako (Carpinteria, CA, USA) for 2 h, followed by an incubation of HRP-conjugated secondary antibody against mouse IgG from the Dako LSAB2 detection kit, and chromogenic reaction using 3-3′-diaminobenzidine (DAB) substrate kit from Dako according to the manufacturer’s instructions. The proliferating tumor cells were stained in brown nuclei and the tissue sections were counterstained with hematoxylin.

TUNEL assay.  The induction of apoptosis in the histocultured tumor cells caused by BPR0C261 treatments was detected with an ApopTag detection kit (Intergen, Purchase, NY, USA).(28) The histocultured tumor sections were deparaffinized, rehydrated, and digested with proteinase K before they were incubated with TdT enzyme, digoxigenin-dUTP, and anti-digoxigenin peroxidase conjugates for TUNEL reactions. TUNEL-positive tumor cells or apoptotic bodies were labeled in brown nuclei by chromogenic reaction using the DAB substrate kit (Dako) followed by hematoxylin counterstain.

Cell growth inhibition assay.  Human cervical cancer KB and KB-derived cells were seeded at 5000 cells/well in 24-well plates and treated with various concentrations of BPR0C261 for 72 h followed by methylene blue dye assay.(24) HUVECs were cultured in M200 medium (Cascade Biologics, Portland, OR, USA) containing the endothelial cell growth supplement (Millipore, Billerica, MA, USA), seeded at 6000 cells/well in 96-well plates, and treated with various concentrations of BPR0C261 for 72 h, followed by the colorimetric 3-(4,5-dimethylthisazol-2-yl)-2,5-diphyenyltetrazolium bromide (MTS)/phenazine methosulfate (PMS) assay.(22) The IC50 concentrations inhibiting the growth of 50% cultured cells were estimated.

Endothelial cell migration assay.  The endothelial migration ability was measured as previously reported(29) with modifications. HUVECs were seeded at 3 × 105 cells/well in 12-well plates and cultured in serum-free medium for 24 h. A narrow area on the cultured monolayer was scratched off using a pipette tip and the cultures were incubated in the presence of BPR0C261 at the concentrations indicated. At the incubation times of 0, 4, 8, and 24 h, the numbers of the migrating cells were counted and photographs were taken.

Endothelial capillary-like tube formation assay.  The assay was carried out as previously described with modifications.(30) HUVECs were seeded at 1.5 × 104 cells/well onto the Matrigel-coated 96-well plates and incubated in the presence of BPR0C261 (0.0025–25 μM) at 37°C for 4 h. The 2D capillary-like tube formations of HUVECs were visualized using MTT staining. The images of the MTT-stained capillary-like tube formations were acquired using a MagnaFire SP digital camera from Optronics (Goleta, CA, USA) and the integrated area intensities of the capillary network were estimated by computer-aided software Angiogenesis Image Analyzer from Kurabo (Osaka, Japan). The IC50 value inhibiting 50% of the capillary tube formation activities was calculated.

Rat aorta tube formation assay.  Rat aorta cultures were set up as previously reported.(31) Six-week-old male Sprague–Dawley rats purchased from the National Laboratory Animal Center (NLAC, Taipei, Taiwan) were killed and the thoracic aortas were harvested, cross-sectioned into rings of 1-mm thickness, and cultured at one ring per well in 48-well plates. After 1 day in culture, the aorta rings were treated with BPR0C261 at the indicated concentrations for 5 days in culture at 37°C in a CO2 incubator. The cultures were subjected to the 3-(4,5-dimethylthisazol-2-yl)-2,5-diphyenyltetrazolium bromide (MTS)/phenazine methosulfate (PMS) colorimetric reaction from which the OD values of the solutions at 490 nm were measured. The IC50 concentration inhibiting 50% of 3D endothelial capillary network growth was determined.

Matrigel plug assay in mice.  Matrigel plug assay for the assessment of in vivo angiogenesis activity in mice was carried out.(32) Matrigel containing 50 ng/mL vascular endothelial growth factor (VEGF) was s.c. implanted at 0.5 mL/mouse into 7-week-old male C57BL/6JNarl mice (NLAC). The mice were orally gavaged with BPR0C261 at 100 and 200 mg/kg/day for five consecutive days per week, for 2 weeks. The mice were then killed and the Matrigel plugs were harvested, which were then extracted for measurements of the hemoglobin contents by spectrophotometry using the Drabkin (Sigma) method.

Permeability assay in human intestinal Caco-2 cells.  Caco-2 cells cultured in monolayer were used to predict in vivo intestinal permeability of BPR0C261.(19) Caco-2 cells seeded at 3 × 105 cells/well and grew in 6-well Transwell plates (Corning, Lowell, MA, USA) to a confluence where the transmonolayer electrical resistance was >2300 Ω cm2, followed by treatments with 20 μM BPR0C261 or doxorubicin in the upper chambers. Aliquots of 200 μL medium in the lower chambers were collected hourly after the drug application and stored at −80°C until HPLC analysis. The medium was spiked with an equal volume of acetonitrile and centrifuged at 20 000g for 20 min. The supernatants of 50 μL were analyzed using a Zorbax Eclipse XDB-C18 column with mobile phase (acetonitrile with 10 mM ammonium acetate in 0.1% formic acid) and flow rate at 0.5 mL/min in an LC/MSD system (Agilent, Palo Alto, CA, USA).

Measurement of BPR0C261 tissue levels in tumors s.c. xeno-grafted in nude mice.  BPR0C261 was orally gavaged in a vehicle mixture of DMSO/Cremophor EL (polyoxyl 35 castor oil from BASF, Ludwigshafen, Germany)/water (5/20/75%: v/v/v) to adult male BALB/c nude mice (NLAC) each bearing an s.c. growing human colorectal Colo205 tumor of approximately 1 cm3. At 2, 6, 24, and 48 h after the dosing, the animals were killed and tumors were harvested and homogenized. A 30-μL homogenate aliquot of each tumor was mixed with 60 μL acetonitrile containing internal standard BPR0L187 (500 ng/mL) followed by centrifugation at 15 000g for 20 min. As previously reported with modifications,(33,34) the supernatants of 15-μL aliquots were subjected to LC-MS/MS analyses for BPR0C261 levels using a Zorbax Eclipse XDB-C8 column with mobile phase: water containing 0.1% formic acid (A) and acetonitrile (B) and gradient profile: 0.0–1.2/5, 1.3–3.9/95, and 4.0–5.0/5 (min/%B) at a flow rate of 1.5 mL/min.

Pharmacokinetic studies in mice and beagles.  BPR0C261 was given i.v. or p.o. to adult male BALB/c mice (= 3 per time point) (NLAC) and beagles (= 4) (Nosan, Yokohama, Japan) and blood samples of 1–2 mL were collected by cardiac puncture (mouse) and cephalic vein catheter (beagle), respectively, at 2, 5, 15, and 30 min and 1, 2, 4, 6, 8, 12, 24, and 30 h after the dosing. Plasma samples were prepared for measurements of BPR0C261 using the same LC-MS/MS condition as described above for measuring BPR0C261 concentrations in the tumors. Plasma concentration time data were analyzed by the non-compartmental model using WinNonlin (Pharsight, Mountain View, CA, USA) for estimation of pharmacokinetic parameters,(19) such as: AUC(0–∞), area under concentration–time curve from 0 to infinity; Cmax, peak concentration; Tmax, time to reach the peak concentration; CL, total body clearance; T1/2, terminal half-life; and Vdss, volume of distribution at the steady state. The absolute bioavailability, F% = (AUCp.o./AUCi.v.) × (Dosei.v./Dosep.o.) ×100, was calculated.

Subcutaneously xenografted human tumors in nude mice.  Ado-pted from the previous report,(28) adult male BALB/c nude mice (NLAC) were s.c. implanted with human gastric MKN-45 (5 × 105), nasopharyngeal TW-039 (5 × 105), or colorectal Colo205 (1 × 106) cancer cells per mouse. Tumor sizes were measured using an electronic caliper and calculated in length × (width)2/2. Tumor size and animal body weight were monitored twice a week. The tumor-bearing mice were orally gavaged with BPR0C261 suspended in the vehicle mixture of DMSO/Cremophor EL/saline (5/20/75%:v/v/v) at the dose regimens as indicated.

Murine leukemia mouse model and combination treatments of BPR0C261 plus cisplatin.  The murine leukemia P388 cell-induced leukemia mouse model was adopted.(22) Adult male inbred DBA/2JNarl mice (NLAC) were i.v. inoculated with 1 × 106 P388 cells per mouse through the tail veins and the compound treatments were started at 24 h after the cell inoculation. The treatment regimens were: (i) oral gavage with BPR0C261 (100 and 200 mg/kg/day for four consecutive days, p.o.); (ii) i.v. (tail vein) administration with a single dose of cisplatin (5 and 7.5 mg/kg, i.v.); and the combination of regimens (i) and (ii). Mice orally dosed with the vehicle for only 4 days were included as a control group. DMSO/Cremophor EL/saline (5/20/75%:v/v/v) was used as the vehicle to deliver both compounds. The P388 cell-inoculated leukemia mice were examined and monitored for survival twice daily. A median survival time is the time at which 50% of the leukemia mice still survive. The median survival times were estimated and used to calculate the percentages of increase (normalized to the median survival time of the vehicle control group) in the lifespan of the treated leukemia mice.

Statistical analysis.  Data were subjected to t-test or anova followed by Student–Newman–Keuls test and the log–rank test was used for survival analysis using spss software (spss, Chicago, IL, USA). A P value <0.05 between groups was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

BPR0C261 inhibits microtubule polymerization.  As indicated in Figure 2(A), both the polymerization rate and plateau level of microtubule polymerization were inhibited in a concentration-dependent manner by BPR0C261 and completely abolished at 10 μM. BPR0C261 and colchicine had an IC50 of 0.14 and 0.03 μM (= 3) against tubulin polymerization, respectively, as shown in Figure 2(B). Furthermore, BPR0C261 at both 5 and 20 μM almost completely prohibited [3H]-colchicine from binding to tubulins as shown in Figure 2(C), indicating that BPR0C261 binds to tubulins and, thus, strongly interferes with colchicine binding to tubulins. Whether this interference on the colchicine binding by BPR0C261 is through a competitive or allosteric inhibition mechanism remains to be further elucidated.

image

Figure 2.  BPR0C261 inhibits tubulin polymerization by interacting with colchicine binding sites and causes aberration of microtubule arrangements. (A) Tubulin polymerization was concentration-dependently inhibited by BPR0C261. (B) The areas under the curves of the tubulin polymerization kinetics were calculated and normalized to the vehicle control to obtain the IC50 value for BPR0C261. (C) [3H]-Colchicine binding to tubulin was interfered by BPR0C261 and a stronger colchicine binding interference in the presence of BPR0C261 is noted. (D) Human gastric cancer NUGC-3 cells were treated with vehicle (left panel) and BPR0C261 at 1 μM (right panel) for 16 h, followed by DNA staining with Hoechst 33258 (blue). Tubulins were immunostained and visualized using IgG-FITC conjugates (green). Disorganized green fluorescent microtubule structures were observed. Scale line = 50 μm.

Download figure to PowerPoint

BPR0C261 disrupts microtubule structure in human cancer cells.  The microtubules of the BPR0C261-treated NUGC-3 gastric cancer cells were visualized by FITC green fluorescence, as shown in Figure 2(D) (right panel). A vehicle control was included, as shown in Figure 2(D) (left panel). Compared to the normally arranged microtubule structures in the vehicle-treated control, the disarrayed microtubule structures in the BPR0C261-treated NUGC-3 cells were observed with diffused and less dense green fluorescent tubulin-immunoreactive activities. It was also noted that the nuclei of the BPR0C261-treated NUGC-3 cells showed irregular shapes. Microtubules are one of the molecular targets mediating the BPR0C261 effects, which was indicated not only by observations of inhibiting tubulin polymerization (Fig. 2A–C) but also changes in the morphology of microtubules (Fig. 2D).

BPR0C261 arrests human cancer cells at the G2/M phase.  As shown in Figure 3(A), BPR0C261 caused cell cycle arrest at the G2/M phase in human gastric cancer NUGC-3 cells in a concentration-dependent manner. BPR0C261 at a low concentration (10 nM) inhibited the cell cycle and caused an accumulation of 69% of the NUGC-3 cells at the G2/M phase (Fig. 3B). The results suggest that BPR0C261 inhibits tumor growth in part by arresting the proliferating cancer cells at the G2/M phase.

image

Figure 3.  BPR0C261 causes cell cycle arrest and induces apoptosis in human cancer cells. (A) BPR0C261 caused G2/M phase arrest in human gastric cancer NUGC-3 cells in a concentration-dependent manner. (B) Percentages of the NUGC-3 cells in the cell cycle phases are shown (= 3). (C) BPR0C261 concentration-dependently inhibited clonogenic growths of human colorectal Colo205 and cervical KB cancer cells. Mean ± SEM (= 3). *< 0.05 versus vehicle. (D) BPR0C261 caused DNA fragmentation in human gastric cancer MKN-45 cells at 0.01 μg/mL or higher. (E) BPR0C261 inhibited proliferation (left panel) and induced apoptosis (right panel) of tumor cells in histocultured human gastric MKN-45 tumor xenografts. Representative tumor tissue sections stained for BrdU-incorporated proliferating and TUNEL-positive apoptotic tumor cells are shown. Scale lines = 30 μm.

Download figure to PowerPoint

BPR0C261 inhibits clonogenic growth and causes DNA frag-mentation in human cancer cells.  BPR0C261 significantly and concentration-dependently reduced the colony forming abilities of both human cervical KB and colorectal Colo205 cancer cells, which showed IC50 values of 10 and 46 nM, respectively (Fig. 3C). Apoptosis, as indicated by DNA fragmentation in the human gastric MKN-45 cancer cells, was caused by a 48-h treatment with BPR0C261 in a concentration-dependent relationship (Fig. 3D). Therefore, in addition to the cell cycle arrest at G2/M phase, apoptotic induction in the cancer cells is one of the underlying mechanisms for the anticancer effects of BPR0C261.

BPR0C261 inhibits tumor cell proliferation and induces apoptosis in histocultured human gastric tumors.  BPR0C261 inhibited, in a concentration-dependent manner, cell proliferation and induced apoptosis in human gastric cancer MKN-45 cells growing ex vivo in histocultured tumors. Representative tumor tissue sections stained for BrdU-labeled proliferating and TUNEL-positive apoptotic tumor cells after BPR0C261 treatments are shown in Figure 3(E). Compared to vehicle controls, the numbers of the proliferating tumor cells were decreased and apoptotic tumor cells were increased in the BPR0C261-treated tumors. The IC50 values for tumor cell cytotoxicity and apoptosis induction were estimated to be 20 ± 8 and 379 ± 58 nM, respectively. These findings showed that BPR0C261 inhibits the growth of, and induces apoptosis of, cancer cells in human solid tumors.

BPR0C261 active against human cancer cells of drug-resistant phenotypes.  BPR0C261 inhibited the growth of cervical cancer KB cells with an IC50 of 3.2 ± 0.1 nM and its drug-resistant sublines, including KB100 (camptothecin-resistant), KB-VIN10 (vincristine-resistant), and KB-7D (VP-16-resistant) with comparable IC50 values of 4.9 ± 0.2, 3.3 ± 0.1, and 3.3 ± 0.1 nM, respectively. The results indicate that these drug-resistant phenotypes do not compromise the anticancer activities of BPR0C261 and, therefore, suggested that BPR0C261 may be explored for treating patients with multidrug-refractory tumors.

BPR0C261 inhibits HUVEC growth and migration.  BPR0C261 inhibited the proliferating activity of HUVECs showing an IC50 of 1.6 ± 0.2 nM (= 3) with approximately 30% of residual proliferating activity (Fig. 4A). The migration activity of HUVECs was also concentration- and time-dependently suppressed by BPR0C261, as shown in Figure 4(B). Treatment of BPR0C261 at ≥2.5 nM for 8 h or longer significantly suppressed endothelial migration and thus inhibited the enclosure of the pipette-tip scratched areas. Representative images are shown in Figure 4(B) (upper panel). Numbers of migrated HUVECs in the presence of BPR0C261 at 2.5, 25, and 250 nM during the 24-h observation period were estimated and summarized (Fig. 4B, lower panel).

image

Figure 4.  BPR0C261 shows in vitro, ex vivo, and in vivo anti-angiogenic activities. (A) BPR0C261 concentration-dependently inhibited HUVEC proliferation (= 4). (B) HUVEC migration was suppressed by BPR0C261 in a concentration- and time-dependent manner (upper panel). Migrated HUVECs were counted and presented as means ± SEM (= 3–4). *P < 0.05 versus vehicle (lower panel). BPR0C261 inhibited the endothelial capillary-like network formations in (C) 2D HUVEC cultures (scale lines = 100 μm), and in (D) 3D rat aorta ring cultures. (E) BPR0C261 given orally showed anti-angiogenic activities in s.c. implanted Matrigel plugs in mice. Hemoglobin (Hb) contents in the plugs are expressed as means ± SEM (= 7–8). *< 0.05 versus control.

Download figure to PowerPoint

BPR0C261 inhibits in vitro/ex vivo endothelial capillary tube formations.  BPR0C261 inhibited the 2D tube formation of HUVECs. Representative images of the MTT-stained capillary networks of HUVECs treated with BPR0C261 at 2.5, 25, and 250 nM are shown in Figure 4(C). These images were digitalized and analyzed to estimate the IC50 inhibiting 50% of the formed capillary networks as 120 ± 56 nM (= 3). Furthermore, BPR0C261 showed concentration-dependent suppressive activity on endothelial growth in the ex vivo 3D rat aorta endothelial tube formation system, as observed in Figure 4(D), with an IC50 of 17 ± 8 nM (= 3).

BPR0C261 suppresses angiogenesis in the Matrigel plugs s.c. implanted in mice.  A much lower density of the infiltrated angiogenic blood vessels was observed in the VEGF-containing Matrigel plugs harvested from the mice orally gavaged with BPR0C261 than those harvested from the vehicle-treated mice (Fig. 4E, upper panel). The hemoglobin contents in the Matrigel plugs collected from the mice orally treated with BPR0C261 at 100 and 200 mg/kg/day for 2 weeks were decreased to 49% and 51% of those collected from the vehicle-treated mice, respectively (Fig. 4E, lower panel).

BPR0C261 is permeable through the monolayer culture of the human intestinal Caco-2 cells.  BPR0C261 showed an optimal permeability through the human intestinal Caco-2 cell monolayer culture (Fig. 5A). As a reference to the assay system, a known non-permeable doxorubicin was included and showed no permeability, indicating the intactness of the monolayer culture. During the 4-h incubation period, the percentage of the penetrated BPR0C261 increased proportionally with the increasing incubation time and reached a fraction close to 20% of the initially loaded BPR0C261. These results indicate that BPR0C261 is orally absorbable in patients.

image

Figure 5.  BPR0C261 is orally absorbable and capable of distributing into growing tumors in mice. (A) BPR0C261 was able to permeate through the monolayer of human intestinal Caco-2 cells in a time-dependent manner (n = 3). A non-permeable doxorubicin was included as a reference agent. (B) The observed tissue concentrations of BPR0C261 in s.c. xenografted human colorectal Colo205 tumors in nude mice decayed exponentially and were positively correlated (< 0.01) to the oral doses given to the mice (n = 3).

Download figure to PowerPoint

Tissue concentrations of orally absorbable BPR0C261 in human tumors s.c. growing in nude mice.  Tissue concentrations of BPR0C261 in the s.c. growing human colorectal Colo205 tumors in nude mice orally gavaged with a single dose of BPR0C261 (10, 50, 100, or 200 mg/kg) are presented in Figure 5(B). At 2 h after it was given orally, the tumor tissue concentrations of BPR0C261 at all four dose levels reached the maximum concentration at approximately 1 μM or higher and declined exponentially. The area under the tumor tissue concentration versus time curves, AUC2-48, were 2.7, 11.5, 16.0, and 30.0 μg/mL × h and the half-lives of the tumor tissue levels, T1/2_Tumors, were 2.2, 8.5, 8.2, and 5.5 h for BPR0C261 at the oral doses of 10, 50, 100, and 200 mg/kg, respectively. The AUC2-48 values of BPR0C261 were positively correlated with the given oral doses (< 0.01). It was noted that the T1/2_Tumor of the mice orally dosed with 10 mg/kg was the shortest. Nevertheless, BPR0C261 levels in the tumor tissues of all four dose levels maintained at a concentration higher than 10 ng/mL for at least 24 h after dosing.

Pharmacokinetic parameters in mice and dogs.  The pharmacokinetic parameters of BPR0C261 given in a single (i.v. or p.o.) dose in BALB/c mice and beagle dogs were determined as summarized in Table 1. The CL of BPR0C261 was less than the liver blood flow in both species. The relatively large Vdss for mice and dogs indicate that BPR0C261 distributes, to a great extent, into the tissue compartment. T1/2 was longer in dogs for both i.v. and p.o. routes. A good oral bioavailability was observed in dogs.

Table 1.   Pharmacokinetic parameters of BPR0C261 in mice and dogs
SpeciesDose† (i.v.) (mg/kg)Dose (p.o.) (mg/kg)CL (mL/min/kg)Vdss (L/kg)T1/2 (i.v.) (h)T1/2 (p.o.) (h)Cmax (ng/mL)Tmax (h)F (%)
  1. †Dosing vehicle: DMSO/polyoxyl 35 castor oil/water = 5/25/70% (v/v/v) for all, except DMSO for i.v. in dogs. CL, total body clearance; Cmax, peak concentration; F, absolute bioavailability; T1/2, terminal half-life; Tmax, time to reach peak concentration; Vdss, volume of distribution at the steady state.

Mouse2.020283.21.84.7245218
Dog0.522817.011.69.269243

BPR0C261 given orally suppresses the growths of s.c. xenografted human tumors in nude mice.  BPR0C261 at various daily oral dose regimens significantly reduced the sizes of the s.c. xenografted human nasopharyngeal TW-039 (Fig. 6A) and colorectal Colo205 (Fig. 6B) tumors to 55% and 47% of the vehicle-treated controls, respectively, in nude mice. Regimens of different dosing intensities given through different dose levels and at an intermittent dosing frequency were further examined. BPR0C261 orally given at three doses per week for 4 weeks showed a dose-dependent growth reduction against the Colo205 tumors by 37% and 64% at 100 and 200 mg/kg, respectively (Fig. 6C). Furthermore, two different 4-week dosing regimens of BPR0C261, that is: (i) continuous administrations given for five consecutive daily oral doses of 200 mg/kg/day per week; and (ii) intermittent administrations given at one dose of 300 mg/kg every other day at three doses per week, showed significant and comparable antitumor effects in reducing the sizes of the human gastric MKN-45 tumors by 63% and 53%, respectively (Fig. 6D). During the BPR0C261 treatments, there was a transient loss (3–15%) of body weight in those tumor-bearing nude mice in which tumor burden may contribute, in part, to the observed body weight loss. Nevertheless, the body weight loss is immediately recoverable right after discontinuation of BPR0C261 treatments (Data S5). BPR0C261 is orally active against human colorectal, nasopharyngeal, and gastric cancer.

image

Figure 6.  BPR0C261 is orally active against the growth of human solid tumors s.c. xenografted in nude mice. BPR0C261 given orally significantly inhibited the growths of human nasopharyngeal (A), colorectal (B,C), and gastric (D) tumors in nude mice. (C) Dose-dependent tumor growth inhibition by BPR0C261. (D) BPR0C261 given orally at two different dosing frequencies and intensities (consecutive daily doses of 200 mg/kg versus intermittent doses of 300 mg/kg on the indicated days) caused comparable activities against tumor growth. Data are the mean ± SEM. *< 0.05 versus control.

Download figure to PowerPoint

Combination treatments of BPR0C261 and cisplatin prolong the lifespans of leukemia mice.  BPR0C261 alone or in combination with cisplatin prolonged the survival fraction and lifespans of DBA/2 inbred mice inoculated with murine leukemia P388 cells (= 8–9 per group), as shown in Figure 7. The increased median lifespans of the mice treated with different regimens were expressed as the percentage of that of the vehicle-treated control mice, as listed in Table 2. BPR0C261 given orally at 100 and 200 mg/kg/day for four consecutive days caused significant increases of 75% and 113% in the median lifespans of the leukemia mice, respectively. A single intravenous dose of cisplatin at 5 or 7.5 mg/kg led to a significant 75% lifespan increase. Compared to the effects caused by either agent used alone, the combination treatments with BPR0C261 and cisplatin significantly potentiated the median lifespans of the leukemia mice in a dose-dependent manner. Notably, a synergistic activity was observed in the combinational use of BPR0C261 (200 mg/kg/day, p.o., days 1–4) plus cisplatin (7.5 mg/kg, i.v., day 1), which caused a lifespan increase of >525% in the leukemia mice.

image

Figure 7.  BPR0C261 given orally, alone or in combination with i.v. cisplatin, dose-dependently prolongs the lifespans of leukemia mice. Inbred DBA/2 mice were systemically inoculated with 1 × 106 murine leukemia P388 cells per mouse into the tail vein followed by, 1 day later, oral gavages of BPR0C261 alone or in combination with i.v. injected cisplatin at the regimens indicated. • Vehicle control; bsl00066 Cisplatin, 5 mg/kg (i.v., day 1); △ Cisplatin, 7.5 mg/kg (i.v., day 1); bsl00001 BPR0C261, 100 mg/kg/day (p.o., days 1–4); □ BPR0C261, 200 mg/kg/day (p.o., days 1–4); ♦ BPR0C261, 100 mg/kg/day (p.o., days 1–4) + cisplatin, 5 mg/kg (i.v., day 1); ⋄ BPR0C261, 100 mg/kg/day (p.o., days 1–4) + cisplatin, 7.5 mg/kg (i.v., day 1); bsl00072 BPR0C261, 200 mg/kg/day (p.o., days 1–4) + cisplatin, 5 mg/kg (i.v., day 1); bsl00083 BPR0C261, 200 mg/kg/day (p.o., days 1–4) + cisplatin, 7.5 mg/kg (i.v., day 1).

Download figure to PowerPoint

Table 2.   BPR0C261 alone and in combination with cisplatin prolong the lifespans of leukemic mice
TreatmentIncreased median lifespan (% of vehicle control)
  1. Percents of the increased median lifespans of mice treated with vehicle control are calculated using the equation: increased lifespan in % = (T − C)/C × 100%, where T and C are the median lifespans of drug-treated and vehicle control groups, respectively. *< 0.05 versus all other drug-treated groups; †< 0.05 versus individual single drug alone-treated groups at the same doses; ‡,§< 0.05 between the indicated two groups; —, not applicable.

Vehicle control*
Cisplatin (5 mg/kg, i.v., day 1)75
Cisplatin (7.5 mg/kg, i.v., day 1)75
BPR0C261 (100 mg/kg/day, p.o., days 1–4)75
BPR0C261 (200 mg/kg/day, p.o., days 1–4)113
BPR0C261 (100 mg/kg/day, p.o., days 1–4) + cisplatin (5 mg/kg, i.v., day 1)125†,‡
BPR0C261 (100 mg/kg/day, p.o., days 1–4) + cisplatin (7.5 mg/kg, i.v., day 1)150†,§
BPR0C261 (200 mg/kg/day, p.o., days 1–4) + cisplatin (5 mg/kg, i.v., day 1)200†,‡
BPR0C261 (200 mg/kg/day, p.o., days 1–4) + cisplatin (7.5 mg/kg, i.v., day 1)>525†,§

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

BPR0C261 is a novel synthetic N-heterocyclic indolyl-2-oxoacetamide that has been shown to have in vitro growth inhibition and antimitotic activities in human cancer cells of different histological origins and organs. The anticancer effects of BPR0C261 are in part due to its binding to tubulins through interaction with colchicine binding sites, which leads to the inhibition of microtubule polymerization and disruption of the normal microtubule arrangements of cancer cells. Proliferating cancer cells are then arrested at the G2/M phase by BPR0C261. The occurrences of tumor cell growth inhibition and apoptosis in the BPR0C261-treated histocultured human solid tumors suggest that both cell cycle inhibition and apoptotic cell death induction contribute to the antitumor effects of BPR0C261. In addition, BPR0C261 suppresses in vitro cell proliferation, migration, and capillary network formations of human and rat endothelial cells, and inhibited in vivo blood vessel formations in mice. Notably, BPR0C261 is orally active in vivo against the growths of several human solid tumor xenografts in nude mice. BPR0C261 also shows similar chemosensitivities between cervical cancer KB cells and its drug-resistant sublines such as KB-7D (decreased expression of topoisomerase II and MRP-overexpressing), KB-VIN10 (P-gp-overexpressing), and KB100 (topoisomerase I-overexpressing), which are resistant to etoposide, vincristine, and camptothecin, respectively. The results suggest that BPR0C261 may be used to treat tumors of multidrug-resistant phenotype in patients.

Angiogenesis is the process of generating new blood vessels from existing vasculature and is essential for tumor growth.(35) BPR0C261 is anti-angiogenic as indicated by in vitro endothelial cell growth, migration, and tube formation, ex vivo capillary-like tube formation, and in vivo VEGF-mediated Matrigel plug angiogenesis. The IC50 of BPR0C261 inhibiting HUVEC growth is estimated as 1.6 nM and is at least 10-fold less than those (IC50s = 17–1711 nM) against human cancer cell growth, as reported previously in the same experimental conditions.(22) The plasma BPR0C261 concentrations in mice within 24 h of oral treatment are higher than the needed IC50s against the in vitro (1.6 nM) and ex vivo (17 nM) endothelial cell growths. These results indicate that the anti-angiogenic effect of BPR0C261 on the endothelial cells can be achieved before its direct cytotoxic effect on the tumor cells is in action. This finding is further rationalized by the observation that the tumor tissue BPR0C261 concentrations are dose-dependent and sustainable at levels of ≥10 ng/mL for at least 24 h after the oral dosing, as shown in Figure 5(B). Most importantly, oral BPR0C261 reproducibly inhibits the growth of xenografted tumors of several human organ types in nude mice, including nasopharyngeal, colorectal, and gastric cancer in the present study, and lung H1299 tumors (data not shown). The antitumor activities of BPR0C261 are, therefore, produced in significant part by the inhibition of endothelial cell proliferation, migration, and phenotypic capillary network formation, in which the VEGF-associated angiogenic processes may be inhibited by BPR0C261.

There are numerous potential advantages of anticancer agents given orally, rather than the most frequently used parenteral injection, such as a better patient compliance, ease of use, and low cost.(17,18,36) BPR0C261 showed a good permeability through the tight junction-formed monolayer cultures of the human intestinal P-gp-expressing Caco-2 cells. The results indicate that BPR0C261 is not a substrate of P-gp efflux pump that is responsible for the lack of efficacy of orally given doxorubicin(37) and paclitaxel(19) due to poor oral bioavailability. Previous reports described the in vitro permeability kinetics of an orally active antipsychotic agent chlorpromazine(38) and indicated good oral bioavailability (32%) of chlorpromazine in humans.(39) BPR0C261 shares similar permeability kinetics to that of chlorpromazine and good oral bioavailability of BPR0C261 in humans is therefore expected. In agreement, we observed the oral bioavailability of 18% in mice and a more satisfactory 43% in beagle dogs. After orally absorbed, it is also able to penetrate the targeted tumors in which the BPR0C261 tissue levels are somewhat oral dose-dependent in nude mice. Furthermore, BPR0C261 exerts dose-dependent antitumor activities (Fig. 6C) and comparable activities between two regimens: (i) given once daily doses continuously; and (ii) given a higher dose in a longer dosing interval, as observed in Figure 6(D). These findings, together with that obtained from the tumor tissue concentration study, have suggested linear oral pharmacokinetic and pharmacodynamic properties for BPR0C261 suspended in the dosing vehicle mixture of DMSO/Cremophor EL/water (5/20/75%: v/v/v) in doses ranging from 10 to 300 mg/kg in mice. BPR0C261 given orally at a dose (20 mg/kg, p.o.) within the linear range had a Cmax = 245 ng/mL (644 nM) and T1/2 = 4.7 h in mice. While the linear pharmacokinetic characteristics are assumed, the plasma concentrations in the tumor-bearing mice orally treated with BPR0C261 (200 mg/kg, p.o.) could be maintained for 24 h at a level of approximately 100 ng/mL (263 nM), which is higher than all IC50s for exerting its anticancer and anti-angiogenic activities observed in the present in vitro studies. The antitumor and anti-angiogenic efficacies of BPR0C261 in the animal models are, therefore, explained.

In addition to uses as a single drug treating naïve and multidrug-resistant tumors, BPR0C261 combined with cisplatin prolonged the survival time of leukemia mice. Agreeably, cisplatin prolonged the survival of cancer-bearing mice when combined with bevacizumab, a mAb against VEGF(40) and the antitumor activity of cisplatin was enhanced when combined with a tubulin-targeting agent interacting on the colchicine binding site.(12) BPR0C261 is a small molecule and distributes more readily to the targeted metastasized tumors located in the deep tissue compartments, than to macromolecules. For example, the Vdss values for bevacizumab were 78 mL/kg in monkeys(41) and 71.8 mL/kg in humans(42), representing a limited distribution in the intravascular space only. BPR0C261 showed a Vdss at approximately 40-fold (3.2 L/kg in mice) and 200-fold (17 L/kg in dogs) higher than those of bevacizumab, which indicates a strong distribution of BPR0C261 into the tissue compartments beyond the circulatory system. Furthermore, BPR0C261 did not inhibit drug metabolizing human liver cytochrome P450 isozymes 2D6, 3A4, and 2C19, up to 100 μM (data not shown) in a preliminary study. This finding suggests that BPR0C261 has a better chance of being used in combination with other anticancer drugs and a good safety margin of BPR0C261 in patients who are treated simultaneously with agents that may be the substrates of these cytochrome P450 isozymes. Combination chemotherapy of cisplatin and BPR0C261 is suggested for further pre-clinical and clinical investigations.

Further development of BPR0C261 is in the preclinical stage, and it did not cause any inhibitory effect on the potassium current of hERG potassium channels in HEK-293 cells in concentrations of 0.001–10 μM (Data S1). There were no animal deaths, loss of body weight, or change in food consumption observed for 14 days in an acute oral toxicity study in both male and female ICR mice treated orally with a single dose of BPR0C261 up to 2000 mg/kg (Data S2). BPR0C261 causes no change of the cellularity and distribution of three lineages of mouse bone marrow cells (Data S3, S4), nor did it cause genetic mutation in Salmonella typhimurium tester strains in the Ames test. In summary, we reported a novel orally active anticancer agent BPR0C261 with dual activities of antimitosis and anti-angiogenesis. Whether it acts on other subcellular molecules to exert its anticancer activities is to be discovered. Further preclinical and clinical developments are needed to explore its possible clinical uses in cancer therapies. The effects of BPR0C261 used as a single agent or in combination with a cytotoxic agent, such as cisplatin, against chemotherapy naïve as well as multidrug refractory tumors remain to be further investigated in patients.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

This work was supported by Grant Nos BP-090-CF03, BP-091-CF03, BP-092-PP-10, BP-093-PP-11, BP-094-PP-11, BP-095-PP-07, and BP-095-PP-13 from The National Health Research Institutes, Zhunan, Miaoli, Taiwan.

Disclosure Statement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

The National Health Research Institutes, Taiwan holds patents (US 6,903,104 B2 and US 7,396,838) on the methods of preparation and uses of BPR0C261. Authors Wen-Tai Li, Der-Ren Hwang, and Chiung-Tong Chen are inventors of the two patents. The other authors declare no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information
  • 1
    Budman D. Vinorelbine (Navelbine): a third-generation Vinca alkaloid. Cancer Invest 1997; 15: 47590.
  • 2
    Kamath K, Jordan MA. Suppression of microtubule dynamics by epothilone B is associated with mitotic arrest. Cancer Res 2003; 63: 602631.
  • 3
    Katsumata N. Docetaxel: an alternative taxane in ovarian cancer. Br J Cancer 2003; 89(Suppl 3): S915.
  • 4
    Goncalves A, Braguer D, Kamath K et al. Resistance to taxol in lung cancer cells associated with increased microtubule dynamics. Proc Natl Acad Sci USA 2001; 98: 1173742.
  • 5
    Kavallaris M, Tait AS, Walsh BJ et al. Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells. Cancer Res 2001; 61: 58039.
  • 6
    Dumontet C, Isaac S, Souquet P et al. Expression of class III beta tubulin in non-small cell lung cancer is correlated with resistance to taxane chemotherapy. Bull Cancer 2005; 92: E2530.
  • 7
    Ferlini C, Raspaglio G, Mozzetti S et al. The seco-taxane IDN5390 is able to target class III b-tubulin and to overcome paclitaxel resistance. Cancer Res 2005; 65: 2397405.
  • 8
    Zhou J, Giannakakou P. Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 2005; 5: 6571.
  • 9
    Carlson RO. New tubulin targeting agents currently in clinical development. Expert Opin Investig Drugs 2008; 17: 70722.
  • 10
    Simoni D, Romagnoli R, Baruchello R et al. Novel combretastatin analogues endowed with antitumor activity. J Med Chem 2006; 49: 314352.
  • 11
    Tron G, Pirali T, Sorba G, Pagliai F, Busacca S, Genazzani A. Medicinal chemistry of combretastatin A4: present and future directions. J Med Chem 2006; 49: 303344.
  • 12
    Gourdeau H, Leblond L, Hamelin B et al. Antivascular and antitumor evaluation of 2-amino-4-(3-bromo-4,5-dimethoxy-phenyl)-3-cyano-4H-chromenes, a novel series of anticancer agents. Mol Cancer Ther 2004; 3: 137584.
  • 13
    Folkman J. Angiogenesis. Annu Rev Med 2006; 57: 118.
  • 14
    Hung H. Bevacizumab plus 5-fluorouracil induce growth suppression in the CWR-22 and CWR-22R prostate cancer xenografts. Mol Cancer Ther 2007; 6: 214957.
  • 15
    Hurwitz H, Fehrenbacher L, Novotny W et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350: 233542.
  • 16
    Dal Lago L, D’Hondt V, Awada A. Selected combination therapy with sorafenib: a review of clinical data and perspectives in advanced solid tumors. Oncologist 2008; 13: 84558.
  • 17
    Aisner J. Overview of the changing paradigm in cancer treatment: oral chemotherapy. Am J Health Syst Pharm 2007; 64: S47.
  • 18
    Bedell CH. A changing paradigm for cancer treatment: the advent of new oral chemotherapy agents. Clin J Oncol Nurs 2003; 7: 59.
  • 19
    Ho PY, Yeh TK, Yao HT et al. Enhanced oral bioavailability of paclitaxel by D-a-tocopheryl polyethylene glycol 400 succinate in mice. Int J Pharm 2008; 359: 17481.
  • 20
    Choy H. Satraplatin: an orally available platinum analog for the treatment of cancer. Expert Rev Anticancer Ther 2006; 6: 97382.
  • 21
    Bacher G, Nickel B, Emig P et al. D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks neurotoxicity. Cancer Res 2001; 61: 3929.
  • 22
    Li WT, Hwang DR, Chen CP et al. Synthesis and biological evaluation of N-heterocyclic indolyl glyoxylamides as orally active anticancer agents. J Med Chem 2003; 46: 170615.
  • 23
    Park S-Y, Lam W, Cheng Y-C. X-ray repair cross-complementing gene I protein plays an important role in camptothecin resistance. Cancer Res 2002; 62: 45965.
  • 24
    Kuo CC, Hsieh HP, Pan WY et al. BPR0L075, a novel synthetic indole compound with antimitotic activity in human cancer cells, exerts effective antitumoral activity in vivo. Cancer Res 2004; 64: 46218.
  • 25
    Li WT, Hwang DR, Song JS et al. Synthesis and biological activities of 2-amino-1-arylidenamino imidazoles as orally active anticancer agents. J Med Chem 2010; 53: 240917.
  • 26
    Singer WD, Hersh RT, Himes RH. Effect of solution variables on the binding of vinblastine to tubulin. Biochem Pharmacol 1988; 37: 26916.
  • 27
    Kato Y, Salumbides BC, Wang XF et al. Antitumor effect of the histone deacetylase inhibitor LAQ824 in combination with 13-cis-retinoic acid in human malignant melanoma. Mol Cancer Ther 2007; 6: 7081.
  • 28
    Yeh TK, Li CM, Chen CP et al. Antitumor activities and pharmacokinetics of silatecans DB-67 and DB-91. Pharmacol Res 2010; 61: 10815.
  • 29
    Huang YC, Chen CT, Chen SC et al. A natural compound (ginsenoside Re) isolated from Panax ginseng as a novel angiogenic agent for tissue regeneration. Pharm Res 2005; 22: 63646.
  • 30
    Myers T, Chengedza S, Lightfoot S et al. Flexible heteroarotinoid (Flex-Het) SHetA2 inhibits angiogenesis in vitro and in vivo. Invest New Drugs 2009; 27: 30418.
  • 31
    Wang H-S, Hwang L-L, Sue H-F, Lee K-M, Chen C-T. A simple quantitative method for evaluation of angiogenesis activity. Assay Drug Dev Technol 2004; 2: 318.
  • 32
    Kwak HJ, Park MJ, Park CM et al. Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation. Int J Cancer 2006; 118: 271120.
  • 33
    Lee SH, Yoo SD, Lee KH. Rapid and sensitive determination of paclitaxel in mouse plasma by high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 1999; 724: 35763.
  • 34
    Chang YW, Chen WC, Lin KT et al. Development and validation of a liquid chromatography-tandem mass spectrometry for the determination of BPR0L075, a novel antimicrotuble agent, in rat plasma: application to a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci 2007; 846: 1628.
  • 35
    Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992; 267: 109314.
  • 36
    Ranade VV. Drug delivery systems 5A. Oral drug delivery. J Clin Pharmacol 1991; 31: 216.
  • 37
    van der Sandt IC, Blom-Roosemalen MC, de Boer AG, Breimer DD. Specificity of doxorubicin versus rhodamine-123 in assessing P-glycoprotein functionality in the LLC-PK1, LLC-PK1:MDR1 and Caco-2 cell lines. Eur J Pharm Sci 2000; 11: 20714.
  • 38
    Brown D, Goosen TC, Chetty M, Hamman JH. Effect of oral contraceptives on the transport of chlorpromazine across the CACO-2 intestinal epithelial cell line. Eur J Pharm Biopharm 2003; 56: 15965.
  • 39
    Dahl SG, Strandjord RE. Pharmacokinetics of chlorpromazine after single and chronic dosage. Clin Pharmacol Ther 1977; 21: 43748.
  • 40
    Mabuchi S, Terai Y, Morishige K et al. Maintenance treatment with bevacizumab prolongs survival in an in vivo ovarian cancer model. Clin Cancer Res 2008; 14: 77819.
  • 41
    Xu L, Zuch CL, Lin YS, Modi NB, Lum BL. Pharmacokinetics and safety of bevacizumab administered in combination with cisplatin and paclitaxel in cynomolgus monkeys. Cancer Chemother Pharmacol 2008; 61: 60714.
  • 42
    Herbst RS, Johnson DH, Mininberg E et al. Phase I/II trial evaluating the anti-vascular endothelial growth factor monoclonal antibody bevacizumab in combination with the HER-1/epidermal growth factor receptor tyrosine kinase inhibitor erlotinib for patients with recurrent non-small-cell lung cancer. J Clin Oncol 2005; 23: 254455.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

Data S1. BPR0C261 does not inhibit hERG potassium channel activity.

Data S2. No body weight loss or food consumption change caused by BPR0C261 in ICR mice.

Data S3. BPR0C261 does not cause morphology changes or distribution of bone marrow cells in mice.

Data S4. BPR0C261 does not cause hemolysis in human erythrocytes.

Data S5. Transient body weight loss of BPR0C261-treated tumor-bearing nude mice is immediately recoverable.

FilenameFormatSizeDescription
CAS_1744_sm_DataS1-S5.doc725KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.