Methods and compositions for treating solid tumor using f16 isoindole small molecules

Abstract

To provide pharmaceutical compositions for treating solid tumor, in particular, glioblastoma multiforme (GBM).SOLUTION: Provided herein is a pharmaceutical composition for use in a method for retarding progression of brain tumor, where the composition comprises a therapeutical effective dose of F16, that is, isoindole (1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl)-amide and a therapeutically effective dose of a chemotherapeutic agent in a pharmaceutical carrier, where the brain tumor is glioblastoma multiforme, and the chemotherapeutic agent is temozolomide (TMZ) or bevacizumab (BVZ) or similar agent.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention falls within the field of cancer therapy and relates generally to the use of small molecules to target solid tumors, and in particular to the use of F16 isoindole small molecules for the treatment of brain tumors. [Background technology]

[0002] Despite significant efforts and resources dedicated to developing newer treatment strategies and therapies, cancer remains a fatal disease of humanity, with millions of people worldwide dying from various types of cancer each year. One of the most widespread types of this deadly disease is brain tumor, which is the leading cause of cancer-related death in children and the third most common cause of cancer-related death in adolescents and young adults between the ages of 15 and 39 [1, 2].

[0003] There are 12 major groups and more than 100 subgroups of brain tumors that share common biological characteristics [Reference 3]. Gliomas include all tumors arising from the supportive tissues of the brain and are the most aggressive form of brain tumor, accounting for 24.7% of all primary brain tumors and 74.6% of all malignant brain tumors [Reference 4]. Glioblastoma multiforme (GBM) is the most commonly diagnosed form of glioma in the United States and the most deadly type worldwide. Despite the use of multidisciplinary treatment approaches, GBM has a very low 5% 5-year survival rate and an average survival rate of approximately 1 year after diagnosis [References 3, 4]. GBM is generally classified as a grade IV glioma, and some of the histological features that distinguish it from other grades are the presence of necrosis and a dramatic increase in vascular growth around the tumor [Reference 5]. Indeed, GBM is one of the most highly vascularized solid tumors, as its growth depends on angiogenesis, as supported by various preclinical studies showing that glioma growth is critically dependent on the development of tumor-associated blood vessels [4, 6]. In addition, GBM tumor vasculature is characterized by a dense vascular network that is tortuous, hyperpermeable, and has an abnormally increased vessel diameter and thickness of the basement membrane. This abnormal tumor vasculature is thought to increase tumor hypoxia, inhibit the delivery of cytotoxic chemotherapy, and thus contribute to treatment failure [5, 7]. Therefore, antagonizing tumor vasculature has emerged as a new strategy for treating brain tumors, particularly GBM.

[0004] The few forms of treatment currently available for GBM are often ineffective, and therefore, the prognosis for GBM remains poor. Current treatment for glioblastoma involves surgery, whenever applicable, followed by radiation and chemotherapy with temozolomide (TMZ). This treatment strategy provides a modest increase in overall survival [Reference 8]. In preclinical and clinical studies, the use of antiangiogenic agents in combination with chemotherapeutic agents has shown promising results across a wide range of cancer types [References 9-12]. In particular, antiangiogenic agents are currently being thoroughly investigated for treating GBM, and various preliminary studies have yielded promising results [References 13-15]. Accordingly, several antiangiogenic agents are currently undergoing clinical trials for the treatment of GBM, either as monotherapy or in combination [Reference 16]. To date, bevacizumab (BVZ), a monoclonal antibody with antiangiogenic effects, has been approved by the FDA for the treatment of recurrent GBM. The FDA approval of BVZ was based on an increase in the overall "objective response rate (ORR)." However, a thorough analysis of BVZ treatment data for GBM patients did not demonstrate an improvement in overall survival (OS) [References 17, 18]. It is worth mentioning that while antiangiogenic agents when used as monotherapy can generally provide cell growth inhibitory effects, the maximum therapeutic effect is achieved when these agents are combined with cytotoxic chemotherapy agents [References 19, 20].

[0005] One of the major obstacles to treating brain tumors is the ability of therapeutic agents to cross the blood-brain barrier (BBB) ​​[Reference 21]. It is known that penetrating the BBB is not easy for drugs with high molecular weights, such as BVZ (~150 kD MW), meaning that BVZ treatment for GBM may not provide optimal delivery and therapeutic results [References 22, 23]. Therefore, recent interest has shifted toward the use of small molecules that can cross the BBB and modulate angiogenesis and similar processes. In this regard, a novel compound, namely, isoindole (1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl)-amide, was developed at Nova Southeastern University (NSU) and codenamed F16. See U.S. Patent No. 7,875,603, Japanese Patent No. 5436544, and Korean Patent No. 10-1538822. F16 Chemical Structure (19, Reference Example 2): TIFF2025166160000002.tif52147

[0006] F16 not only exhibits strong vascular endothelial growth factor receptor-2 (VEGFR-2) binding and inhibition of VEGFR-2 phosphorylation in human umbilical vein endothelial cells (HUVECs), but also demonstrates significant in vivo growth inhibition in mice bearing breast cancer and colorectal cancer xenografts (data not published). More importantly, preclinical pharmacokinetic studies have shown that F16 can cross the BBB and accumulate in brain regions (data not published). Furthermore, results from preclinical safety studies have so far demonstrated that F16-treated experimental animals remain healthier compared with groups treated with other FDA-approved anticancer drugs, such as paclitaxel (data not published).

[0007] The small molecule F16 (isoindole) exerts antiangiogenic effects by blocking vascular endothelial growth factor receptor 2 (VEGFR2), which is necessary for the development of new blood vessels within solid tumors such as breast cancer (Figure 1). Studies conducted at the Rumbaugh-Goodwin Institute for Cancer Research have demonstrated that the patented small molecule F16 demonstrates both antiangiogenic and proapoptotic (programmed cell death) effects against solid tumors. This novel compound demonstrated promising anticancer efficacy in both cell culture and in vivo experiments, and demonstrated relatively low toxicity compared to some existing FDA-approved anticancer drugs. Studies using breast cancer xenograft mouse models demonstrated significant anticancer efficacy comparable to that of the commonly used chemotherapy agent, paclitaxel (Taxol), due to its antiangiogenic properties and tumor-suppressing function. Furthermore, in these mouse model studies, F16 exhibited significantly less toxicity than Taxol. Xenograft studies also demonstrated that F16 was effective in suppressing tumor growth when used alone or in combination with paclitaxel. When both F16 and Taxol were used in combination in in vivo studies, it not only resulted in nearly 85% tumor growth inhibition but also prevented the significant toxicity often associated with Taxol monotherapy. These findings provided substantial evidence supporting the use of F16 as an anticancer agent for treating cancers with angiogenic potential. In addition to therapeutic studies using subcutaneous xenografts, the tissue distribution of F16 was analyzed, showing accumulation in the brain in the range of 5,000 ng / g of tissue (Figure 2). This study prompted the inventors to believe that F16, exhibiting angiogenic potential for its own survival and growth, could be useful for the treatment of brain tumors. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,875,603 [Patent Document 2] Japanese Patent No. 5436544 [Patent Document 3] Korean Patent No. 10-1538822 [Patent Document 4] US Patent No. 7,939,557 B2 Summary of the Invention [Problem to be solved by the invention]

[0009] Because the inventive methods (and compositions) described herein demonstrate the effectiveness of F16 in slowing the progression of glioblastoma through its anti-angiogenic and pro-apoptotic functions, it (F16) can potentially be used as the basis for generating new avenues for the effective treatment of brain tumors, especially those that exhibit angiogenic capabilities that enable their growth and survival. [Means for solving the problem]

[0010] The small molecule F16 (isoindole) offers promising new cancer therapeutic potential. Based on preliminary in vitro and in vivo experiments, its cytotoxic effects in monolayer and 3D cultures were confirmed. To gain a better understanding of the effects of F16 on the migration and invasion capabilities of cancer cells, scratch assays, trans-well migration assays, and invasion assays were performed. The anti-migration and anti-invasive capabilities of U87MG cells, which typically coincide with its anti-angiogenic properties during cancer metastasis, were determined through the above-mentioned assays. Results confirmed that the invasive ability of U87MG cells was significantly reduced in a dose-dependent manner relative to untreated control cells 24 hours after treatment with F16. The results were compared with those of the FDA-approved drug TMZ (temozolomide). Thus far, F16 has shown consistent inhibitory effects on cell migration and cancer cell invasion, as presented in the results, which are significantly better than those of TMZ. Changes in pro-apoptotic gene expression were also analyzed, and F16 appeared to be better able to arrest the cell cycle and induce apoptosis in the U87MG cell line than TMZ.

[0011] To evaluate the efficacy of F16 in the treatment of glioblastoma and monitor tumor growth inhibition through optical imaging, luciferase-transduced U87MG-luc tumor cells were established. First, a xenograft model was generated by injecting U87MG-Luc cells intraperitoneally (i.p.). Animals were treated with F16, TMZ, and a combination of both drugs. Studies using the U87MG-Luc glioblastoma cell line showed favorable results with the F16 compound. Although F16 reduced tumor volume, it did not alter body weight during the treatment period. Analysis of blood parameters such as RBCs, WBCs (Figure 14A-B), hemoglobin levels, and hematocrit (Figure 14C-G) in F16-treated animals showed no signs of toxicity. While examining liver markers during blood chemistry analysis, TMZ significantly elevated ALT (alanine transaminase) levels, while they remained near normal in F16-treated cells. Neither F16 nor JFD showed an increase in BUN (blood urea nitrogen) levels, suggesting that renal function was not affected by either drug. Similarly, blood glucose, calcium, phosphorus, and protein levels remained within the normal range (Figure 14C-G).

[0012] After completing efficacy testing using subcutaneous tumor models and confirming the safety of F16, intracranial implant studies were initiated. In the intracranial experiments, F16 was able to block brain tumor growth in 50% of the animals. This confirmed that F16 was able to cross the BBB and inhibit the growth of U87MG-derived tumors in the brain. It was also noted that KP (Kolliphor®), used as an excipient, slightly increased the brain delivery of F16 but also caused some side effects.

[0013] In its most basic aspect, the present invention provides methods for the manipulation of malignant cells, particularly interactions within malignant cells characterized by uncontrolled growth.

[0014] In another basic aspect, the present invention provides a new treatment modality for cancer.

[0015] In a general aspect, the present invention provides methods and compositions for the treatment of cancers that manifest as solid tumors, particularly, but not limited to, solid tumors that exhibit angiogenic capacity.

[0016] In a general aspect, the present invention provides methods and compositions for the treatment of cancer, particularly brain tumors such as, but not limited to, gliomas.

[0017] In aspects, the present invention provides methods and compositions for the treatment of aggressive and / or late stage brain tumors, particularly, but not limited to, glioblastoma multiforme (GBM).

[0018] In an aspect, the present invention provides a pharmaceutical composition for the treatment of solid tumors and / or brain tumors having angiogenic capacity, particularly, but not limited to, GBM, which comprises an F16 (isoindole) small molecule. The terms "F16" and "isoindole" are used interchangeably herein.

[0019] In another aspect, the present invention provides a pharmaceutical composition for the treatment of solid tumors and / or brain tumors, particularly, but not limited to, GBM, (the pharmaceutical composition) comprising a therapeutically effective dose of F16 in a pharmaceutical carrier. A "pharmaceutical carrier" can be any inert and non-toxic agent useful in the preparation of medicines. The phrase "therapeutically effective dose" or "therapeutically effective amount" refers to the amount of a composition necessary to achieve a desired function, e.g., inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) in malignant cells. Malignant cells are cells characterized by uncontrolled growth. The terms "malignant cell," "cancer cell," and "tumor cell" are used interchangeably herein.

[0020] In embodiments, in addition to a therapeutically effective dose of F16, the pharmaceutical composition can include a therapeutically effective dose of a chemotherapeutic agent, particularly, but not limited to, temozolomide (TMZ) or bevacizumab (BVZ) or similar agents.

[0021] In embodiments, the present invention provides various methods of using F16 compositions to treat malignant cells, such as, but not limited to, brain tumor malignant cells. These methods include providing an F16 composition described herein and administering the composition to the malignant cells. These methods include, but are not limited to, inhibiting VEGFR-2 in the malignant cells, inhibiting VEGFR-2 phosphorylation in the malignant cells, inhibiting the migration and invasion of the malignant cells into surrounding tissues, inhibiting cell cycle in the malignant cells, arresting the cell cycle in the malignant cells, and inducing apoptosis in the malignant cells.

[0022] In another aspect, the present invention provides a method for inhibiting and / or preventing angiogenesis in tissue exhibiting abnormal vasculature. The method comprises providing an F16 composition as described herein and administering the composition to tissue exhibiting abnormal vasculature. The method can be used as a treatment for highly vascularized solid tumors or for any tumor capable of generating new blood vessels. A non-limiting example of such a tumor is a brain tumor.

[0023] In yet another aspect, the present invention provides a method of treating glioblastoma multiforme (GBM) in a subject in need thereof. The method comprises providing an F16 composition described herein and administering the composition to the subject. The term "subject" refers to any human or animal that would benefit from the use of the compositions, methods, and / or treatments described herein. A preferred, but non-limiting, example of a subject is a human patient with a brain tumor.

[0024] Other objects and advantages of this invention will become apparent from the following description which sets forth, by way of example, certain embodiments of the invention. [Brief explanation of the drawings]

[0025] A more complete understanding of the present invention can be obtained by reference to the accompanying drawings when considered in conjunction with the following detailed description. The embodiments illustrated in the drawings are intended to be exemplary of the present invention only and should not be construed as limiting the present invention to the illustrated embodiments.

[0026] [Figure 1] FIG. 1 is a schematic diagram of the mechanism of F16 binding to vascular endothelial growth factor receptor-2 (VEGFR2), which prevents vascular endothelial growth factor (VEGF) from binding to the receptor, thus achieving an anti-angiogenic effect. [Figure 2] 1 is a bar graph showing the tissue distribution of F16. [Figure 3A] FIG. 1 is a graph showing the results of a cytotoxicity assay in which cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) and the trypan blue dye exclusion method (TBDE), and IC50 is the concentration of drug required for 50% inhibition. [Figure 3B] FIG. 1 is a graph showing the results of a cytotoxicity assay in which cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) and the trypan blue dye exclusion method (TBDE), and IC50 is the concentration of drug required for 50% inhibition. [Figure 3C] FIG. 1 is a graph showing the results of a cytotoxicity assay in which cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) and the trypan blue dye exclusion method (TBDE), and IC50 is the concentration of drug required for 50% inhibition. [Figure 4A] 10 shows images showing the morphology of U87MG cells during treatment with F16 or TMZ (prior to cell death). [Figure 4B]10 shows images showing the morphology of U87MG cells during treatment with F16 or TMZ (prior to cell death). [Figure 5A] FIG. 1 shows the migration ability of U87MG cells using a scratch assay. [Figure 5B] FIG. 1 shows the migration ability of U87MG cells using a scratch assay. [Figure 5C] FIG. 1 shows the migration ability of U87MG cells using a scratch assay. [Figure 5D] FIG. 1 shows the migration ability of U87MG cells using a scratch assay. [Figure 6A] FIG. 1 shows the migration ability of U87MG cells using a trans-well assay. [Figure 6B] FIG. 1 shows the migration ability of U87MG cells using a trans-well assay. [Figure 6C] FIG. 1 shows the migration ability of U87MG cells using a trans-well assay. [Figure 6D] FIG. 1 shows the migration ability of U87MG cells using a trans-well assay. [Figure 7A] FIG. 1 shows the invasive ability of U87MG cells using a cell invasion assay. [Figure 7B] FIG. 1 shows the invasive ability of U87MG cells using a cell invasion assay. [Figure 7C] FIG. 1 shows the invasive ability of U87MG cells using a cell invasion assay. [Figure 7D] FIG. 1 shows the invasive ability of U87MG cells using a cell invasion assay. [Figure 8A] FIG. 1 shows the effect of F16, TMZ, and the combination on anchorage-independent growth of U87MG cells using a soft agar colony formation assay. [Figure 8B] FIG. 1 shows the effect of F16, TMZ, and the combination on anchorage-independent growth of U87MG cells using a soft agar colony formation assay. [Figure 9]FIG. 1 shows gene expression in U87MG cells using reverse transcription polymerase chain reaction (RT-PCR) analysis. [Figure 10A] FIG. 1 shows protein expression in U87MG cells using Western blot analysis. [Figure 10B] FIG. 1 shows protein expression in U87MG cells using Western blot analysis. [Figure 10C] FIG. 1 shows protein expression in U87MG cells using Western blot analysis. [Figure 11A] FIG. 1 shows results from the development of a glioblastoma xenograft animal model. [Figure 11B] FIG. 1 shows results from the development of a glioblastoma xenograft animal model. [Figure 11C] FIG. 1 shows results from the development of a glioblastoma xenograft animal model. [Figure 11D] FIG. 1 shows results from the development of a glioblastoma xenograft animal model. [Figure 11E] FIG. 1 shows results from the development of a glioblastoma xenograft animal model. [Figure 12A] FIG. 1 shows the results of selection and measurement of luciferase signals in U87MG-Luc cells. [Figure 12B] FIG. 1 shows the results of selection and measurement of luciferase signals in U87MG-Luc cells. [Figure 13] 1 is a bar graph recording changes in mouse weight. [Figure 14A] 1 is a bar graph showing hematological parameters of mice. [Figure 14B] 1 is a bar graph showing hematological parameters of mice. [Figure 14C] 1 is a bar graph showing hematological parameters of mice. [Figure 14D] 1 is a bar graph showing hematological parameters of mice. [Figure 14E] 1 is a bar graph showing hematological parameters of mice. [Figure 14F]1 is a bar graph showing hematological parameters of mice. [Figure 14G] 1 is a bar graph showing hematological parameters of mice. [Figure 15] FIG. 1 shows Table 1 referencing hematological parameters of mice. [Figure 16A] 1 is a bar graph showing biochemical parameters of mice. [Figure 16B] 1 is a bar graph showing biochemical parameters of mice. [Figure 16C] 1 is a bar graph showing biochemical parameters of mice. [Figure 16D] 1 is a bar graph showing biochemical parameters of mice. [Figure 16E] 1 is a bar graph showing biochemical parameters of mice. [Figure 16F] 1 is a bar graph showing biochemical parameters of mice. [Figure 16G] 1 is a bar graph showing biochemical parameters of mice. [Figure 16H] 1 is a bar graph showing biochemical parameters of mice. [Figure 17] FIG. 1 shows Table 2 referencing biochemical parameters of mice. [Figure 18A] FIG. 1 shows data demonstrating the inhibition of U87MG-derived xenograft tumor growth by F16 in mice. [Figure 18B] FIG. 1 shows data demonstrating the inhibition of U87MG-derived xenograft tumor growth by F16 in mice. [Figure 18C] FIG. 1 shows data demonstrating the inhibition of U87MG-derived xenograft tumor growth by F16 in mice. [Figure 18D] FIG. 1 shows data demonstrating the inhibition of U87MG-derived xenograft tumor growth by F16 in mice. [Figure 19A] FIG. 1 shows survival rates (of mice) and signs of toxicity (within mice). [Figure 19B] FIG. 1 shows survival rates (of mice) and signs of toxicity (within mice). [Figure 20A] 10 is an image showing the results of microvascular density assessment. [Figure 20B] 10 is an image showing the results of microvessel density assessment. [Figure 20C] 10 is an image showing the results of microvascular density assessment. [Figure 20D] 10 is an image showing the results of microvessel density assessment. [Figure 20E] 10 is an image showing the results of microvessel density assessment. [Figure 20F] 10 is an image showing the results of microvessel density assessment. DETAILED DESCRIPTION OF THE INVENTION

[0027] For the purposes of facilitating an understanding of the principles of the invention, reference will now be made hereinafter to the embodiments exemplified herein, and specific language will be used hereinafter to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. All alterations and further modifications of the compositions and methods described, as well as any further applications of the principles of the invention as described herein, are contemplated as would normally occur to one skilled in the art to which the invention pertains.

[0028] Glioblastoma multiforme (GBM) is one of the most aggressive and lethal types of cancer, with a very low 5-year survival rate. Therefore, the development of effective treatments for GBM is urgently needed. Because GBM is highly vascularized and its growth is angiogenesis-dependent, antagonizing tumor angiogenesis using antiangiogenic agents appears to be a promising approach undergoing various stages of evaluation. In this regard, intensive preclinical evaluation of the novel small molecule F16 has demonstrated potent antiangiogenic and antitumor activity through selective antagonism of vascular endothelial growth factor receptor-2 (VEGFR-2). More importantly, pharmacokinetic evaluation of F16 by tissue distribution analysis showed that F16 was transported across the blood-brain barrier (BBB) ​​and accumulated in brain regions without signs of neurotoxicity. Therefore, further studies were conducted to determine the efficacy of F16 in delaying glioblastoma progression through the inhibition of tumor angiogenesis. In vitro studies clearly demonstrated the inhibition of migration and invasion of U87MG cells, and TMZ (IC 50 We observed a potent cytotoxic effect on these cells compared with F16 (26 μM vs. 430 μM). In addition, F16 inhibited VEGF receptors through competitive binding, blocked VEGFR-2 phosphorylation, and activated p53-mediated pathways, thereby inducing cell cycle arrest and apoptosis. Furthermore, in vivo studies using subcutaneous (sc) xenograft models have shown that F16 treatment is effective in delaying tumor growth. Thus far, results suggest that F16 treatment can induce substantial cell cycle arrest and cause tumor regression. F16 can also cross the BBB and reach the brain, thus emerging as a viable drug for targeting glioblastoma. [Example]

[0029] Example 1: Glioblastoma xenograft model Materials and Methods Cell lines and reagents U87MG, a human glioblastoma cell line, was purchased from ATCC (Manassas, VA, USA) and maintained in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, and 1% penicillin / streptomycin. Cells were cultured at 37°C in a humidified incubator with 95% air and 5% CO2. U87MG cells were used for the assay when they were between passages 3 and 9. F16 and TMZ (Sigma-Aldrich, St. Louis, MO, USA) were prepared as solutions in dimethyl sulfoxide (DMSO). Antibodies against VEGFR-2, p-VEGFR-2 (Tyr 1175), AKT, p-AKT (Ser473), ERK1 / 2, p-ERK1 / 2, p53, p21, Bax, Bcl2, MMP-2, and MMP-9 were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals used in these experiments were of research grade.

[0030] Cytotoxicity assay Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA) and trypan blue dye exclusion method (TBDE). For the MTT assay, U87MG cells were cultured at 5 × 10 per well. 3Cells were cultured in 6-well plates at a density of 100 μM and incubated under 5% CO2 for 24 hours. Next, the cells were treated with various concentrations of F16 (0.1-100 μM) and TMZ (0.1-500 μM) for 24 hours. At the end of the treatment, the old medium was aspirated, 10 μL of MTT (0.5 mg / mL in PBS) was added to each well, and the cells were incubated at 37°C for an additional 3 hours. Finally, the MTT solution was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The plates were gently rotated on an orbital shaker for 10 minutes to completely dissolve the precipitate, and the absorbance was measured at 570 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA). For the TBDE method, U87MG cells were cultured at 5 × 10 per well. 4 Cells were plated in 24-well plates at a density of 1000 μM and incubated at 37°C under 5% CO for 48 hours. Cells were then treated with various concentrations of F16 (0.1-100 μM) and TMZ (10-1000 μM). After 24, 48, and 72 hours of treatment, aliquots (50 μL) of cell suspension from each treatment were mixed with a 1:1 (v / v) volume of 0.4% trypan blue. Viable cells were counted using a Bio-Rad TC20® automated cell counter (Hercules, CA, USA).

[0031] Morphological observations U87MG cells were grown in 6-well culture plates to 70%-80% confluency. Various concentrations of F16 (0.1-100 μM) and TMZ (10-1000 μM) were then added to the medium. After 24 hours of treatment, morphological changes were recorded using a Leica microscope (100x magnification). At least three fields were photographed from each treatment well to observe changes in cell morphology.

[0032] Migration assay The migration ability of U87MG cells was determined using both scratch and trans-well assays. For the scratch assay, monolayers of U87MG cells were grown on 6-well plates at near 80% confluence. A single linear scratch was made in each well using a sterile 200 μL tip. The wells were washed with phosphate-buffered saline (PBS) and replenished with growth medium containing various concentrations of F16 (0.1-20 μM) and TMZ (10-400 μM). Images were taken 12 and 24 hours after scratching using a Leica microscope. For the trans-well migration assay, polycarbonate membrane inserts in 6.5 mm trans-well plates with 8 μm pore size (Corning, NY, USA) were used. After an initial equilibration period, 5 × 10 cells were suspended in 100 μL of basal medium without FBS. 4 Cells were added to the upper compartment of the trans-well insert and exposed to various concentrations of F16 (0.1-20 μM) and TMZ (10-400 μM). The lower chamber was filled with 600 μL of EMEM medium supplemented with 10% fetal bovine serum. The trans-well plate was then incubated at 37°C under 5% CO2 for 24 hours to allow migration of U87MG cells across the porous membrane. Non-migrating cells on the upper chamber were gently removed with a cotton swab. Migrating cells at the bottom of the chamber were fixed in 70% ethanol and stained with crystal violet for 20 minutes at room temperature. The trans-well insert was then rinsed with distilled water until excess dye was removed, and the trans-well insert was then allowed to dry. Five different fields per well were photographed with a Leica microscope (DMI 3000 B, IL, USA) using 10x magnification, and the number of cells that penetrated the membrane was counted using ImageJ software (NIH Image, Bethesda, MD, USA).

[0033] Invasion assay While the cell migration assay described above measures the number of cells crossing a porous membrane, the cell invasion assay monitors the movement of cells through an extracellular matrix such as Matrigel®. Cell invasion assays of U87MG cells were performed using Corning® BioCoat™ Matrigel® invasion chambers pre-coated with BD Matrigel substrate (Corning, NY, USA). The 8 μm pores of the 24-well membrane inserts allow single cells to invade. After rehydration of the Matrigel with growth medium, 5 × 10 cells suspended in 500 μL of basal medium without FBS were used. 4 Cells were added to the upper chamber of Corning® BioCoat™ Matrigel® inserts and exposed to various concentrations of F16 (0.1-20 μM) and TMZ (10-400 μM). The lower chamber was filled with 750 μL of EMEM medium supplemented with fetal bovine serum. The assay plate was then incubated at 37°C under 5% CO2 for 24 hours to allow invasion of U87MG cells across the porous membrane. Non-invaded cells remaining in the upper chamber were gently removed with a cotton swab. Invaded cells found at the bottom of the chamber were fixed in 70% ethanol and stained with crystal violet at room temperature for 20 minutes. The inserts were then rinsed with distilled water until excess dye was removed and allowed to dry. Five different fields per well were photographed under a Leica microscope (10x magnification), and the number of cells that had penetrated the membrane was counted using ImageJ software.

[0034] Soft agar colony formation assay The assay was performed in a 6-well plate coated with 0.6% agarose containing EMEM. Five thousand U87MG cells suspended in EMEM containing 0.3% low-melting agarose were added to each well of solidified 0.6% agarose. Cells were treated with F16 (10 and 20 μM), TMZ (200 and 400 μM), or a combination of both (20 μM F16 + 400 μM TMZ). After two weeks, cells were washed with PBS, fixed with methanol for 15 minutes, and stained with 0.005% crystal violet for 15 minutes. Five different fields per well were photographed under a Leica microscope (2.5x magnification), and the number of colonies was counted. Three independent experiments were performed for each assay.

[0035] Reverse transcription polymerase chain reaction (RT-PCR) analysis For RT-PCR analysis, total RNA was extracted from treated and untreated U87MG cells using the RNeasy Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The RT-PCR reaction mixture (50 μL) contained 1x AMV / Tfl, 1 mM MgSO, 0.2 mM dNTPs. S The reaction mixture consisted of 1 μM each of the forward and reverse primers (listed in Table 1), and 0.1 μL each of Tfl DNA polymerase and AMV reverse transcriptase. The RT-PCR products from this reaction were electrophoresed on a 1.5% agarose gel (VWR Life Sciences, Radnor, PA, USA) containing a non-mutagenic fluorescent DNA dye. The cDNA bands were visualized and photographed using a bioimaging system (UVP, Upland, CA, USA). RT-PCR products were compared by measuring band intensity using ImageJ software.

[0036] Western blot analysis Western blotting was performed using proteins from both the cell lysates and cell supernatants. After 24 hours of treatment, U87MG cells were extracted from both the control and treatment groups using RIPA (radioimmunoprecipitation assay) lysis buffer containing a protease inhibitor cocktail (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Upon supernatant collection, the cell culture medium was separated and centrifuged at 5,000 rpm for 5 minutes at 4°C to remove cell debris. After centrifugation, the cell culture medium was concentrated using an Amicon Ultra-15® filter with a molecular weight cutoff of 10 kDa at 4,000 rpm for 15 minutes at 4°C. Total protein content was determined using the bicinchoninic acid (BCA) assay (ThermoFisher Scientific, Rockford, IL, USA). For protein separation, 5-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli

[26] . Equal amounts of protein samples were loaded and electrophoresed, then transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). After blocking with 5% nonfat dry milk solution, the membranes were probed with the appropriate primary antibodies (1:1000 dilution) for VEGFR-2, p-VEGFR-2 (Tyr 1175), AKT, p-AKT (Ser 473), ERK1 / 2, p-ERK1 / 2, p53, p21, Bax, Bcl-2, MMP-2, and MMP-9. The membrane was subsequently incubated with a secondary antibody conjugated to horseradish peroxidase (HRP) enzyme and developed using the LumiGLO chemiluminescent substrate system (KPL Biosolutions, USA). As a loading control, β-actin Western blot was used for analysis. Protein band intensity was quantified using ImageJ software.

[0037] Animal models The glioblastoma xenograft model was developed using 8-10 week-old male athymic nude (Nu / Nu) mice (Charles Rivers, USA) weighing approximately 25 g. All animals were housed in pathogen-free ventilated cages under environmentally controlled conditions of humidity and temperature (22°C, 12:12 h light / dark cycle) with free access to pathogen-free food and water. All animal care and experiments were performed in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) at Nova Southeastern University (NSU), Fort Lauderdale, Florida, USA. Animals were cultured in 4 x 10 mice suspended in 100 μL of PBS mixed with Matrigel (BD Biosciences). 6 Each mouse was subcutaneously injected with 100 μg of U87MG glioblastoma cancer cells into the right flank. After 3 weeks, when the mice developed sufficiently palpable tumors, they were randomly divided into four groups: Group I was an untreated control; Group II was treated with F16 (100 mg / kg); Group III was treated with temozolomide (50 mg / kg); and Group IV was treated with F16 (100 mg / kg) and temozolomide (50 mg / kg) 3 hours later. The experimental mice were treated once every two days for a period of 16 days. At the end of the treatment, the tumors were isolated, and the length (L) and width (W) of the tumor were measured and calculated using the formula: TV = 1 / 2 × (L × W). 2 ) to calculate tumor volume (TV). To determine the tumor inhibitory effect of F16 and TMZ treatment, the inhibition ratio (IR) was calculated using the formula: IR (%) = [1-(ΣTV in treatment group / TV in control group) × 100. At the end of treatment, all animals in the control and experimental groups were sacrificed, and tumors were excised and weighed.

[0038] statistical analysis The data presented herein represent the mean ± SD values ​​from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance, and differences between means were tested by Tukey's multiple comparison test. A value of p<0.05 was considered statistically significant. Graphs were generated and statistical analysis was performed using Prism GraphPad (Mac OS X version 7.0b).

[0039] result Effect of F16 and TMZ on U87MG cell viability The inhibitory effect of F16 on U87MG cell growth was confirmed using the MTT assay and TBDE. Figure 3A shows the percentage of viable cells obtained by the MTT assay after 24 hours of treatment with various concentrations of F16 (0.1-100 μM) and TMZ (0.1-500 μM). U87MG cell growth was significantly reduced after F16 treatment in a concentration-dependent manner. After 24 hours of culture, a 50% reduction in U87MG cell viability was found to be achieved at concentrations of 26 ± 4 μM for F16 and 430 ± 10 μM for TMZ. In addition, the TBDE method was performed to confirm the MTT results. U87MG cell growth was significantly reduced after F16 treatment in a concentration- and time-dependent manner. The maximum percentages of U87MG cell death after treatment with F16 (100 μM) for 24, 48, and 72 hours were 58%, 82%, and 95%, respectively (Figure 3B).The maximum percentages of U87MG cell death after treatment with TMZ (1000 μM) for 24, 48, and 72 hours were 68%, 95%, and 82%, respectively (Figure 3C).

[0040] F16 changed cell morphology in a concentration-dependent manner In addition to cell death, F16 was able to induce changes in cell morphology in U87MG cells in a concentration-dependent manner, preceding cell death (Figure 4A). Therefore, it was proposed that F16 may inhibit cell migration and invasion in U87MG cells. Given the observation that morphological changes were observed without significant cell death upon treatment with 10 and 20 μM F16 for 24 h, these concentrations were selected for further studies. Similarly, 200 and 400 μM TMZ, which were below its IC50 value, were selected for further studies. As expected, both F16 and TMZ altered the cell morphology of U87MG cells, exhibiting concentration-dependent effects up to 100 μM and 1000 μM, respectively (Figure 4B).

[0041] F16 inhibited migration within U87MG cells To further confirm the antiangiogenic properties and effects of F16 on U87MG cell migration, a commonly used scratch assay (wound healing assay) was performed. The results showed that F16 was able to significantly inhibit the migratory ability of U87MG cells in a concentration-dependent manner (Figure 5A-B). At 12 and 24 h after scratching, migration was not observed when cells were treated with 20 μM F16, clearly indicating that F16 has a strong ability to inhibit U87MG cell migration. However, cells treated with 400 μM TMZ showed inhibition of migration until 12 h after scratching, but they began to migrate thereafter (Figure 5C-D). Similarly, F16 exhibited a consistent inhibitory effect on cell migration, as shown by the results obtained from trans-well migration assays. Approximately 80% of U87MG cells treated with 20 μM F16 for 24 h were trapped in the upper compartment compared to untreated cells, indicating the strong anti-migratory effect of F16 (Figure 6A-B). Consequently, approximately 80% of U87MG cells were trapped in the upper compartment upon treatment with 400 μM TMZ compared to untreated cells (Fig. 6C-D).

[0042] F16 inhibited invasion in U87MG cells To determine whether F16 attenuates cell invasion ability, a Matrigel® invasion assay was performed using a trans-well plate. The results showed that U87MG cells invading through the Matrigel® substrate were significantly reduced in a concentration-dependent manner after 24 hours of treatment with F16 compared to untreated control cells (Figures 7A-B). As shown in Figures 7A-B, F16 significantly reduced cell invasive ability. However, at a much higher concentration (400 μM), similar results were obtained with TMZ treatment, confirming that TMZ can slightly inhibit the invasion ability of U87MG cells in a concentration-dependent manner (Figures 7C-D).

[0043] F16 reduced anchorage-independent growth in U87MG cells To explore the effect of F16 on the anchorage-independent growth of U87MG cells, a soft agar colony formation assay was performed. The results showed that the number of anchorage-independent colonies was significantly reduced after treatment with F16 compared to untreated control cells (Figure 8A-B). Similarly, similar results were obtained with TMZ and combination (F16 + TMZ) treatment (Figure 8A-B). However, there was no significant reduction with combination (20 μM F16 + 400 μM TMZ) compared to F16 (20 μM) and TMZ (400 μM).

[0044] Determination of gene expression in U87MG cells using RT-PCR To further strengthen our findings, Figure 9 shows the expression levels of selected genes in U87MG-treated and untreated cells. Differences in band intensity obtained through RT-PCR indicate differences in the mRNA levels of the corresponding genes. VEGFR-2 and AKT mRNA levels were downregulated by TMZ (400 μM) and the F16 + TMZ combination (20 and 400 μM) compared with the control. Interestingly, p53 and Bax mRNA levels were significantly upregulated in F16 (10 and 20 μM)-treated cells, as well as in TMZ (200 and 400 μM) and F16 + TMZ combination-treated cells. Furthermore, a slight increase in p21 mRNA levels was observed in F16- and TMZ-treated cells compared with the control. Notably, Bcl2, MMP-2, and MMP-9 mRNA levels were significantly downregulated by individual and combined treatment with F16 and TMZ.

[0045] Inhibition of VEGFR-2 phosphorylation and downstream signaling Previous studies have clearly demonstrated that preventing VEGFR-2 activation can significantly limit the angiogenesis process, which plays a key role in tumor progression

[27] . The level of phospho-VEGFR-2 (Tyr1175), the active form of VEGFR-2, was significantly reduced after F16 treatment (Figure 10A). Furthermore, p-AKT expression at Ser473, an important downstream target of VEGFR-2, was also significantly suppressed by F16 in U87MG cells (Figure 10A). These results indicate that F16 has the ability to attenuate AKT-dependent cell viability. Similarly, similar results were obtained with TMZ and combined (F16 + TMZ) treatment.

[0046] F16 induced cell cycle arrest and apoptosis To better understand the role of F16 in cell cycle arrest and apoptosis, the expression of proteins p53, p21, Bax, and Bcl2 was analyzed. Expression of p53, a well-established tumor suppressor gene, was upregulated after F16 and combination treatment, but showed a smaller increase in expression level after treatment with TMZ alone (Figure 10B). In addition, p21 expression was significantly upregulated after F16 and combination treatment (Figure 10B). Surprisingly, p21 expression was significantly downregulated with TMZ treatment (Figure 10B). Furthermore, Bax expression was also increased after F16, TMZ, and combination treatment, whereas Bcl2 expression was suppressed by the same treatment (Figure 10B). These findings suggest that p53 induces cell cycle arrest and apoptosis through p21- and Bax-dependent pathways in U87MG cells.

[0047] Effects of F16 on ERK1 / 2, MMP-2, MMP-9, and cell invasion ERK1 / 2 are important subfamily of mitogen-activated protein kinases that regulate a wide range of cellular activities and physiological processes. p-ERK1 / 2 expression was upregulated after F16, TMZ, and combination treatment (Figure 10C). Furthermore, MMP-2 and MMP-9 expression was downregulated after F16 treatment (Figure 10C). These results demonstrate the ability of F16 to activate ERK1 / 2 in a sustained manner, which likely contributes to the downregulated expression of MMP-2, resulting in the suppression of cell invasion. Interestingly, similar results were obtained with TMZ and combination treatment.

[0048] Inhibition of U87MG-derived xenograft tumor growth by F16 To further investigate the in vivo tumor growth inhibitory effect of F16, a subcutaneous glioblastoma xenograft model using U87MG cells was established as described above in the Materials and Methods section. Previous studies have shown that the U87MG xenograft model is considered one of the most widely utilized experimental models available for preclinical testing of glioblastoma [References 28, 29]. Therefore, once tumors were fully established, mice were randomized into four groups as described above and treated intraperitoneally with F16, TMZ, and the combination of F16 and TMZ for 16 days. Representative photographs of excised tumors are shown in Figure 11A. The results clearly showed that mice implanted with U87MG tumors exhibited 58%, 53%, and 70% tumor growth inhibition after 16 days of treatment with F16 (100 mg / kg), TMZ (50 mg / kg), and F16 (100 mg / kg) + TMZ (50 mg / kg), respectively (Figure 11B). Interestingly, the tumor growth inhibitory effect of F16 monotherapy was comparable to that of TMZ at the doses shown, with no signs of toxicity in the F16 group. However, the combination of F16 with TMZ, the standard of care for the treatment of glioblastoma cancer, did not result in a significant reduction in tumor volume (70%) compared with either F16 (58%) or TMZ (53%) monotherapy.

[0049] The changes in the experimental mice's body weight were also examined during the treatment period (Figure 11C). Consistent with previous studies, F16 treatment was well tolerated at the dose used for treatment (100 mg / kg). However, symptoms of toxicity, such as weight loss, general weakness, and accumulation of ascites, were observed 1 week after treatment in the TMZ group and the combination group, which lost one animal from the TMZ group. At the end of the treatment period, tumors were excised for comparison. As shown in Figure 11D, tumor weights were significantly lower in the F16 and TMZ, and combination treatment groups compared with the control group. IR% was calculated as described in the Methods section and shown in Figure 11E.

[0050] Discussion The prognosis for glioblastoma multiforme (GBM) remains poor, and available treatment options currently offer only marginal benefits with only modestly significant increases in patient survival. The current standard of care for newly diagnosed GBM patients is surgical resection followed by a course of cytotoxic therapy with radiation plus temozolomide (TMZ) chemotherapy

[30] . The addition of TMZ to radiation therapy extends overall mean survival by 2.6 months (total 14.6 months) compared with the mean survival of 12 months with radiation therapy alone

[31] . However, TMZ administration is clinically associated with severe toxicities such as genotoxicity, myelosuppression, teratogenicity, and severe intestinal damage

[32] . Previous studies have generally reported that TMZ, like several other cytotoxic chemotherapy agents, possesses cytotoxic effects on normal cells, often associated with the development of secondary malignancies

[33] . All these drawbacks associated with TMZ have prompted scientists to develop more effective treatment options for GBM. Furthermore, the high expression of VEGF found in GBM is also associated with poor prognosis, providing a rationale for evaluating antiangiogenic agents as suitable agents for treating GBM. In this context, the inventors have discovered a novel small molecule, F16, which competitively blocks VEGF binding to its receptor, blocks the ligand that induces phosphorylation of VEGFR-2 (Tyr1175) in HUVECs, and exhibits in vitro antiangiogenic activity. The aforementioned VEGFR-2-specific binding agent has been shown to inhibit endothelial cell growth, migration, and tube formation

[24] .

[0051] Initially, VEGFR-2 was thought to be expressed only at high levels and exclusively in endothelial cells. However, several studies conducted in recent years have revealed that certain cancer cells, such as glioblastoma cells, also express VEGFR-2 at relatively high levels

[34] . Interestingly, the U87MG cell line is one of the glioblastoma cell lines that express high levels of VEGFR-2

[34] and are highly sensitive to TMZ treatment

[35] . Therefore, the U87MG cell line was selected as a model representing glioblastoma, and the efficacy of F16 was tested and compared with standard TMZ. Initial experiments were aimed at comparing the anti-growth effects of F16 and TMZ on U87MG glioblastoma cancer cells using MTT and TBDE assays. In in vitro experiments, F16 exhibited an IC value higher than that of TMZ (430 μM). 50 The IC of 26 μM was 15-fold lower than the value (Figure 3A). 50 The data show higher efficacy against U87MG cells with TMZ (172-700 μM) than reported in the literature [References 36-38]. 50 Furthermore, the concentration- and time-dependent effects of F16 in inducing cytotoxicity in U87MG using the TBDE method were also consistent with the IC values ​​achieved in the MTT assay. 50 Furthermore, the effects of F16 and TMZ on the anchorage-independent growth (the ability of cells to grow independently on a solid surface) of U87MG cells were examined using a soft agar colony formation assay

[39] . In vitro colony formation of U87MG cells in soft agar was significantly inhibited by F16 and TMZ compared with the control (Figure 8A-B), confirming the ability of F16 to inhibit the anchorage-independent growth of U87MG cells.

[0052] To investigate the underlying molecular mechanisms mediating F16-induced cytotoxicity in U87MG cells, the phosphorylation of VEGFR-2 after F16 treatment was investigated. VEGFR-2 has seven phosphorylation sites, including Tyr1175, which regulates cell growth and migration

[40] . Results showed a significant suppression of p-VEGFR-2 (Tyr1175) levels in U87MG cells after F16 treatment (Figure 10A). Recent studies have also confirmed the antagonistic effect of F16 through competitive binding with VEGFR-2

[24] .

[0053] Blockade of VEGFR-2 phosphorylation by F16 led to the exploration of the PI3K-AKT pathway, one of the downstream targets of VEGFR-2 that plays an important role in facilitating cell viability and cell cycle progression [Refs. 40, 41]. Previous studies have shown that activation of AKT is involved in suppressing apoptosis by interfering with transcription factors that facilitate the expression of pro-apoptotic genes and enhancing the transcription of anti-apoptotic genes [Refs. 41, 42]. Furthermore, AKT has been shown to indirectly suppress p53-mediated apoptosis by phosphorylating mouse double minute 2 (MDM2), a negative regulator of p53 [Ref. 43]. On the other hand, inhibition of AKT phosphorylation has been shown to facilitate cancer cell death and apoptosis through the p53-mediated pathway [Refs. 41, 43]. Therefore, the results suggested that F16 could facilitate cell death through the inhibition of AKT phosphorylation at Ser473 and activation of the p53 pathway, ultimately inducing cell cycle arrest and apoptosis by upregulating p21 and Bax. As expected, F16 was able to induce the expression of p53, p21, and Bax and reduce the expression of Bcl2 after 24 hours of treatment (Figure 10B). These results clearly demonstrate that F16 can suppress AKT-mediated viability of U87MG cells and induce apoptosis through activation of the p53 pathway.

[0054] A distinct pathological feature of GBM cells is their ability to extensively invade surrounding areas, including normal brain tissue [Ref. 44]. GBM cell invasion is a complex, multistep process that typically begins with the degradation of the extracellular matrix (ECM) by MMPs, which allows cancer cells to migrate from the primary tumor and form secondary tumors [Ref. 44, 45]. Numerous studies have reported that MMP-2, along with MMP-9, is highly expressed in various human glioblastoma cell lines, including U87MG [Ref. 46-48]. Both MMP-2 and MMP-9 degrade type IV collagen, the most abundant component of the basement membrane. Thus, collagen degradation is a critical step for the initiation of migratory progression in most cancers [Ref. 46]. Therefore, downregulation of MMP-2 and MMP-9 expression is closely associated with the suppression of GBM cell migration and invasion [Ref. 48]. Results with U87MG cells suggest that F16 inhibits IC 50 The results clearly showed that F16 significantly inhibited both migration and invasion at concentrations lower than the 2000 ng / mL values ​​(Figures 5A-B, 6A-B, and 7A-B). While blocking migration and invasion, F16 treatment also downregulated the expression of MMP-2 and MMP-9 (Figure 10C). Furthermore, several studies have reported sustained activation of ERK1 / 2 signaling, which suppresses tumor cell invasion in many human glioblastoma cancer cells, including U87MG cells [Reference 49] and human prostate cancer cells [Reference 50]. ERK1 / 2 enzymes are an important subfamily of mitogen-activated protein kinases that have shown substantial roles in regulating cell growth, apoptosis, and invasion, depending on the cell type and mode of activation [References 51-53]. It has been shown that transient activation of ERK1 / 2 (stimulation for <15 min) can induce cancer cell growth, migration, and invasion. On the other hand, the opposite effect was observed with sustained activation (>15 min of stimulation) of ERK1 / 2 [Refs. 53-55] (Figure 10C), which appears to be consistent with the results obtained after treatment of U87MG cells with F16 and TMZ.

[0055] To support the in vitro results, an in vivo model was considered to examine the efficacy of F16 in delaying glioblastoma progression. A subcutaneous glioblastoma xenograft model was successfully established using athymic nude mice treated with F16, TMZ, and the combination. The in vivo results showed that F16 significantly suppressed xenograft tumor growth, suggesting that VEGFR-2 blockade using F16 treatment is effective in delaying glioblastoma cancer growth (Figure 11B). Unlike mice treated with TMZ alone, F16 treatment up to 16 days showed no signs of toxicity [References 24, 25], consistent with previous studies conducted in our laboratory on various cancer models. Unexpectedly, mice treated with the combination of F16 and TMZ showed no significant difference in tumor volume reduction compared to mice treated with monotherapy (Figure 11B). Furthermore, signs of increased toxicity and intolerance were observed in the combination group. Such toxicity may be reduced if lower doses of TMZ are used or if the interval between administration of the two drugs is increased.

[0056] In conclusion, Example 1 shows that the in vitro and in vivo results clearly demonstrate the high efficacy of F16 treatment in inhibiting the viability, migration, and invasion of U87MG cells. Compared to TMZ, F16 significantly reduced the IC 50 F16 has potent cytotoxicity against U87MG cells at 26 μM (Figure 3A) and is well tolerated in mice. It also exhibits potent anticancer effects by slowing tumor growth in xenograft-implanted athymic nude mice. [Example]

[0057] Example 2: Intracranial model of glioblastoma While promising results were obtained with F16, Example 1 used a single cell line in a subcutaneous xenograft model that responded to drug treatment. Therefore, the use of an alternative in vivo model, such as an intracranial brain tumor xenograft, would provide further validation of its therapeutic efficacy against GBM. Therefore, the primary focus of Example 2 was to determine the efficacy of F16 in delaying glioblastoma progression using an intracranial GBM xenograft model, and to evaluate the tolerability of F16 in a KP formulation and establish its safety profile using nude mice.

[0058] Cancer remains the second leading cause of death worldwide, despite significant efforts and resources being devoted to developing newer treatment strategies and diagnostic methods [Reference 1]. Millions of people worldwide are diagnosed with cancer each year, and the survival rate for these patients is extremely low, primarily in the later stages. Among cancer types, glioblastoma multiforme (GBM) is one of the most aggressive and deadly types of brain tumors with a poor prognosis, with fewer than 5% of patients surviving 5 years after diagnosis [Reference 2]. As described above in Example 1, the current standard of care for newly diagnosed GBM patients is surgical resection followed by a course of chemotherapy, such as radiation plus temozolomide (TMZ), whenever applicable [Reference 3]. The addition of TMZ provides a modest increase in overall survival (OS) of 12.1 to 14.6 months compared with adjuvant radiation therapy followed by surgical debulking [References 4, 5]. However, TMZ administration can lead to the development of resistance and is clinically associated with severe toxicities, such as genotoxicity, teratogenicity, bone marrow suppression, and severe intestinal damage. [6] Therefore, the development of more effective and safer treatments for GBM is urgently needed.

[0059] One of the defining features of GBM is its abundant and abnormal vasculature [Reference 7]. Unlike normal cerebral vasculature, GBM vasculature is disorganized, poorly connected, and tortuous, associated with prominent endothelial growth, resulting in areas of hypoxia [Reference 8]. Furthermore, vascular endothelial growth factor (VEGF) is elevated in GBM, along with increased vascular permeability, vessel diameter, and abnormalities in endothelial wall and basement membrane thickness [References 9, 10]. High VEGF expression in GBM is also associated with poor prognosis, providing a rationale for evaluating antiangiogenic agents as appropriate drugs to treat GBM [Reference 11].

[0060] In preclinical and clinical studies, the use of antiangiogenic agents in combination with chemotherapy has shown promising results against a wide range of cancer types [12-15]. Recently, the use of antiangiogenic agents has emerged as a new strategy for the treatment of glioblastoma (GBM) due to the significant angiogenesis that occurs in GBM. To date, bevacizumab (BVZ) is the only antiangiogenic agent approved by the FDA for the treatment of recurrent GBM. However, BVZ treatment has not resulted in an improvement in overall survival (OS), and FDA approval was based on an increase in overall objective response rate (ORR) [16, 17].

[0061] As mentioned above, one of the major challenges in treating brain tumors is the presence of the blood-brain barrier (BBB). The BBB is a highly selective barrier, and crossing it is difficult for large molecules; small (molecular weight less than 400–500 Da) lipophilic molecules are required

[18] . Therefore, recent interest has shifted to the search for small molecules that can cross the BBB and modulate angiogenesis and similar processes. In this regard, F16, a novel small molecule (molecular weight 301.2 g / mol), has shown potent antiangiogenic and antitumor activity through selective antagonism of vascular endothelial growth factor receptor-2 (VEGFR-2) in both in vitro and in vivo models

[19] . More importantly, preclinical pharmacokinetic studies have shown that F16 has the potential to cross the BBB and accumulate in brain regions

[20] . Therefore, in Example 1, the direct effect of F16 to inhibit the growth, angiogenesis, and migration ability of U87MG glioblastoma cells (known to express high levels of VEGFR) was examined. In vitro studies confirmed the potent inhibitory effect of F16 on the migration and invasion of U87MG cells, and compared it with temozolomide (IC 50 430 μM) treatment on U87MG cells. 50 In addition, F16 inhibited VEGFR-2 phosphorylation through competitive binding and induced cell cycle arrest and apoptosis by activating the p53 pathway in U87MG cells. Furthermore, in vivo results using an ectopically implanted xenograft model confirmed the fact that F16 could significantly suppress tumor growth in mice implanted with the U87MG glioblastoma cell line.

[0062] Example 2 provides further validation of the efficacy of F16 treatment on GBM using another in vivo model, such as intracranial brain tumor xenografts. Therefore, the primary focus of Example 2 was to determine the efficacy of F16 in delaying glioblastoma progression using an intracranial GBM xenograft model, and to evaluate the tolerability of F16 in a KP formulation and establish its safety profile using a mouse model.

[0063] Materials and Methods Cell lines and reagents U87MG, a human glioblastoma cell line, was purchased from ATCC (Manassas, VA, USA) and maintained in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, and 1% penicillin / streptomycin. Cells were cultured at 37°C in a humidified incubator with 95% air and 5% CO2. U87MG cells were used for the assay when they were between passages 3 and 9. F16 and TMZ (Sigma-Aldrich, St. Louis, MO, USA) were prepared as solutions in dimethyl sulfoxide (DMSO). All other chemicals used in these experiments were of research grade.

[0064] U87MG cells transfected with luciferase gene (pcDNA3.1-Luc) For the purpose of developing cell lines for xenograft imaging experiments, U87MG cell lines at 90-95% confluency (6-well plates) were used for transfection with Lipofectamine 2000. On the day of transfection, cells were replenished with fresh medium without any antibiotics. For the transfection process, complex A (10 μg of pcDNA3.1-Luc in 100 μL of serum-free medium + 15 μL of PLUS Reagent) and complex B (12 μL of Lipofectamine 2000 in 100 μL of serum-free medium) were prepared separately and incubated at room temperature for 15 minutes. Complexes A and B were combined and incubated at room temperature for an additional 15 minutes. This solution (200 μL) was added to the fixed cells containing 800 μL of the appropriate medium (serum- and antibiotic-free) and incubated for an additional 5 hours at 37°C in a 5% CO2 incubator. Additionally, 1 mL of growth medium containing 20% ​​serum without antibiotics was added to the transfection wells, and the culture was continued for another 72 hours to allow stable transfection (with U-87MG cells).

[0065] Measurement of luciferase signals in U87MG-Luc cells To measure the cultured luciferase gene-transfected cells (U87MG-Luc cells), the inventors cultured various cell numbers (1 x 10 cells) by adding phosphate-buffered saline with D-luciferin (Fisher Scientific, USA) at a concentration of 0.15 mg / ml. 4 -3x10 5 Luciferase signals were imaged using a Bruker Xtreme II (Bruker, Billerica, MA, USA). U87MG-Luc cells were imaged 10 minutes after incubation with D-luciferin at room temperature. Luciferase signal measurements were analyzed using a Bruker Xtreme II (Bruker, Billerica, MA, USA).

[0066] Animal models For tolerability studies, 8-10 week old male BALB / c mice weighing approximately 25 g were used (Charles Rivers, USA). For intracranial studies, 8-10 week old female athymic nude (Nu / Nu) mice weighing approximately 25 g were used (Taconic Biosciences, USA). All animals were housed in pathogen-free ventilated cages under environmentally controlled conditions of humidity and temperature (22°C, 12:12 h light / dark cycle) with free access to pathogen-free food and water. All animal care and experiments were performed in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) at Nova Southeastern University (NSU), Fort Lauderdale, Florida, USA.

[0067] Drug preparation F16 (100 mg / kg) was dissolved in 10% DMSO + 90% KolliphorEL (KP). TMZ (50 mg / kg) was dissolved in 10% DMSO + 90% phosphate-buffered saline (PBS). All drugs were prepared fresh before the scheduled injection

[21] . The total injection volume was 100 μL / mouse for all experiments administered intraperitoneally.

[0068] Experimental procedure For the tolerability study, BALB / c mice were randomly assigned to four different treatment groups (Figure 15; Table 1). At the end of the treatment period, blood samples were collected from all mice and sent to the Department of Comparative Pathology at the University of Miami (Miami, FL, USA) for analysis of hematological and biochemical parameters.

[0069] For intracranial studies, a glioblastoma xenograft model was developed using athymic nude (Nu / Nu) mice. Briefly, mice were placed under general anesthesia (intraperitoneal injection of 100 mg / kg ketamine and 10 mg / kg xylazine) and positioned in a stereotaxic device. A ~1 cm median incision was made, and a burr hole was drilled into the right striatum of the skull (1.0 mm anterior and 2.0 mm lateral to the bregma). Subsequently, U87MG cells (2 x 10 in 3 μL of PBS) expressing the luc reporter gene were transfected. 5 Tumor cells were injected using a 10 μL Hamilton syringe at a rate of 1 μL / min to a depth of 3 mm. After the injection, the needle was held in place for 2 minutes, then slowly removed, and the hole was sealed with sterile bone wax. The incision was closed, and triple antibiotic ointment was applied. One week after tumor cell implantation, mice were randomly divided into five groups (n=5 per group): 1) controls treated with DMSO in PBS, 2) controls treated with DMSO in KP, 3) controls treated with F16 (100 mg / kg), 4) controls treated with temozolomide (50 mg / kg), and 5) controls treated with F16 (100 mg / kg) followed by temozolomide (5 mg / kg) 3 hours later. One or more groups without tumor implants were included in the study as negative controls (n=5). Experimental mice were treated twice per week for three weeks. After treatment was completed, the mice were maintained without any treatment until they showed severe illness, and then euthanized using a Euthanex CO2 smart box. The brains and tumors of the euthanized mice were isolated for histology and immunohistochemistry (IHC) studies.

[0070] In vivo bioluminescence imaging Bioluminescence imaging (BLI) was used to assess and confirm tumor growth in intracranial xenografts. BLI was performed in vivo using a Bruker Xtreme, a sensitive optical X-ray instrument designed for preclinical in vivo studies based on the BLI concept. Briefly, mice were intraperitoneally injected with D-luciferin (Sigma) dissolved in saline at a dose of 150 mg / kg body weight. Immediately after injection, mice were anesthetized with isoflurane, and a series of bioluminescence images were acquired at 3-minute acquisition intervals over approximately 20 minutes until the luciferin washed out. The image with peak BLI intensity was used for quantification in photon count units.

[0071] Histology and immunohistochemistry Histological analysis was performed to evaluate tumor histology and the effects of experimental drugs on tumors. Surgically resected tumors in brain tissue were rinsed with 1X PBS to remove blood for histology and IHC preparation. Samples from each experimental group were fixed in 10% neutral buffered formalin (NBF) and shipped to the University of Florida Molecular Pathology Core for further histology and IHC preparation. Microscopic images and data were received from the facility. For IHC, samples were incubated with a primary mouse monoclonal anti-CD31 antibody (1:100 dilution; Cell Signaling Tech. Inc.), and a secondary antibody, biotin-labeled rabbit anti-mouse IgG (1:500; Nichirei, Tokyo, Japan), was used with a DAB staining kit. Sections were counterstained with hematoxylin. For H&E (hematoxylin and eosin) staining, samples were stained with Harris' hematoxylin solution followed by eosin solution from the University of Florida Molecular Pathology Core.

[0072] statistical analysis The data presented here represent the mean ± SD values ​​from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance, and differences between means were tested by Tukey's multiple comparison test. A value of p<0.05 was considered statistically significant. Graphs were generated and statistical analysis was performed using Prism GraphPad (Mac OS X version 7.0b).

[0073] result Selection and measurement of luciferase signals in U87MG-Luc cells For selection, cells were treated with various concentrations of G418 antibiotics (0.1-0.8 mg / mL) for 14 days. After antibiotic selection, cells were examined for luciferase expression using the Steady-Glo Luciferase Assay System (Promega, USA). U87MG cells treated with 0.8 mg / mL of G418 antibiotic generated maximum luminescence. Luciferase-transduced optical imaging enabled us to monitor the response to anticancer treatment in tumor xenografts. In addition, luciferase images of fixed U87MG-luc cells showed a steady increase in BLI signal as cell numbers increased (Figures 12A-B).

[0074] Toxicity evaluation A comprehensive toxicity study using BALB / c mice was performed to evaluate the toxicity profiles of F16, TMZ, and the F16 + TMZ combination. Mice injected with KP were used as controls. All drugs were administered as ip injections twice weekly for 4 weeks. Independent toxicity assessments, serum biochemistry, postmortem macroscopic examination, and histopathological examination of major organs were performed by the Comparative Pathology Department at the University of Miami, Florida.

[0075] During the treatment period, mice were monitored weekly for changes in body weight, and no significant fluctuations were observed (Figure 13). Similarly, mice were monitored for general behavior, physical appearance, convulsions, drug-induced diarrhea, salivation, and mortality. In general, F16 treatment was associated with no observable signs of toxicity. However, some symptoms of sensitivity or discomfort were noted in the F16, combination, and control (KP) groups immediately after injection, and then the symptoms disappeared the next day. The same symptoms were observed in the control group. Since F16 was well tolerated and no signs of toxicity or discomfort were observed in all previous animal studies, KP is suspected as the reason for these symptoms.

[0076] Complete blood counts (CBCs) were performed to measure hemoglobin (HB), hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell count (RBC), and white blood cell count (WBC). HB, MCH, MCHC, and MCV levels did not change significantly in the various treatment groups (Figure 15: Table 1). Slight increases in hematocrit and RBC were observed in the F16 and TMZ treatment groups, but not in the KP and F16 + TMZ treatment groups (Figure 15: Table 1). These results do not indicate signs of myelosuppression resulting in anemia, thrombocytopenia, or neutropenia (Figure 15: Table 1). Analysis of WBC counts showed no significant changes in the KP, F16, and F16+TMZ treatment groups, while TMZ-treated mice showed a significant increase in WBC counts (Figure 15: Table 1).

[0077] Total protein levels were analyzed to assess the effect of the treatment regimen on protein metabolism. No significant changes were observed in total protein levels in any treatment group (Figure 17: Table 2).

[0078] Assessment of major organ function Liver function was assessed by measuring ALT levels. A significant increase in ALT was observed in the TMZ-treated group (Table 2 in Figure 17). Renal function was also assessed by measuring blood urea nitrogen (BUN), creatine, and BUN / creatine ratio levels. No significant changes in BUN were detected in the KP and TMZ-treated groups. However, a significant decrease in BUN levels was observed in the F16 and F16 + TMZ-treated groups (Table 2 in Figure 17). As shown in Table 2 in Figure 17, no significant changes in creatine and the ratio of BUN / creatine levels were found in any of the treatment groups. Additionally, the effect of F16 on the pancreas was assessed by measuring glucose, an essential energy source. No significant changes were observed in blood glucose in any of the treatment groups (Table 2 in Figure 17).

[0079] Inhibition of U87MG-derived xenograft tumor growth by F16 To further investigate the in vivo tumor growth inhibitory effect of F16 and confirm previous studies using a subcutaneous model, an intracranial glioblastoma xenograft model using U87MG cells was established as described above in the Materials and Methods section. U87MG-luc cells were implanted into the brains of mice, and tumor growth was monitored by BLI. One week after cell implantation, animals were randomly divided into five groups (control-PBS, control-KP, F16, TMZ, and F16 + TMZ). Tumor growth was monitored weekly by BLI, and representative mice from the five groups are shown in Figure 18A. The results clearly showed that the BLI signal intensity of F16-treated mice was 60% lower than that of control mice (Figure 18B). However, the BLI signal intensity of TMZ- and combination-treated mice was the lowest among the five groups (Figure 18B). These results showed that administration of F16 either as monotherapy or in combination therapy reduced tumor growth, but TMZ treatment was more efficient than F16 treatment, which would be expected since F16 is cytostatic rather than cytoreductive. Furthermore, after mouse death, the tumor-bearing brains were excised, and the length (L) and width (W) of the brain tumors were then measured, and TV = 1 / 2 × (L × W). 2The tumor volume (TV) was calculated by the formula (Figure 18C). The brains of the athymic nude mice before euthanasia and the same mice after euthanasia are shown in Figure 18D.

[0080] Survival and signs of toxicity The survival rates of mice with glioma xenografts after treatment with vehicle-PBS, vehicle-KP, F16, TMZ, and the combination were examined. Tumor-bearing mice treated with F16 showed a significant increase in survival time with a mean survival rate of 39 days compared with mice treated with vehicle-PBS and vehicle-KP, which had mean survival rates of 34 and 36 days, respectively (Figure 19A). Furthermore, 60% of mice in the TMZ and combination groups survived until 50 days after transplantation with a mean survival rate of 47 days (Figure 19B). However, brains excised from the TMZ and combination groups were fragile and damaged.

[0081] The changes in body weight of the experimental mice were monitored weekly from the day of implantation until the end of the experiment (Figure 19B). Consistent with previous studies, F16 treatment was well tolerated at the dose used for treatment (100 mg / kg). No significant changes in body weight were observed in mice treated with F16, TMZ, or the combination compared to mice treated with the vehicle.

[0082] Microvascular density assessment Xenograft brains and tumors were excised and subjected to IHC analysis. The expression of the glioblastoma marker CD31 in F16-, TM-, and F16 and TMZ-combined tumor sections was compared with tumors extracted from the control group (Figures 20A-F). High levels of CD31 were expressed in control-PBS and control-KP tumor sections, indicating that exponential growth of GBM is associated with angiogenesis (Figures 20B-C). In contrast, a significant decrease in CD31 expression was observed in F16 tumor sections compared with control and TMZ tumor sections, indicating that F16 treatment substantially blocked angiogenesis in vivo (Figures 20D-E). These results indicate that the antitumor activity of F16, exerted through reducing the vascular density of xenograft tumors, was more pronounced and effective.

[0083] Discussion As mentioned above, glioblastoma multiforme (GBM) treatment is extremely challenging, as evidenced by the low survival rate of GBM patients, who generally do not survive longer than one year [Reference 22]. The current standard of care for GBM patients is diverse and begins with extensive surgical resection of the tumor mass. Patients then undergo radiation therapy (RT) and combination chemotherapy with temozolomide (TMZ). In fact, the TMZ plus RT treatment regimen is considered the most effective, as it increases the mean overall survival rate by 2.6 months to 14.6 months compared to 12 months with RT alone, and the percentage of patients surviving at two years increases from 10.4% to 26.5% [Reference 4]. Unfortunately, 60-75% of TMZ-treated patients do not respond to TMZ treatment, and more than 50% of patients fail treatment after six months due to tumor progression [References 23, 24]. The drawbacks of this response are due to the overexpression of O6-methylguanine methyltransferase (MGMT) and / or DNA damage repair systems in GBM cells

[25] . Furthermore, 15-20% of TMZ-treated patients develop significant toxicity, which may lead to treatment failure

[23] . All these drawbacks associated with TMZ have prompted scientists to develop more effective treatment options. In this regard, new treatment strategies targeting vascular endothelial growth factor (VEGF) or its downstream signaling pathways have shown promising results as an add-on to standard treatments

[26] .

[0084] The dependence of tumor growth and migration on angiogenesis supports the idea of ​​using antiangiogenic approaches to treat cancer. Furthermore, angiogenesis inhibitors have been clinically proven to improve patients' quality of life and prolong progression-free survival (PFS) and / or overall survival (OS) in several advanced cancers, prompting scientists to investigate the use of angiogenesis inhibitors for GBM treatment. In 2009, BVZ was approved by the FDA for the treatment of recurrent GBM [Reference 27]. Indeed, the use of BVZ for the treatment of recurrent GBM failed to improve OS but improved PFS [References 17, 28]. Furthermore, angiogenesis inhibitors have been proposed to be useful in alleviating intracranial pressure associated with brain tumors by reducing vascular permeability through normalization of the existing vasculature [Reference 29]. Unfortunately, the use of angiogenesis inhibitors for GBM treatment faces two hurdles: most angiogenesis inhibitors are unable to cross the blood-brain barrier (BBB) ​​

[30] , and some angiogenesis inhibitors are associated with severe toxicity that limits their clinical benefit

[31] . Thus, there is a critical need to develop new angiogenesis inhibitors that can cross the BBB with little or no toxicity.

[0085] In 2011, F16, a novel antiangiogenic agent, was disclosed in U.S. Patent No. 7,939,557 B2. F16 not only exhibited strong binding and inhibition of vascular endothelial growth factor receptor-2 (VEGFR2) phosphorylation in human umbilical vein endothelial cells (HUVECs), but also demonstrated significant in vivo tumor growth inhibition in mice bearing GI-101A (breast cancer) xenografts and Colo-320DM (colon cancer) xenografts [Reference 19]. Additionally, preclinical pharmacokinetic studies demonstrated substantial accumulation of F16 in major organs of mice after a single i.p. administration [Reference 20]. An unexpected finding was that 12 hours after injection, F16 concentrations were highest in the brain compared with the liver and kidney. The F16 concentrations in the brain approached those observed in plasma, which were 1.3-fold and 6.1-fold higher than those in the liver and kidney, respectively. These results indicate that F16 is easily transported across the BBB and slowly accumulates in brain regions without evidence of clinical behavioral toxicity. Indeed, lipophilicity and molecular weight, two important factors, play a key role in facilitating BBB penetration of any drug [Reference 32]. Consistent with these criteria, F16 possesses a higher lipophilicity and a smaller molecular weight (301.2 g / mol), which may explain its penetration across the BBB. All these results prompted the inventors to test the efficacy of F16 in the treatment of GBM.

[0086] Treatment-related toxicity is generally one of the most common limitations of clinically available drugs for cancer treatment. Hepatotoxicity and nephrotoxicity are common toxicities associated with chemotherapeutic agents, including angiogenesis inhibitors. In this toxicity study, mice treated with TMZ showed signs of hepatotoxicity, as evidenced by increased ALT (Figure 17; Table 2). The results are also consistent with previous reports of TMZ in rodent models [Reference 24]. In humans, TMZ treatment is associated with hepatotoxicity as well as bone marrow suppression, including neutropenia and thrombocytopenia [Reference 33]. Previous results from safety evaluation studies have shown that F16-treated experimental animals remained healthy compared with groups treated with other FDA-approved chemotherapeutic drugs, such as Sutent® and Taxol [Reference 20]. Similarly, in the current study, F16 was well tolerated with no mortality events in the experimental animals. Furthermore, there were no significant changes in body weight, food intake, or behavior in the experimental groups (Figure 13). Even though F16 accumulated in the brain, no signs of cognitive changes were observed in the treatment group. Furthermore, evaluation of biochemical parameters reflecting vital organ function showed no signs or increases in the levels of injury-related biomarkers in the liver, kidney, and pancreas after F16 treatment.

[0087] Xenograft models using human cancer cells offer tremendous benefits to the oncology field. Initially, subcutaneous xenograft models, called heterotopic models, are the most commonly used preclinical procedures for establishing tumor xenografts because they are fast, inexpensive, and easily reproducible [34, 35]. However, it has consistently been noted that some treatment regimens that are curative in heterotopic models have no significant effect on human disease. Therefore, emphasis has shifted to establishing orthotopic xenografts, such as intracranial brain tumor xenografts. In orthotopic models, tumor xenografts are implanted into the same anatomical site or organ where the cancer develops, providing a convenient location for tumor-host interactions, the ability to study site-specific dependence of treatments, and organ-specific gene expression, and sufficient preclinical testing for anticancer drugs [35, 36]. Furthermore, tumor progression and migration are known to depend on the formation of new blood vessels in most situations

[37] . Similarly, biochemical imbalances in the tumor microenvironment contribute to pathological angiogenesis and tumor growth progression through the continuous secretion of growth factors

[38] .

[0088] To mimic tumor growth in a suitable tumor microenvironment, an intracranial GBM xenograft model was established, which provides a better representation of the clinical characteristics of tumor angiogenesis and is more relevant to the real-life situation in the human brain. The results showed that F16 significantly suppressed xenograft tumor growth (Figure 18B) and prolonged median survival (Figure 19A), suggesting that VEGFR-2 blockade using F16 treatment is effective in slowing glioblastoma cancer growth. In previous in vitro and in vivo studies (subcutaneous xenografts, Example 1), the effect of F16 was comparable to that of TMZ. However, in the intracranial xenograft model, TMZ demonstrated much better tumor inhibition (99%) than F16 (60%), which is expected because F16 is cytostatic rather than cytoreductive like TMZ. Another possible reason behind the difference in results is the difference in drug concentration reaching the brain after penetrating the BBB. While full drug concentrations reach cancer cells when in vitro models are used, substantial drug concentrations reach tumor sites when subcutaneous xenograft models are used. Conversely, drug delivery to the brain is affected by several factors, such as lipophilicity and small molecular weight due to the presence of the BBB. TMZ is a lipophilic small molecule with a molecular weight of 194.15 g / mol, so it can easily cross the BBB [Reference 39]. Previous studies using rats and monkeys to test the penetration of TMZ into the CNS have shown that TMZ levels in the brain are approximately 30–40% of the plasma concentration, which is significant [Reference 40]. Undoubtedly, the TMZ and combination treatment groups outlived the F16 treatment group. However, excised brains from the TMZ and combination groups were fragile and damaged, implying that TMZ treatment affected surrounding normal tissue and ultimately caused death. Consistent with the inventor's observations, a recent study concluded that TMZ treatment may affect the extracellular matrix structure of normal brain tissue, leading to disease progression [Reference 41].

[0089] F16's antitumor activity is substantially mediated through the inhibition of angiogenesis

[19] . IHC results confirmed the in vivo antiangiogenic activity of F16 using CD31 expression as a biomarker, indicating the presence of endothelial cells within tumor tissue

[42] . As expected, F16 treatment was associated with low levels of CD31 expression, representing a significant reduction in tumor microvessel density (Figure 20D).

[0090] In conclusion to Example 2, the in vivo results clearly demonstrated the high efficacy of F16 treatment in suppressing tumor growth and prolonging the mean survival rate of mice intracranially implanted with U87MG-luc cells. Compared to TMZ, F16 was well tolerated in mice without evidence of significant preclinical or laboratory toxicity. The use of the KP formulation improved the intracerebral delivery of F16 by 40% compared to the PBS formulation (data not shown), and the KP preparation caused some hypertensive reactions that could lead to more severe side effects when used for longer periods (Reference 43). Finally, these findings provide a new means for GBM treatment that could benefit a large number of patients by prolonging their overall survival and improving their quality of life.

[0091] conclusion The discoveries disclosed herein provide new avenues for the treatment of solid tumors with angiogenic potential, particularly for the treatment of brain tumors such as glioblastoma multiforme (GBM). Such novel treatments could benefit a large number of patients by extending their overall survival and / or improving their quality of life.

[0092] All patents and publications described herein are indicative of the level of skill of those skilled in the art to which this invention pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. While specific forms of the invention have been illustrated, it is to be understood that the invention is not intended to be limited to the specific forms or arrangements described and illustrated herein. It will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention, and the invention should not be construed as limited to that shown and described herein. One skilled in the art will readily appreciate that the present invention is adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods using F16 described herein are representative of presently preferred embodiments and are intended to be illustrative and not limiting in scope. Modifications thereof and other uses within the spirit of the invention will occur to those skilled in the art. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as ultimately claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the present invention.

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[0094] References (Example 2) 1. Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2018. CA Cancer J Clin, 2018. 68(1): p. 7-30. 2. Kim, W.Y. and H.Y. Lee, Brain angiogenesis in developmental and pathological processes: mechanism and therapeutic intervention in brain tumors. FEBS J, 2009. 276(17): p. 4653-64. 3. Anjum, K., et al., Current status and future therapeutic perspectives of glioblastoma multiforme (GBM) therapy: A review. Biomed Pharmacother, 2017. 92: p. 681-689. 4. Stupp, R., et al., Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med, 2005. 352(10): p. 987-96. 5. Stupp, R., et al., Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol, 2009. 10(5): p. 459-66. 6. Neyns, B., et al., Dose-dense temozolomide regimens: antitumor activity, toxicity, and immunomodulatory effects. 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[0095] announcement 1. Mohammad Algahtani1, Khalid Alhazzani2, Thiagarajan Venkatesan, Ali Alaseem, Sivanesan Dhandayuthapani and Appu Rathinavelu (2019), Direct cytotoxic effect of a novel anti-angiogenic drug F16 towards U87MG glioblastoma cell line, Presented at the AACR Annual Meeting 2019, March 29 - April 3 Atlanta, GA. 2. Mohammad Algahtani, Khalid Alhazzani, Sivanesan Dhandayuthapani, Thanigaivelan Kanagasabai, Appu Rathinavelu, (2017) F16 is a novel new candidate for brain tumors, Presented at Cancer Research and Targeted Therapy (CRT) Oct 26-28, Miami FL, USA. 3. Sivanesan Dhandayuthapani, Thanigaivelan Kanagasabai, Khadija Cheema and Appu Rathinavelu (2017), Bioavailability, pharmacokinetics and safety profile of a novel anti-angiogenic compound JFD in pre-clinical models. Presented at the AACR Annual Meeting 2017, April 1-5 Washington, DC. 4. Thanigaivelan Kanagasabai, Khalid Alhazzani, Thiagarajan Venkatesan, Sivanesan Dhandayuthapani, Ali Alaseem, Appu Rathinavelu (2017), impact of MDM2 inhibition on cell cycle regulation through Aurora Kinase B-CDK1 axis in prostate cancer cells, Presented at the Annual Conference of the American Association for Cancer Research (AACR) April 1-5, Washington, DC, USA. 5. Ali Alaseem, Thiagarajan Venkatesan, Thanigaivelan Kanagasabai, Khalid Alhazzani, Saad Alobid, Priya Dondapati, Appu Rathinavelu (2017), Increased MMPs activity in MDM2 overexpressing cancer cell lines 6. Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Thanigaivelan Kanagasabai, Appu Rathinavelu (2017), Effects of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells 7. Khalid Alhazzani, Ali Alaseem, Thiagarajan Venkatesan, Appu Rathinavelu (2017), Angiogenesis-related gene expression profile of a novel antiangiogenic agent F16 in human vascular endothelial cells, Presented at the Annual Conference of the American Association for Cancer Research (AACR) April 1-5, Washington, D.C., USA 8. Saad Ebrahim Alobid, Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Appu Rathinavelu (2017), analysis of human hypoxia related miRNA in MDM2 transfected prostate cancer cells, Presented at the Annual Conference of the American Association for Cancer Research (AACR) April 1-5, Washington, D.C., USA 9. Mohammad Algahtani, Khalid Alhazzani, Thiagarajan Venkatesan, Appu Rathinavelu (2017), apoptosis pathway-focused gene expression profiling of a novel VEGFR2 inhibitor, Presented at the Annual Conference of the American Association for Cancer Research (AACR) April 1-5, Washington, D.C., USA0. 10. Paramjot Kaur, Sivanesan Dhandayuthapani, Shona Joseph, Syed Hussain, Miroslav Gantar, Appu Rathinavelu. Evaluation of the cell surface binding of phycocyanin and associated mechanisms causing cell death in prostate cancer cells. Presented at the American Association for Cancer Research (AACR) 2017 Apr 1-4; Washington DC, USA 11. Khalid Alhazzani, Sivanesan Dhandayuthapani, Khadijah Cheema, Thanigaivelan Kanagasabai, Ali Alaseem, Thiagarajan Venkatesan, Appu Rathinavelu (2016), Pharmacokinetic and Safety Profile of a Novel Anti-angiogenic Agent F16 with High Levels of Distribution to the Brain. Presented in: 2016 AAPS Annual Meeting and Exposition at Colorado, Denver, on Nov 16th 2016. 12. Thanigaivelan Kanagasabai, Sivanesan Dhandayuthapani, Khalid Alhazzani, Ali Alaseem and Appu Rathinavelu (2016), The pharmacodynamics profile and tissue distribution of a novel anti-angiogenic compound JFD in pre-clinical models. Presented in: Molecular and Cellular Basis of Breast Cancer Risk and Prevention at Tampa, Florida on Nov, 12th - 15th 2016. 13. Appu Rathinavelu (2016), Novel VEGFR2 Inhibitors for Treating Solid Tumors and Brain Metastasis (2016), Presented at the International Conference on Cancer Research and Targeted Therapy, in Baltimore, Maryland on October 21-23 of 2016. 14. Thanigaivelan Kanagasabai, Rohin Chand, Amy Aman Kaur, Sivanesan Dhandayuthapani, Olena Bracho, Appu Rathinavelu. MDM2 stabilizes and induces HIF-1α levels during reoxygenation of cancer cells. Presented at the Annual Conference of the American Association for Cancer Research (AACR), April 16-20, New Orleans, LA., USA 15. Thiagarajan Venkatesan, Ali Alaseem, Aiyavu Chinnaiyan, Sivanesan Dhandayuthapani, Thanigaivelan Kanagasabai, Khalid Alhazzani, Priya Dondapati, Saad Alobid, Umamaheswari Natarajan, Ruben Schwartz, Appu Rathinavelu (2018). MDM2 Overexpression Modulates the Angiogenesis-Related Gene Expression Profile of Prostate Cancer Cells. Cells, 2018, 7(5), 41. 16. Appu Rathinavelu, Thanigaivelan Kanagasabai, Sivanesan Dhandayuthapani, Khalid Alhazzani (2018), The anti-angiogenic and pro-apoptotic effects of a small molecule JFD-WS in in vitro and breast cancer xenograft mouse model. Oncology Reports. Published online on: February 9, 2018, Pages:1711-1724; https: / / doi.org / 10.3892 / or.2018.6256 17. Rathinavelu. A, Alhazzani. K, Dhandayuthapani. S and Kanagasabai. T. (2017) Anti-cancer effects of F16 - A novel vascular endothelial growth factor receptor specific inhibitor, Tumor Biology, Nov; 39 (11):1010428317726841. https: / / doi: 10.1177 / 1010428317726841.

Claims

1. 1. A pharmaceutical composition for use in a method for slowing the progression of brain tumors, comprising a therapeutically effective dose of F16, i.e., isoindole (1,3-dioxy-2,3-dihydro-1H-isoindol-4-yl)-amide, in a pharmaceutical carrier, and a therapeutically effective dose of a chemotherapeutic agent, wherein the brain tumor is glioblastoma multiforme and the chemotherapeutic agent is temozolomide (TMZ) or bevacizumab (BVZ) or a similar agent.

2. The pharmaceutical composition of claim 1, wherein the therapeutically effective dose of F16 is 100 mg / kg body weight.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the therapeutically effective dose of the chemotherapeutic agent is 50 mg / kg body weight.

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