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Abstract

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

We recently reported that compound 20d (comp.20d), a novel pyrrolo[3, 2-d]pyrimidine derivative, is a potent and selective inhibitor of tumor angiogenesis-related kinases, including vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). In this study, we show that comp.20d potently blocks the VEGF- and PDGF-stimulated cellular phosphorylation (IC50 = 2.5 and 3.6 nM, respectively) and proliferation of HUVECs and human coronary artery smooth muscle cells with IC50 values of 2.8 and 9.6 nM, respectively, and potently inhibits the VEGF-induced tube formation of endothelial cells cocultured with fibroblasts (IC50 = 3.3 nM). Given orally twice daily, comp.20d at the doses of 1.5–6 mg/kg showed antitumor effects in mice bearing various human cancer xenografts. Consistent with the anti-angiogenic mechanism of action, histological examination of tumors from comp. 20d-treated mice indicated a decrease in microvessel density and inhibition of pericyte recruitment to microvessels, and these were concomitant with decreased interstitial fluid pressure that allowed for therapeutic intratumoral uptake of CPT-11 (irinotecan hydrochloride). In conclusion, comp.20d is an extremely potent inhibitor of VEGFR/PDGFR kinases whose activities suggest therapeutic potential for the treatment of solid tumors that rely on angiogenesis for their survival. (Cancer Sci 2012; 103: 939–944)

Angiogenesis is one of the most attractive and well-validated targets for anticancer therapy because it is essential for the progression, invasion, and metastasis of tumors.[1] It is well established that VEGF plays a major signaling role in angiogenesis and is expressed by most solid tumors. VEGF signaling through its receptors is required for the growth and survival of endothelial cells within tumor vasculature. Therefore, blockade of VEGF signaling is a rational strategy for cancer treatment. Bevacizumab, a neutralizing humanized mAb targeting VEGF, and several small molecular weight inhibitors of VEGFR tyrosine kinase have already been approved for clinical use in the treatment of solid tumors, either alone or in combination therapy.[2-6]

The VEGF/VEGFR pathway is mainly involved in the initiation of angiogenesis, whereas the PDGF/PDGFR pathway is related to the remodeling, maturation, and stabilization of blood vessels. Secretion of PDGF by VEGF-stimulated endothelial cells is required for the proliferation, recruitment, and differentiation of pericytes[7, 8] that are essential for the function and survival of endothelial cells. Furthermore, PDGF/PDGFR signaling has been reported to play an important role in tumor angiogenesis.[8] Accordingly, simultaneous inhibition of these two angiogenesis-related signaling pathways would enhance antitumor activity through more potent blockade of angiogenesis.

During our attempts to generate VEGFR2 and PDGFR dual kinase inhibitors, we discovered a novel pyrrolo[3,2-d]pyrimidine derivative, comp.20d, that strongly inhibits VEGFR1, VEGFR2, PDGFRα, and PDGFRβ kinases with IC50 values of less than 100 nM.[9] In this study, we describe the cellular and antitumor activities of comp.20d as wells as the anti-angiogenic activities accompanied with the reduction of tumor IFP.

Materials and Methods

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

Test compound

Comp.20d, N-{2-fluoro-4-[(5-methyl-5H-pyrrolo[3,2-d]pyrimidin-4-yl)oxy] phenyl}- N’-[3-(trifluoromethyl) phenyl] urea, was synthesized by Takeda Pharmaceutical (Fujisawa, Japan) (Fig. 1).[9] For in vitro experiments, comp.20d was initially prepared as a 10 mM stock solution in DMSO. For in vivo studies, the mono hydrochloride salt form of comp.20d was suspended in 1% citric acid (Wako Pure Chemical, Osaka, Japan) and 1% gum Arabic (Suzu Pharmaceutical, Osaka, Japan) vehicle solution. Free form of comp.20d was suspended in 0.5% methylcellulose solution (Shinetsu Chemical, Tokyo, Japan).

image

Figure 1. Chemical structure of compound 20d.

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Receptor phosphorylation assay

Both HUVECs and CASMCs were obtained from Cambrex (Walkersville, MD, USA). The cells were seeded in type Ι collagen-coated plates for 2 days. After treatment with comp.20d for 2 h, the cells were stimulated by recombinant human VEGF (100 ng/mL; R&D Systems, Minneapolis, MN, USA) or PDGF-BB (20 ng/mL; PeproTech, London, UK) for 5 min then lysed. Lysates were analyzed by Western blotting with antibodies for phospho-Tyr951-VEGFR2 (Cell Signaling Technology, Beverly, MA, USA), VEGFR2 (Cell Signaling Technology), and PDGFRβ (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Phospho-PDGFRβ was detected with an antiphosphotyrosine antibody (4G10; Upstate Systems, Charlottesville, VA, USA) after immunoprecipitation with the anti-PDGFRβ antibody. After treatment with enhanced chemiluminescence reagent, the membranes were scanned with a LAS lumino-image analyzer (Fujifilm, Kanagawa, Japan) and the density of each blot was quantified using Multi Gauge version 3.3 software (Fujifilm). The concentration causing 50% inhibition (IC50) was calculated from the dose–response curve generated by least-squares linear regression analysis of the response using SAS software and the NLIN procedure (version 5.0; SAS Institute Japan, Tokyo, Japan).

Cell proliferation assay

The HUVECs were seeded in 96-well plates and cultured overnight. Comp.20d and VEGF (60 ng/mL) were added and the cells were cultured for 5 days. The CASMCs were cultured in serum-free medium overnight, then treated with comp.20d and PDGF-BB (200 ng/mL) and cultured for 6 days. Cancer cell lines were obtained from ATCC (Manassas, VA, USA) or the European Collection of Animal Cell Cultures (Salisbury, UK). Cells were seeded in 96-well plates and cultured overnight. Then comp.20d was added and the plates were incubated for 3 days. Cell proliferation was assessed using the Cell Counting Kit-8 reagent (10 μL/well; Dojindo Laboratories, Kumamoto, Japan) as directed by the manufacturer. The IC50 values were calculated as described above.

Tube formation assay

Both HUVECs and normal human dermal fibroblasts (Cambrex) were seeded and cocultured with VEGF (10 ng/mL) and various concentrations of comp.20d for 7 days. The cells were fixed and treated with mouse anti-human CD31 mAb (R&D systems). After washing with PBS, the cells were incubated with Alexa Fluor 488-conjugated chicken anti-mouse IgG (H+L) polyclonal antibody (Invitrogen, Carlsbad, CA, USA). Tube formation by endothelial cells was visualized using fluoroscopic imaging (Discovery-1 system; Molecular Devices, Sunnyvale, CA, USA) with excitation and emission wavelengths of 470 and 535 nm, respectively, according to the manufacturer's protocol. The tube area was quantified using MetaMorph software (Molecular Devices).

Tumor xenograft models

All animal experiments were carried out according to the guidelines of the Takeda Experimental Animal Care and Use Committee. Athymic nude mice (BALB/cA Jcl-nu/nu) and severe combined immune-deficiency mice (C.B17/Icr-scid/scid Jcl-nu/nu) were purchased from CLEA (Tokyo, Japan). Animals were housed in a barrier facility with 12:12 light:dark cycle and were provided with sterilized food and tap water ad libitum. Mice (6–8 weeks old) received s.c. injections into the hind flank with 100 μL cultured cancer cell suspension in Hanks’ balanced salt solution (Invitrogen). After the tumor xenografts were established, the animals were randomly grouped (Day 0). From the following day (Day 1), the mice were orally given the vehicle or comp.20d b.i.d. Some animals were treated with CPT-11 (irinotecan hydrochloride; Yakult, Tokyo, Japan) either alone or in combination with comp.20d. CPT-11 was given i.p. on Day 1 followed by four additional injections at 3-day intervals. Tumor volumes were assessed by measurement of two dimensions with vernier calipers twice weekly and were calculated as length × width2 × 1/2, where length was taken to be the longest diameter across the tumor and with the corresponding dimensions perpendicular to it. As an index of antitumor activity, the mean change in tumor volume during the treatment period was compared between the control and treated groups, and the T/C ratio was calculated as a percentage. Body weight was also measured on the day of tumor volume assessment. Statistical comparisons were carried out using the one-tailed Williams’ test, one-tailed Shirley–Williams test, or Dunnett's multiple comparison test (P ≤ 0.025, P ≤ 0.025, or P ≤ 0.05 was considered statistically significant, respectively).

Immunohistochemistry

Nude mice bearing A549 tumors were treated with comp.20d. Cryosections of tumor tissues dissected from the xenografts of mice were prepared at a thickness of 7 μm. For visualization of blood vessels, immunohistochemical staining for CD31 was carried out by incubation with a rat anti-CD31 mAb (BD Biosciences, Bedford, MA, USA) followed by the immunoperoxidase technique with 3,3′-diaminobezidine tetrahydrochloride as the chromagen. The slides were scanned with a NanoZoomer Digital Pathology instrument (Hamamatsu Photonics, Shizuoka, Japan), and the number of CD31-positive filaments per 0.66 mm2 of viable tumor area was quantified in five randomly selected fields of each tumor using WinROOF software (Mitani, Tokyo, Japan). To assess pericyte recruitment, comp.20d or vehicle-treated tumor tissues were prepared by perfusion fixation with paraformaldehyde and embedding in paraffin. Endothelial cells and pericytes were stained with anti-CD31 (Spring Bioscience, Fremont, CA, USA) and anti-αSMA antibodies (Dako Cytomation, Glostrup, Denmark), respectively. The fluorescent immunohistochemical staining for CD31 and α-SMA was carried out using Alexa Fluor 594-labeled donkey anti-rabbit IgG and Alexa Fluor 488-labeled donkey anti-mouse IgG (Invitrogen), respectively. Subsequently, the slides were scanned and the pericyte area was quantified by analysis of five randomly selected fields from each tumor, as mentioned above.

Monitoring tumor IFP

In anesthetized tumor-bearing nude mice, tumor IFP was measured by the wick-in-needle technique. In brief, a 23-gauge needle was connected to a pressure transducer (DX-100; Nippon Becton Dickinson, Tokyo, Japan) through a polyethylene tube filled with heparinized saline (70 units/mL). The transducer was connected to an amplifier (AP-610J; Nihon Kohden, Tokyo, Japan) and the amplified signal was imported into a PowerLab system (ML846 PowerLab 4/26; AD Instruments, Sydney, Australia). The IFP was measured at the center of the s.c. tumors on Day 0 (the day before starting treatment), and on Days 2, 4, 8, and 15 of treatment with comp.20d.

Pharmacokinetic analysis

Mice with s.c. HT-29 xenografts were given comp.20d (1.5 mg/kg) or vehicle p.o. twice daily for 2 weeks. CPT-11 (30 mg/kg) was injected i.p. on Days 2, 4, 7, or 14. One hour after injection of CPT-11, whole blood and tumor tissue samples were collected. The blood was centrifuged to collect plasma, and tumor tissues were snap frozen before homogenization in saline. The plasma and tumor homogenate samples were deproteinized with acetonitrile containing an internal standard. After centrifugation, the supernatant was diluted with LC mobile phase and centrifuged again. The concentration of SN-38, the main active metabolite of CPT-11, in the supernatant was measured by LC/MS/MS systems (AB Sciex, Foster City, CA, USA).

Results

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

Comp.20d selectively inhibits cellular VEGF and PDGF signaling

Comp.20d markedly inhibited VEGF-induced phosphorylation of VEGFR2 in HUVECs and PDGF-BB-induced phosphorylation of PDGFRβ in CASMCs with IC50 values of 2.5 and 3.6 nM, respectively (Fig. 2A). Comp.20d also caused concentration-dependent inhibition of the VEGF- and PDGF-BB-induced proliferation of HUVECs and CASMCs with IC50 values of 2.8 and 9.6 nM, respectively (Fig. 2B). Furthermore, comp.20d dose-dependently blocked the VEGF-stimulated cell migration of HUVECs (Fig. S1). The effect of comp.20d was also assessed in tube formation assay to mimic neovascularization. Endothelial cells cocultured with fibroblasts produced tube-like structures in the presence of VEGF. Comp.20d strongly inhibited this tube formation in agreement with its antiproliferative effect on HUVECs, with an IC50 value of 3.3 nM (Fig. 2C). In contrast, comp.20d showed weaker inhibition of cancer cell proliferation (IC50 = 3.5–7.2 μM, Fig. 2D). The relative lack of antiproliferative activity on cultured cancer cells suggests that any antitumor effect of the compound would be mediated through its effects on angiogenesis rather than a direct effect on cancer cell proliferation or survival. These results suggest that comp.20d selectively inhibits the VEGF and PDGF signaling pathways and blocks processes fundamental to blood vessel establishment and maintenance.

image

Figure 2. Inhibitory activity of compound 20d (comp.20d) against cellular vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) signaling. (A) Inhibition of VEGF receptor 2 (VEGFR2) and PDGF receptor β (PDGFRβ) phosphorylation by comp.20d. Both HUVECs and coronary artery smooth muscle cells (CASMCs) were treated with comp.20d for 2 h then stimulated with VEGF or PDGF-BB, respectively. The VEGFR2 phosphorylation was detected by immunoblotting with an antiphosphorylated VEGFR2 antibody; PDGFRβ phosphorylation was detected by immunoblotting with an antiphosphotyrosine antibody after immunoprecipitation with anti-PDGFRβ antibody. Each experiment was carried out in triplicate and representative blots are shown. (B) Inhibition of VEGF- and PDGF-driven proliferation by comp.20d. The HUVECs and CASMCs were incubated with VEGF or PDGF-BB, respectively, in the presence of comp.20d. The results are shown as a percentage of the control wells without comp.20d. The mean and SD of quadruplicate determinations were calculated. (C) The HUVECs cocultured with normal human dermal fibroblasts were treated with comp.20d in the presence of VEGF for 7 days. Endothelial cells were visualized and quantified with anti-CD31 staining and fluorescent imaging; representative images are shown (magnification ×20). Duplicate wells were assayed for each concentration. (D) Effect of comp.20d on the proliferation of four human cancer cell lines. Results are shown as a percentage of control without comp.20d (mean and SD of quadruplicate determinations).

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Comp.20d shows antitumor and anti-angiogenesis activity

The effect of comp.20d on the A549 human non-small-cell lung cancer cell line was investigated in a mouse xenograft model. As shown in Figure 3(A), comp.20d significantly suppressed tumor growth in a dose-dependent manner. Given orally b.i.d. for 1 week, 1.5 mg/kg comp.20d inhibited tumor growth with a T/C ratio of 59%, whereas tumor stasis was seen at 6 mg/kg (T/C = 5%). There were no significant differences in body weight between the vehicle group and the treated groups (Fig. S2), suggesting that the compound was relatively well-tolerated in this model. Comp.20d also showed potent antitumor activity against various xenografts derived from lung, colon, breast, prostate, ovary, pancreas, and skin cancer (Table 1). To assess whether tumor growth inhibition was associated with a reduction of tumor angiogenesis, we examined the histology of implanted A549 tumors after treatment with comp.20d. For visualization of endothelial cells and pericytes, tumor tissue sections were immunostained for CD31 (a marker of endothelial cells) and α-SMA (a marker of pericytes), respectively. It was found that the number of vessels (CD31-positive filaments) and pericyte recruitment to tumor blood vessels were significantly reduced in a dose-dependent manner by treatment with comp.20d (Fig. 3B,C).

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Figure 3. Antitumor (A) and anti-angiogenic (B) activity of compound 20d (comp.20d) in a mouse xenograft model. (A) Comp.20d was given orally at doses of 1.5 or 6 mg/kg twice daily for 7 days to A549 tumor-bearing mice. Data represent the mean and SD (n = 4). (B) A549 xenograft mice were treated with comp.20d twice daily at doses of 1.5 or 6 mg/kg for 7 days. A549 tumors were resected 1 day after the last dose and cryosections were immunostained for CD31 to visualize blood vessels. Data are shown as the mean and SD (n = 4). Representative tumor sections are shown. (C) Comp.20d was given orally once daily for 3 days at 3 or 10 mg/kg to A549 xenograft mice. Tumors were resected and processed for immunohistochemical analysis 4 h after the last dose. Sections were simultaneously stained for CD31 and α-smooth muscle actin (α-SMA) to detect endothelial cells and pericytes, respectively. The region positive for α-SMA surrounding the CD31-positive vessels was quantified. Data represent the mean and SD (n = 3). *≤ 0.025 versus the vehicle control group by the one-tailed Williams’ test.

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Table 1. Antitumor activity of compound 20d against various human cancer cell lines
CellTumor typeTreatment period (days)Comp.20d formDose (mg/kg)T/C (%)
  1. a

    P ≤ 0.025 versus the vehicle group by the one-tailed Williams’ or Shirley Williams test. T/C, treated/control ratio.

A549Lung21Salt1.539a
6.04a
HT-29Colon21Free1.544a
5.016a
COLO 205Colon14Free1.550a
5.017a
MDA-MB-231Breast21Salt1.541a
6.05a
DU-145Prostate21Salt1.525a
5.0−10a
SK-OV-3Ovary18Free1.554a
5.020a
CFPAC-1Pancreatic14Salt1.570a
5.0−12a
A431Skin14Free1.569a
5.011a

Comp.20d shows combined antitumor effect associated with a decrease of tumor IFP

The antitumor effect of comp.20d in combination with CPT-11 (a topoisomerase inhibitor that is one of the standard agents for colorectal cancer) was examined in the HT-29 human colorectal cancer xenograft model. Treatment with comp.20d alone (1.5 mg/kg p.o. twice daily) or CPT-11 alone (30 mg/kg i.p. every 3 days) for 2 weeks significantly inhibited tumor growth with T/C ratios of 58% and 55%, respectively (Fig. 4A). Compared to either agent alone, combined treatment with CPT-11 and comp.20d had more potent antitumor activity with a T/C ratio of 35%. We next evaluated whether these changes of tumor vessel structure and function affected tumor IFP or uptake of combined anticancer drugs to enhance antitumor activity. Tumor IFP showed a significant decrease by 49%, 55%, 46%, and 38% on Days 2, 4, 8, and 15, respectively, compared with the IFP of the vehicle group (Fig. 4B). Next, mice with HT-29 tumor xenografts were given comp.20d (1.5 mg/kg) orally b.i.d. as well as CPT-11 (30 mg/kg i.p.) on Days 2, 4, 7, and 14. One hour post dose, SN-38 (the active metabolite of CPT-11) was measured in plasma and tumor. Although there were no significant differences in plasma SN-38 concentrations between the control group and the comp.20d-treated groups (Fig. S3), SN-38 concentration in the tumor tissue was higher in the comp.20d-treated group than in the control group on Day 4 (P < 0.05), Day 7 (P = 0.081), and Day 14 (= 0.071) (Fig. 4C).

image

Figure 4. Combined effect of compoound 20d (comp.20d) plus CPT-11 on interstitial fluid pressure (IFP) and drug uptake. (A) Combined antitumor effect of comp.20d and CPT-11. Comp.20d (1.5 mg/kg given orally twice daily) alone, CPT-11 (30 mg/kg i.p. every 3 days) alone, or comp.20d plus CPT-11 were used to treat mice with HT-29 xenografts. Data points show the mean and SD (n = 5). Arrows indicate treatment date with CPT-11. Statistical analysis of antitumor activity was carried out using Dunnett's multiple comparison test. *≤ 0.05 versus vehicle control; **≤ 0.05 versus combined therapy. (B) Decrease in tumor IFP with comp.20d treatment. HT29 tumor-bearing mice were given comp.20d orally at 1.5 mg/kg twice daily for 2 weeks. Tumor IFP was measured by the wick-in-needle technique. Data are shown as the mean and SD (n = 8). ***≤ 0.001 versus vehicle by Student's t-test. (C) Effect of comp.20d on tumor uptake of SN-38. HT29 tumor-bearing mice were given comp.20d orally at 1.5 mg/kg twice daily for 2 weeks. One hour post dose of CPT-11 on the indicated days, SN-38 (the active metabolite of CPT-11) was measured in tumor. Data are shown as the mean and SD (n = 3). *≤ 0.05 versus vehicle by Student's t-test.

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Discussion

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

In our previous study, we showed that comp.20 inhibits VEGFR1 and VEGFR2 kinases with IC50 values of 15 and 6.2 nM, respectively.[9] Comp.20d also potently inhibited VEGFR3 kinase with an IC50 value of 10 nM (Fig. S4). Similarly, comp.20d inhibits other tumor angiogenesis-related kinases, PDGFRα, PDGFRβ, and Tie-2 with IC50 values of 35, 96, and 20 nM, respectively.[9] Notably, comp.20d showed moderate or weak inhibition of c-Kit, FAK, BRAF, and Aurora A, with IC50 values of over 10 μM. Comp.20d potently inhibited VEGF and PDGF signaling in cellular assays along with its enzymatic inhibition profile. We could not technically confirm the effect of comp.20d on angiopoietin-1-induced Tie-2 phosphorylation in cellular assay. Therefore, comp.20d was regarded as a tyrosine kinase inhibitor specific for VEGFR and PDGFR families. In the mouse A549 tumor xenograft model, almost complete suppression of tumor growth was observed in the animals receiving comp.20d at 6 mg/kg twice daily, despite its relatively modest effect on the proliferation of A549 cells in vitro. Histological examination of A549 tumors showed that microvessels (detected by CD31 immunostaining) and pericyte coverage towards tumor vasculature (detected by α-SMA immunostaining) were markedly decreased in comp.20d-treated tumor tissues. Therefore, the antitumor effect of comp.20d is likely to be mediated through an anti-angiogenic mechanism rather than a direct effect on tumor cell proliferation.

The PDGF/PDGFR signaling is also known to be necessary for tumor vessels to undergo maturation and become stable. Combined inhibition of VEGFR and PDGFR signaling targeting both endothelial cells and pericytes leads to regression of tumor vessels by interfering with pericyte-mediated endothelial cell survival.[10, 11] Therefore, inhibition of PDGFR signaling in addition to VEGFR signaling by comp.20d should potently enhance antitumor activity by suppressing the proliferation and survival of endothelial cells. Consistent with this idea, comp.20d showed potent antitumor activity against various human cancer xenografts, indicating that the inhibitory activity of comp.20d on the angiogenesis-related kinases may be suitable for a wide range of solid tumors, even as monotherapy. Moreover, the inhibitory effect of comp.20d on VEGFR3 kinase associated with lymphangiogenesis may show efficacy against metastasis during long-term treatment.[12]

In general, solid tumors possess abnormal and highly permeable vessels surrounded by an impermeable interstitium.[13] Thus, fluid that leaks out of tumor vessels accumulates in the interstitium and causes the tumor IFP to increase.[14] The interstitium contains a dense network of collagen fibers and activated cancer-associated fibroblasts that contribute further to the elevation of IFP. This elevation of IFP leads to a decrease of tumor transcapillary transport that acts as a barrier to pharmacological treatment by decreasing drug uptake. Conversely, a decrease of IFP restores the pressure gradient across blood vessel walls as well as into the interstitium, thus increasing drug uptake by tumors.[15, 16] In our experiments using mice with HT-29 xenografts, combined treatment with comp.20d and CPT-11 showed more potent antitumor activity than either agent alone. We also observed that comp.20d decreased the IFP of xenograft tumors, which influences the intratumoral uptake of SN-38. Blockade of VEGF signaling by treatment with anti-VEGF or VEGFR2 antibody was reported to decrease tumor IFP in a xenograft model,[17, 18] and bevacizumab was also reported to decrease tumor IFP in patients with renal cell carcinoma.[17, 19] These anti-VEGF treatments reduce the leakiness of tumor vasculature and thus decrease tumor IFP, probably by vascular normalization.[20, 21] In contrast, blockade of PDGF signaling by a PDGFR tyrosine kinase inhibitor, imatinib, is also reported to decrease tumor IFP in animal models.[22-24] Imatinib shows a combined antitumor effect with chemotherapeutic agents by selectively increasing tumor drug uptake,[23, 24] indicating that PDGF plays a role in activating stromal fibroblasts along with ECM molecules. Therefore, inhibition of VEGF and PDGF signaling by comp.20d would decrease tumor IFP by normalization of tumor vessels and suppression of tumor fibroblast activation, thereby having a combined antitumor effect along with an increase in drug uptake.

In conclusion, we showed that comp.20d is a VEGFR and PDGFR tyrosine kinase inhibitor with potent antitumor activity through the inhibition of angiogenesis. We also showed that comp.20d reduces tumor IFP, probably through the inhibition of VEGF and PDGF signaling, and by increasing the tumor uptake of CPT-11. Accordingly, comp.20d may be useful in combination with other anticancer drugs as well as bevacizumab for targeting solid tumors that depend on angiogenesis.

Acknowledgments

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

We thank Dr Takayasu Ito, Dr Takeshi Watanabe, Ms Michiko Tawada, and Mr Naoki Miyamoto for their contributions to this project, as well as Mr Yuichi Kakoi, Ms Midori Yamasaki, and Mr Osamu Kitahara for pharmacological assistance, and Mr Seiji Yamasaki and Mr Kunihiko Mizuno for pharmacokinetic analysis. We also thank Drs Masami Kusaka, Osamu Nakanishi, Shuichi Furuya, and Hideaki Nagaya for their support during this project.

Abbreviations
α-SMA

α-smooth muscle actin

CASMC

coronary artery smooth muscle cell

comp.20d

compound 20d

IFP

interstitial fluid pressure

LC

liquid chromotography

MS

mass spectrometry

PDGF

platelet-derived growth factor

PDGFR

PDGF receptor

p.o.

oral(ly)

T/C

treated/control ratio

VEGF

vascular endothelial growth factor

VEGFR

VEGF receptor

References

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

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cas2238-sup-0001-figS1.tifimage/tif2663KFig. S1. Compound 20d (comp.20d) inhibits vascular endothelial growth factor (VEGF)-mediated cell migration.
cas2238-sup-0002-figS2.tifimage/tif2165KFig. S2. Body weight change during compound 20d (comp.20d) treatment shown in Figure 3(A).
cas2238-sup-0003-figS3.tifimage/tif2625KFig. S3. Plasma concentration of SN-38 during the combination treatment shown in Figure 4(C).
cas2238-sup-0004-figS4.tifimage/tif2822KFig. S4. Inhibition of compound 20d (comp.20d) towards vascular endothelial growth factor receptor 3 (VEGFR3) kinase activity.

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