Patent Application
20220409581
The invention is encompassed within the field of cancer therapy and generally relates to therapies using small molecules to target solid tumors, particularly to therapies using F16 isoindole small molecules for treatment of brain cancers.
Despite the significant efforts and resources that are being devoted for developing newer treatment strategies and cures, cancer remains a fatal disease of mankind and millions of people around the world die every year from various types of cancers. One of the most prevailing types of this deadly disease is brain cancer, which is the leading cause of cancer-related deaths in children and the third most common cause of cancer related death in adolescents and young adults between age 15 and 39 years [1, 2].
There are 12 main groups of brain tumors and more than 100 subgroups that share common biological features [3]. Gliomas include all tumors arising from the supportive tissues of the brain and are the most aggressive form of brain cancers, account for 24.7% of all primary brain tumors, and 74.6% of all malignant brain tumors [4]. Glioblastoma multiforme (GBM) is the most diagnosed form of glioma in the United States and is the most lethal type worldwide. Despite use of multidisciplinary treatment approaches, GBM has a very low 5-year survival rate of 5%, and a median survival of about one year post-diagnosis [3, 4]. Generally, GBM is classified as a grade IV glioma, and some of the histologic features that distinguish it from other grades are the presence of necrosis and the dramatic increase of blood vessel growth around the tumor [5]. In fact, GBM is one of the most highly vascularized solid tumors since its growth depends on angiogenesis as supported by various preclinical studies that have indicated that the glioma growth critically depends on the generation of tumor-associated blood vessels [4, 6]. In addition, GBM tumor vasculature is characterized by a dense network of vessels that are tortuous and hyper-permeable and have abnormally increased vessel diameter and thickness of basement membranes. This aberrant tumor vasculature is believed to enhance tumor hypoxia and impair the delivery of cytotoxic chemotherapy thus contributing to the treatment failure [5, 7]. Therefore, antagonizing tumor vascular is emerging as a novel strategy for brain tumor treatment, particularly for treatment of GBM.
Several forms of treatments that are currently available for GBM are non-effective in many cases, therefore, prognosis for GBM remains poor. The current treatment of glioblastoma involves surgery, whenever it is applicable, followed by radiation and chemotherapy with Temozolomide (TMZ). This treatment strategy provides a modest increase in overall survival [8]. In preclinical and clinical studies, the use of angiogenesis inhibitors in combination with chemotherapeutic agents has shown promising results against a wide range of cancer types [9-12]. Particularly, antiangiogenic agents are currently under intense investigation for treating GBM and various preliminary studies have yielded promising outcomes [13-15]. Therefore, several antiangiogenic agents are now in clinical trials for the treatment of GBM in monotherapy or in combination [16]. So far, Bevacizumab (BVZ), a monoclonal antibody with anti-angiogenic effects, has been approved by 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, in depth analysis of BVZ treatment data of patients with GBM showed no improvement in overall survival (OS) [17, 18]. It is worth mentioning that angiogenesis inhibitors, when used in monotherapy, can generally produce cytostatic effect and maximum therapeutic efficacy is achieved if these agents are combined with cytotoxic chemotherapeutic agents [19, 20].
One of the major obstacles of treating a brain tumor is the ability of the therapeutic agent to cross the blood-brain barrier (BBB) [21]. It is well known that penetrating BBB is not easy for agents with high molecular weight such as BVZ (˜150 kD M.W.), implying that BVZ treatment for GBM may not provide optimal delivery and treatment outcomes [22, 23]. Therefore, recent interest has shifted towards exploring small molecules that can cross BBB to modulate angiogenesis and similar processes. In this context, a novel compound, isoindole (1, 3-dioxy-2, 3-dihydro-1H-isoindol-4-yl)-amide, was developed at Nova Southeastern University (NSU) and code named as F16. See U.S. Pat. No. 7,875,603; Japanese Patent 5436544; and Korean Patent 10-1538822. F16 chemical structure (19, references of Example 2):
F16 is not only showing strong vascular endothelial growth factor receptor-2 (VEGFR-2) binding and inhibition of VEGFR-2 phosphorylation in human umbilical vein endothelial cells (HUVEC) but F16 also exhibits a significant in vivo tumor growth inhibition in mice implanted with breast and [24] colorectal cancer xenografts (data not published). More importantly, the preclinical pharmacokinetics studies have shown that F16 can cross BBB and accumulate into brain regions [25]. Furthermore, results from preclinical safety studies have proven so far that F16 treated experimental animals remain healthy compared to the groups that were treated with other FDA approved anticancer agents such as Paclitaxel [24] and Sunitinib [25].
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 in a solid tumor such as breast cancer (
Since the inventive methods (and compositions) described herein show the efficacy of F16 in delaying glioblastoma progression via its anti-angiogenic and pro-apoptotic abilities, it (F16) can potentially be used as a basis to create new avenues for effective treatment of brain cancers, particularly those exhibiting angiogenic ability which enables their growth and survival.
The small molecule F16 (isoindole) offers the potential for promising new cancer therapy. Based on preliminary in vitro and in vivo experiments, the cytotoxic effects in the monolayer culture and in 3D culture were confirmed. To achieve a good understanding about the effects of F16 on the migration and invasive ability of cancer cells, a Scratch assay, a Trans-well migration assay, and an invasion assay were performed. The anti-migratory effects and the anti-invasive capacity of the U87MG cells, that typically coincide with the anti-angiogenic properties during cancer metastasis, were determined through the above-mentioned assays. The results confirmed that invading abilities of U87MG cells were significantly decreased after 24 h of treatment with F16 versus untreated control cells in a dose-dependent manner. The results were compared with TMZ (Temozolomide) which is an FDA approved drug. So far, F16 has shown consistent inhibitory effects on the cell migration as well as cancer cell invasion, as presented in the results, which are significantly better than the TMZ effects. The changes in the pro-apoptotic gene expressions were also analyzed and it appeared that F16 can inhibit cell cycle and induce apoptosis in the U87MG cell line better than the TMZ.
The luciferase gene transfected U87MG-luc tumor cells were established, to monitor the tumor growth inhibition through optical imaging while assessing the effectiveness of F16 for the treatment of glioblastoma. Initially, the xenograft model was created by injecting the U87MG-Luc cells using intra-peritoneal injection (i.p.). The animals were treated with F16, TMZ, and a combination of both drugs. The studies with U87MG-Luc glioblastoma cell line have shown good results with the F16 compound. While reducing the tumor volume, F16 did not alter the body weight during the treatment period. Analysis of the blood parameters such as RBC, WBC (
After completing testing, the effect with the subcutaneous tumor models, and after confirming the safety of F16, the intracranial implant studies were initiated. In the intracranial experiments, F16 was able to block the tumor growth of the brain in 50% of the animals. This conformed that F16 was able to cross the BBB and inhibit the growth of the U87MG derived tumor in the brain. It has been also noted that KP (Kolliphor®) that was used as a vehicle was slightly increasing the brain delivery of F16 but was also causing some side effects.
In a most basic aspect, the invention provides methods for manipulation of malignant cells, particularly interactions within malignant cells characterized by uncontrolled growth.
In another basic aspect, the invention provides a new treatment modality for cancer.
In a general aspect, the invention provides methods and compositions for treatment of cancers manifesting as solid tumors, particularly, but not limited to, solid tumors exhibiting angiogenic ability.
In a general aspect, the invention provides methods and compositions for treatment of cancer, particularly, but not limited to, brain cancers, such as gliomas.
In an aspect, the invention provides methods and compositions for treatment of aggressive and/or late stage brain cancer, particularly, but not limited to, glioblastoma multiforme (GBM).
In an aspect, the invention provides compositions for treatment of solid tumors having angiogenic ability and/or brain cancer, particularly, but not limited to, GBM, (the compositions) including F16 (isoindole) small molecules. The terms “F16” and “isoindole” are used interchangeably herein.
In another aspect, the invention provides pharmaceutical compositions for treatment of solid tumors and/or brain cancer, particularly, but not limited to, GBM, (the pharmaceutical compositions) including a therapeutically effective dosage of F16 in a pharmaceutical carrier. The “pharmaceutical carrier” can be any inactive and non-toxic agent useful for preparation of medications. The phrase “therapeutically effective dosage” or “therapeutically effective amount” refers to the amount of a composition required to achieve the desired function; for example, inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) in malignant cells. Malignant cells are cells characterized by uncontrolled growth. The terms “malignant cells”, “cancer cells,” and “tumor cells” are used interchangeably herein.
In an aspect, in addition to the therapeutically effective dosage of F16, the pharmaceutical composition can include a therapeutically effective dosage of a chemotherapeutic agent, particularly, but not limited to, temozolomide (TMZ) or bevacizumab (BVZ) or similar agents.
In an aspect, the invention provides various methods of using F16 compositions for treating malignant cells, such as, but not limited to, malignant cells of a brain cancer. These methods include steps of providing the F16 compositions described herein and administering the compositions to the malignant cells. These methods include, but are not limited to, inhibiting VEGFR-2 in malignant cells, inhibiting phosphorylation of VEGFR-2 in malignant cells, inhibiting migration and invasion of malignant cells into surrounding tissues, inhibiting a cell cycle in malignant cells, arresting a cell cycle in malignant cells, and inducing apoptosis in malignant cells.
In another aspect, the invention provides a method for inhibiting and/or arresting angiogenesis in tissue exhibiting aberrant vasculature. This method includes the steps of providing the F16 compositions described herein and administering the compositions to the tissue exhibiting aberrant vasculature. This method can be used as a treatment for highly vascular solid tumors or for any tumor having the ability to produce new blood vessels. A non-limiting example of such a tumor is brain cancer.
In yet another aspect, the invention provides a method for treating glioblastoma multiforme (GBM) in a subject in need thereof. This method includes the steps of providing the F16 compositions described herein and administering the compositions to the subject. The term “subject” refers to any human or animal who will 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 having brain cancer.
Other objectives and advantages of this invention will become apparent from the following description, wherein are set forth, by way of example, certain embodiments of this invention.
A more complete understanding of the present invention may be obtained by references to the accompanying drawings when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments.
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments illustrated herein and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modification in the described compositions and methods along with any further application 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 relates.
Glioblastoma multiforme (GBM) is one of the most aggressive and lethal types of cancer having an exceptionally low 5-year survival rate. Therefore, development of effective treatment for GBM is urgently desired. Since GBM is a highly vascularized tumor and its growth is angiogenesis-dependent, antagonizing tumor angiogenesis by using angiogenesis inhibitors seems to be a promising approach that is undergoing various stages of evaluation. In this context, intensive preclinical evaluation of F16, a novel small molecule, has exhibited potent anti-angiogenic and anti-tumor activities via selectively antagonizing vascular endothelial growth factor receptor-2 (VEGFR-2). More importantly, pharmacokinetic evaluation with tissue distribution analysis of F16 showed that F16 transported across the blood brain barrier (BBB) and accumulated into the brain regions with no signs of neurotoxicity. Therefore, further studies were conducted to determine the efficacy of F16 in delaying glioblastoma progression via inhibiting tumor angiogenesis. In vitro studies have clearly demonstrated inhibition of migration and invasion of U87MG cells and confirmed a potent cytotoxic effect against these cells in comparison to TMZ (IC50 26 μM vs 430 μM). In addition, F16 inhibited the VEGF receptor via competitive binding and blocked the phosphorylation of VEGFR-2, to induce cell cycle arrest and apoptosis by activating p53 mediated pathway. Furthermore, in vivo studies with the subcutaneously implanted (s.c.) xenograft model indicated that F16 treatment is efficacious in delaying tumor growth. So far, results suggest that F16 treatment could effectively induce cell cycle arrest and cause tumor reductive effect. F16 can also cross the BBB to reach the brain and therefore is emerging as a viable agent for targeting glioblastoma.
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 incubated at 37° C. with 95% air and 5% CO2 in a humidified incubator. U87MG cells were used in assays, when the cell passages were between 3 and 9. The F16 and TMZ (Sigma-Aldrich, St. Louis, Mo., USA) were prepared as a solution in dimethyl sulfoxide (DMSO). The 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, Mass., USA). All other chemicals used in these experiments were of research grade.
The cell viability was assessed using a 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 in a 96 well-plate at a density of 5×103 per well and incubated at 37° C. under 5% CO2 for 24 h. Then, the cells were treated with different concentrations of F16 (0.1-100 μM) and TMZ (0.1-500 μM) for 24 h. At the end of treatment, the old medium was aspirated, and 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 h. Finally, the MTT solution was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was gently rotated on an orbital shaker for 10 min to completely dissolve the precipitation and the absorbance was measured at 570 nm using Microplate Reader (VersaMax, Molecular Devices, Sunnyvale, Calif., USA). For the TBDE method, U87MG cells were cultured in a 24 well-plate at a density of 5×104 per well and incubated at 37° C. under 5% CO2 for 48 h. Then, the cells were treated with different concentrations of F16 (0.1-100 μM) and TMZ (10-1000 μM). After 24, 48, and 72 h of treatments, an aliquot (50 μL) of the cell suspension from each treatment was mixed with 1:1 (v/v) volume of 0.4% trypan blue. The viable cells were counted with a Bio-Rad TC20™ Automated Cell Counter (Hercules, Calif., USA)
U87MG cells were grown to 70%-80% confluence in 6-well culture plates. Then various concentrations of F16 (0.1-100 μM) and TMZ (10-1000 μM) were added to the media. After 24 h of treatments, morphological changes were documented with a Leica microscope (100× magnification). At least, 3 vision fields from each treatment wells were captured to see the changes in the cell morphology.
The migration ability of U87MG cell was determined using both scratch and trans-well assays. For the scratch assay, monolayer of U87MG cells was grown on 6-well plates close to 80% confluency. Using a sterile 200 μL tip, a single straight line scratch was made in each well. The wells were washed with phosphate-buffered saline (PBS) and refilled with growth medium containing various concentrations of F16 (0.1-20 μM) and TMZ (10-400 μM). The images were captured using Leica microscope at 12 h and 24 h post-scratch. For the trans-well migration assay, 6.5 mm trans-well plates polycarbonate membrane inserts (Corning, N.Y., USA) with 8 μm pore size were used. After an initial equilibrium period, 5×104 cells suspended in 100 μL basal medium without FBS were added to the upper compartment of the trans-well inserts and exposed to different 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. Then, trans-well plates were incubated at 37° C. under 5% CO2 for 24 h to allow for the migration of U87MG cells across the porous membrane. The non-migrating cells on the top chamber were removed gently with a cotton swab. The migrated cells at the bottom of the chamber were fixed in 70% ethanol and stained with crystal violet at room temperature for 20 min. Then, the trans-well inserts were rinsed with distilled water, until excess dye was removed, and then the trans-well inserts were allowed to dry. Five different fields per well were captured with a Leica microscope (DMI 3000 B; IL, USA) using 10× magnification, and the number of cells that penetrated the membrane was counted using ImageJ software (NIH Image, Bethesda, Md., USA).
The above-described Cell Migration Assay measures the number of cells traversing a porous membrane, while the Cell Invasion Assays monitor cell movement through extracellular matrix such as Matrigel®. The U87MG cell invasion assay was performed using Corning® BioCoat™ Matrigel® Invasion Chamber that was pre-coated with BD Matrigel matrix (Corning, N.Y., USA). The 8 μm pores of the 24-well membrane inserts allow the single cells to invade. After rehydration of the Matrigel with growth medium, 5×104 cells suspended in 500 μL basal medium without FBS were added to the upper chamber of the Corning® BioCoat™ Matrigel® inserts and exposed to different 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 10% fetal bovine serum. Then, the assay plates were incubated at 37° C. under 5% CO2 for 24 h to allow for invasion of U87MG cells across the porous membrane. The non-invading cells remaining on the top chamber were removed gently with a cotton swab. The invaded cells found at the bottom of the chamber were fixed in 70% ethanol and stained with crystal violet at room temperature for 20 min. Then, the inserts were rinsed with distilled water until excess dye was removed and let to dry. Five different fields per well were captured with a Leica microscope (10× magnification), and the number of cells that penetrated the membrane was counted using ImageJ software.
The assay was carried out in 6-well plates coated with 0.6% agarose containing EMEM. Five thousand cells of U87MG suspended in EMEM containing 0.3% low melting agarose were added to the solidified 0.6% agarose of each well. Cells were treated with F16 (10 & 20 μM), TMZ (200 & 400 μM) and a combination of both (F16 20 μM+TMZ 400 μM). After two weeks, the cells were washed with PBS, fixed in methanol for 15 min, and stained with 0.005% crystal violet for 15 min. Five different fields per well were captured with a Leica microscope (2.5× magnification), and the number of colonies counted. Three independent experiments were carried out for each assay.
For the RT-PCR analysis, total RNA was extracted from the treated and non-treated U87MG cells using RNeasy Kit according to the manufacturer's instructions (Qiagen, Valencia, Calif., USA). The RT-PCR reaction mixture (50 μL) consists of 1×AMV/Tfl, 1 mM MgSO4, 0.2 mM dNTPs, 1 μM each of forward and reverse primers (list in Table 1) and 0.1 u/μL of each Tfl DNA polymerase and AMV Reverse Transcriptase. The RT-PCR products obtained from this reaction were electrophoresed on 1.5% agarose gels containing non-mutagenic fluorescent DNA dye (VWR Life sciences, Radnor, Pa., USA). The cDNA bands were visualized and captured using Bioimaging system (UVP, Upland, Calif., USA). RT-PCR products were compared by measuring the band intensity using ImageJ software.
Proteins from both cell lysates and cell supernatants were used to conduct western blotting. After 24 h of treatment, U87MG cells extracted from both the control and treated groups by using RIPA (Radio immunoprecipitation assay) lysis buffer containing protease inhibitor cocktail (Santa Cruz Biotechnology, Inc. Dallas, Tex., USA). For supernatant collection, the cell culture media were separated and centrifuged at 5000 rpm for 5 min at 4° C. to remove the cell debris. After centrifugation the cell culture media were concentrated using Amicon Ultra-15® centrifugal filter, with a molecular weight cut-off limit of 10 kDa, at 4,000 rpm for 15 min at 4° C. Total protein content was determined using bicinchoninic acid (BCA) assay method (ThermoFisher Scientific, Rockford, Ill., USA). For protein separation 5-12% of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared as described by Laemmli [26]. Equal amounts of protein samples were loaded and subjected to electrophoresis, and then transferred to the nitrocellulose membrane (GE Healthcare BioSciences, Pittsburgh, Pa., USA). After blocking with 5% non-fat dry milk solution, the membranes were probed with suitable VEGFR-2, p-VEGFR-2 (Tyr 1175), AKT, p-AKT (Ser473), ERK1/2, p-ERK1/2, p53, p21, Bax, Bcl-2, MMP-2 and MMP-9 primary antibodies (1:1000 dilution). Membranes were subsequently incubated with a secondary antibody that was conjugated to horseradish peroxidase (HRP) enzyme and developed using the LumiGLO, chemiluminescence, substrate system (KPL biosolutions, USA). As a loading control, β-actin western blot was used in the analysis. The protein band intensity was quantified using ImageJ software.
The glioblastoma xenograft model was developed using 8-10 weeks old male athymic nude (Nu/Nu) mice weighing approximately 25 g (Charles Rivers, US). 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) of Nova Southeastern University (NSU), Ft. Lauderdale, Fla. Animals were subcutaneously injected into the right flank of each mouse, with 4×106 of U87MG glioblastoma cancer cells suspended in 100 μL of PBS mixed with Matrigel (BD Biosciences). Three weeks later, once the mice developed well-palpable tumors, they were divided randomly into four groups: group I was the 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 3 h later Temozolomide (50 mg/kg). The experimental mice were treated once in every 2 days for the period of 16 days. At the end of the treatment, the tumors were isolated and then the tumor length (L) and width (W) were measured to calculate the tumor volume (TV) according to the formula: TV=1/2×(L×W2). To determine the tumor inhibitory effects of F16 and TMZ treatments, the inhibition ratio (IR) was calculated using the formula:
At the end of the treatment, all the animals in the control and experimental groups were sacrificed and tumors were excised and weighed.
The data presented herein represent mean±SD values from at least three independent experiments. Statistical analyses were performed using a one-way analysis of variance and the differences between means were tested by Tukey's multiple comparison test. The value of p<0.05 was considered as statistically significant. Prism GraphPad (Mac OS X version 7.0b) was used to generate graphs and perform statistical analysis.
The inhibitory effects of F16 on the proliferation of U87MG cells using MTT assay and TBDE were confirmed. The percentage of viable cells obtained in the MTT assay after 24 h of treatment with varying concentrations of F16 (0.1-100 μM) and TMZ (0.1-500 μM) was shown in
In addition to the cell death, F16 was able to induce changes in the cellular morphology of U87MG cells, preceding the cell death, in a concentration-dependent manner (
To further confirm the anti-angiogenic property and the effects of F16 on the migration of U87MG cells, the commonly used scratch assay (wound healing assay) was performed. The results showed that F16 was able to significantly inhibit the migration ability of U87MG cells in a concentration-dependent manner (
To determine whether F16 weakens the cell invasive potential, Matrigel® invasion assays were conducted using the trans-well plates. The results showed that U87MG cells invading through the Matrigel® matrix was significantly decreased in a concentration-dependent manner after 24 h of treatment with F16 versus untreated control cells (
To explore the effect of F16 on 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 (
In order to further strengthen the findings,
Previous studies have clearly indicated that prevention of VEGFR-2 activity could significantly limit the angiogenesis process which plays a critical role in tumor progression [27]. The level of phospho-VEGFR-2 (Tyr 1175), which is the active form of VEGFR-2, was significantly decreased after F16 treatment (
To better understand the role of F16 in cell cycle arrest and apoptosis, expression of proteins p53, p21, Bax and Bcl2 was analyzed. The expression of p53, a well-established tumor suppressor gene, was upregulated after F16 treatment and combination treatment, but showed lesser increase in the expression levels after treatment with TMZ alone (
ERK1/2 is an important subfamily of mitogen-activated protein kinases that controls a broad range of cellular activities and physiological processes. The expression of p-ERK1/2 was upregulated after F16, TMZ and combination treatments (
To further investigate the in vivo tumor growth inhibitory effects of F16, a subcutaneous glioblastoma xenograft model using U87MG cells was established as described earlier in the materials and methods section. Previous studies have indicated that U87MG xenograft model is considered to be one of the most widely utilized experimental models available for pre-clinical testing of glioblastoma [28, 29]. Therefore, once the tumor was fully established mice were randomized into four groups, as described before and treated intraperitoneally with F16, TMZ, and F16+TMZ combination for 16 days. Representative pictures of excised tumors are shown in
Changes in body weight of the experimental mice were also examined during the treatment period (
The prognosis of Glioblastoma multiforme (GBM) remains poor, and the available treatment options currently provide only modest benefits with a barely significant increase in patient survival. The current standard of care for newly diagnosed patients with GBM is surgical resection followed by a course of radiation plus cytotoxic therapy with chemotherapeutic agent such as Temozolomide (TMZ) [30]. The addition of TMZ to radiotherapy increases the overall median survival by 2.6 months (total of 14.6 months) compared to 12 months of median survival for radiotherapy alone [31]. However, TMZ administration was clinically associated with severe toxicities such as genotoxicity, bone marrow suppression, teratogenicity, and severe intestinal damage [32]. Earlier studies have reported that, similar to several other cytotoxic chemotherapeutic agents in general, TMZ possess cytotoxic effects on normal cells, which are often associated the onset of secondary cancers [33]. All these shortfalls associated with TMZ have prompted scientists to develop more effective therapeutic options for the treatment of GBM. Moreover, high expression of VEGF found in GBM is also associated with poor prognosis, which provides a logical rationale to evaluate angiogenesis inhibitors as preferred drugs to treat GBM. In this context, F16, a novel small molecule that competitively blocks VEGF binding to its receptors and blocks ligand induced phosphorylation of VEGFR-2 (Tyr1175) in HUVEC and exhibits in vitro anti-angiogenic activity, was found by the instant inventors. The above-mentioned VEGFR-2 specific binding agent was shown to inhibit endothelial cell proliferation, migration, and tube formation [24].
Initially, VEGFR-2 was thought to be exclusively expressed at high levels only in endothelial cells. However, several studies conducted in the last few years have demonstrated that certain cancer cells, such as glioblastoma cells, also express the VEGFR-2 in relatively high levels [34]. Interestingly, the U87MG cell line is one of the glioblastoma cell lines that expresses high levels of VEGFR-2 [34] with high sensitivity towards TMZ treatment [35]. Because of that, the U87MG cell line was chosen as a model representing glioblastoma to test and compare the efficacy of F16 with the standard TMZ. The initial experiments were directed towards comparing the anti-proliferative effects of F16 and TMZ against U87MG glioblastoma cancer cells using MTT and TBDE assays. In the in vitro experiments, F16 exhibited higher potency against U87MG cells with an IC50 of 26 μM which was 15 folds lower than IC50 value (
To study the underlying molecular mechanisms that mediate F16 induced cytotoxicity in U87MG cells, phosphorylation of VEGFR-2 after F16 treatment was studied. VEGFR-2 has seven phosphorylation sites, including Tyr1175, which regulates cell proliferation and migration [40]. The results showed a significant inhibition in the level of p-VEGFR-2 (Tyr1175) in U87MG cells after F16 treatment (
As a consequence of the blockade in VEGFR-2 phosphorylation by the F16, the PI3K-AKT pathway was explored, one of the downstream targets of VEGFR-2 that plays an important role in promoting cell survival and cell cycle progression [40, 41]. Previous studies have shown that activation of AKT is involved in inhibiting apoptosis by hindering transcription factors that promote expression of pro-apoptotic genes, and enhancing transcription of anti-apoptotic genes [41, 42]. Furthermore, AKT was shown to suppress p53 mediated apoptosis in indirect manner by phosphorylation of murine double minute 2 (MDM2), which is a negative regulator of p53 [43]. On the other hand, inhibition of AKT phosphorylation was shown to promote cancer cell death and apoptosis through p53 mediated pathway [41, 43]. Thus, the results suggested that F16 could promote cell death through inhibition of AKT phosphorylation at Ser473 site and activating of p53 pathway, to eventually induce cell cycle arrest and apoptosis by up-regulation of p21 and Bax. As anticipated, F16 was able to induce expression of p53, p21, Bax and decrease expression of Bcl2 following 24 h treatment (
A distinctive pathological feature of GBM cells is their ability to extensively invade surroundings containing normal brain tissues [44]. GBM cell invasion is a complex multistep process that typically starts with the degradation of extracellular matrix (ECM) by MMPs, which allows cancer cells to migrate out of the primary tumor to form secondary metastases [44, 45]. Many studies have reported that MMP-2 along with MMP-9 are highly expressed in various human glioblastoma cell lines including U87MG [46-48]. Both MMP-2 and MMP-9 degrade type IV collagen, which is the most abundant component of the basement membrane.
Therefore, degradation of collagen is a crucial step for the initiation of metastatic progression of most cancers [46]. Thus, downregulation of MMP-2 and MMP-9 expression is closely associated with inhibition of GBM cell migration and invasion [48]. The results with U87MG cells clearly showed that F16 significantly inhibited both migration and invasion at concentrations that are below the IC50 values (
To support the in vitro results, the efficacy of F16 in delaying glioblastoma progression using in vivo model was examined. The subcutaneous glioblastoma xenograft model (using the athymic nude mice and treat them with F16, TMZ and combinations) was successfully established. The in vivo results show that F16 significantly inhibited xenograft tumor growth suggesting that VEGFR-2 blockade using F16 treatment is efficacious in delaying glioblastoma cancer growth (
In conclusion to Example 1, the in vitro and in vivo results clearly demonstrate high potency of F16 treatment in inhibiting U87MG cells survival, migration, and invasion. In comparison to TMZ, F16 has a potent cytotoxicity against U87MG cells with an IC50 26 μM (
Though promising results with F16 were obtained, Example 1 used a single cell line in a subcutaneous xenograft model that was responsive to the drug treatment. Therefore, utilization of another in vivo model such as intracranial brain tumor xenograft will provide further validation for its therapeutic effects towards GBM. Hence, the main focus of Example 2 was to determine the efficacy of F16 in delaying glioblastoma progression using intracranial GBM xenograft model, and to evaluate the tolerability of F16 in KP formulation to establish its safety profile using a mouse model.
Cancer remains the second leading cause of death worldwide despite great efforts and resources that are being devoted for developing newer treatment strategies and diagnostic methods [1]. Every year millions of people are diagnosed with cancer around the world, and the survival rate for those patients becomes exceptionally low mainly in the late stages. Among cancer types, glioblastoma multiforme (GBM) is one of the most aggressive and lethal types of brain cancer with a poor prognosis and only less than 5% of patients survive 5-years following diagnosis [2]. As noted above in Example 1, the current standard of care for newly diagnosed patients with GBM is surgical resection, whenever it is applicable, followed by a course of radiation plus chemotherapy such as Temozolomide (TMZ) [3]. Addition of TMZ provides a modest increase in overall survival (OS) from 12.1 to 14.6 months compared to surgical debulking followed by adjuvant radiation therapy [4, 5]. However, TMZ treatment is developed resistance and clinically is associated with severe toxicities such as genotoxicity, teratogenicity, bone marrow suppression, and severe intestinal damage [6]. Therefore, development of more effective and safer treatments for GBM is urgently needed.
One of the defining features of GBM is an abundant and aberrant vasculature [7]. Unlike normal brain vasculature, GBM vasculature is disorganized, poorly connected, tortuous, and associated with marked endothelial proliferation, resulting in regions of hypoxia [8]. Moreover, vascular endothelial growth factor (VEGF) is elevated in GBM with increased vessel permeability, vessel diameter, and abnormality in endothelial wall and basement membrane thickness [9, 10]. High expression of VEGF found in the GBM is also associated with poor prognosis, which provided a logical rationale to evaluate angiogenesis inhibitors as preferred drugs to treat GBM [11].
In preclinical and clinical studies, the use of angiogenesis inhibitors in combination with chemotherapeutic agents has shown promising results against a wide range of cancer types [12-15]. Recently, use of angiogenesis inhibitors has been emerging as a novel strategy for glioblastoma treatment due to the prominent angiogenesis that occur in GBM. So far, bevacizumab (BVZ) is the only antiangiogenic drug that has been approved by FDA for treatment of recurrent GBM. However, BVZ treatment has yielded no improvement in the overall survival (OS) and the FDA approval was based on the increase in the overall Objective Response Rate (ORR) [16, 17].
As noted above, one of the major challenges of treating brain cancers is the presence of the blood brain barrier (BBB). The BBB is a highly selective barrier and crossing this barrier is not easy for large molecules and is required small (molecular mass less than 400-500 Da) lipophilic molecules [18]. Therefore, recent interest has shifted towards exploring small molecules that can cross BBB to modulate angiogenesis and similar processes. In this context, F16, a novel small molecule (molecular weight 301.2 g/mol), has exhibited potent anti-angiogenic and anti-tumor activities via selectively antagonizing vascular endothelial growth factor receptor-2 (VEGFR-2) in both in vitro and in vivo models [19]. More importantly, the preclinical pharmacokinetics studies have shown that F16 can cross BBB and accumulate into brain regions [20]. Therefore, in Example 1, the direct effects of F16 for inhibiting the growth, angiogenesis and the migratory abilities of the U87MG glioblastoma cells (which are known to express high levels of VEGFR) were tested. The in vitro studies confirmed potent inhibitory effects of F16 towards the migration and invasion of U87MG cells and revealed potent cytotoxic effects (IC50 26 μM) against U87MG cells in comparison to Temozolomide (IC50 430 μM) treatment. In addition, F16 inhibited the phosphorylation of VEGFR-2 through competitive binding and induced cell cycle arrest and apoptosis by activating p53 pathway in U87MG cells. Furthermore, the in vivo results with ectopically implanted xenograft model confirm the fact that F16 can significantly inhibit tumor growth in the mice implanted with U87MG glioblastoma cell line.
Example 2 utilizes another in vivo model, such as intracranial brain tumor xenograft, to provide further validation for F16 therapeutic effects towards GBM. Hence, a main focus of Example 2 was to determine the efficacy of F16 in delaying glioblastoma progression using intracranial GBM xenograft model, and to evaluate the tolerability of F16 in KP formulation to establish its safety profile using a mouse model.
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 incubated at 37° C. with 95% air and 5% CO2 in a humidified incubator. U87MG cells were used in assays, when the cell passages were between 3 and 9. The F16 and TMZ (Sigma-Aldrich, St. Louis, Mo., USA) were prepared as a solution in dimethyl sulfoxide (DMSO). All other chemicals used in these experiments were of research grade.
U87MG Cells Luciferase Gene Transfection (pcDNA3.1-Luc)
For the purpose of developing a cell line for xenograft imaging experiments, the U87MG cell line with 90-95% confluency (6 well plates) was 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 pcDNA3.1-Luc+15 μl of PLUS reagent in 100 μl of serum free medium) and complex B (12 μl Lipofectamine 2000 in 100 μl of serum free medium) were prepared separately and incubated for 15 min at room temperature. Complex A and B were combined and incubated for further 15 min at room temperature. This solution (200 μl) added to the plated cells containing 800 μl appropriate medium (serum and antibiotic free) and incubated for further 5 hrs in a 5% CO2 incubator at 37° C. Furthermore, 1 mL of growth medium containing 20% serum without antibiotic was added on transfected wells and incubation was further continued for another 72 hrs (with U-87MG cells) to allow stable transfection.
To measure the cultured luciferase gene transfected cells (U87MG-Luc cells), we imaged the luciferase signal with different cell numbers (1×104-3×105), by adding phosphate buffer saline with D-luciferin (Fisher Scientific, USA) at the concentration of 0.15 mg/ml. U87MG-Luc cells were imaged 10 minutes after incubation with D-luciferin at room temperature. The measurement of the luciferase signal was analyzed using the Bruker Xtreme II (Bruker, Billerica, Mass.).
For tolerability study, 8-10 weeks old male BALB/c mice weighing approximately 25 g were used (Charles Rivers, US). For intracranial study, 8-10 weeks old female athymic nude (Nu/Nu) mice weighing approximately 25 g were used (Taconic Biosciences, US). 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) of Nova Southeastern University (NSU), Ft. Lauderdale, Fla.
F16 (100 mg/kg) was dissolved in a 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 volume of injection was 100 μL/mouse for all experiments which was administered intraperitoneally.
For tolerability studies, BALB/c mice were randomly assigned to 4 different treatment groups (
For intracranial study, the 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 were positioned in the stereotaxic device. A median incision of ˜1 cm was made, and a burr hole was drilled into the right striatum of the skull (1.0 mm forward and 2.0 mm lateral to the bregma). Subsequently, U87MG cells expressing the luc reporter gene (2×105 cells in 3 μL PBS) was injected using a 10-μl Hamilton syringe at the rate of 1 μL/min at a depth of 3 mm. Once injection was completed, the needle was kept in place for 2 minutes and then slowly removed, and the hole was sealed with a sterile bone wax. The incision was closed, and triple antibiotic ointment was applied. One week after tumor cell transplantation, mice were divided randomly into five groups (n=5 in each group): 1) control treated with DMSO in PBS, 2) control treated with DMSO in KP, 3) treated with F16 (100 mg/kg), 4) treated with temozolomide (50 mg/kg) and 5) treated with F16 (100 mg/kg) and 3 h later treated with temozolomide (50 mg/kg). One more group with no tumor implant was added to the study as a negative control (n=5). The experimental mice were treated twice per week for 3 weeks. After the treatment was completed, mice were maintained without any treatment until they showed serious illnesses and then euthanized using the Euthanex CO2 smart box. Brains and tumors of the euthanized mice were isolated for histology and immunohistochemistry (IHC) studies.
Bioluminescence imaging (BLI) was used to assess and confirm tumor growth in intracranial xenograft. BLI was carried out in vivo using the Bruker Xtreme which is a sensitive optical X-ray machine designed for preclinical in vivo study based on BLI concept. Briefly, mice were injected intraperitoneally with D-luciferin (Sigma) dissolved in saline at a dose of 150 mg/kg body weight. Immediately after the injection, mice were anesthetized by isoflurane and series of bioluminescent images was acquired with 3-minutes acquisition intervals for approximately 20 minutes, by which time, the luciferin had been washed out. The image with the peak BLI intensity was used for quantification in units of photon counts.
Histological analyses to evaluate the tumor histology and the effect of the experimental drug against the tumor was performed. Surgically resected tumors in brain tissues were rinsed in 1×PBS to remove blood for the histology and IHC preparations. The specimens from each experimental group were fixed in 10% Neutral Buffered Formalin (NBF) and shipped to Molecular Pathology Core, University of Florida to process the samples for further histology and IHC preparations. The microscopic images and data were received from the facility. Samples of IHC were incubated with primary mouse monoclonal anti-CD31 antibody (1:100 dilution; Cell Signaling Tech. Inc) and the secondary antibody biotin-labeled rabbit anti-mouse IgG (1:500; Nichirei, Tokyo, Japan) was performed using a DAB staining kit. The sections were counterstained with hematoxylin. For H & E (hematoxylin and eosin) staining, samples were stained with Harris' hematoxylin solution and were followed by eosin solution in Molecular Pathology Core, University of Florida.
The data presented here represent mean±SD values from at least three independent experiments. Statistical analyses were performed using a one-way analysis of variance and the differences between means were tested by Tukey's multiple comparison test. The value of p<0.05 was considered as statistically significant. Prism GraphPad (Mac OS X version 7.0b) was used to generate graphs and perform statistical analysis.
For selection, the cells were treated for 14 days with different concentrations of G418 antibiotic (0.1-0.8 mg/mL). After the antibiotic selection, the cells were screened for Luciferase expression using Steady-Glo Luciferase Assay System (Promega, USA). The U87MG cells treated with 0.8 mg/mL of G418 antibiotic was yielded the maximum luminescence. The luciferase transfection optical imaging made it possible to monitor response to anticancer therapies in tumor xenografts. In addition, luciferase images of the plated U87MG-luc cells showed a steady increase in the BLI signal as the number of cells increases (
In order to evaluate the toxicity profile of F16, TMZ and F16+TMZ combination, a comprehensive toxicity study using BALB/c mice was performed. Mice injected with KP were used as controls. All drugs were administered as i.p. injections twice a week for 4 weeks. Independent toxicity evaluations, serum biochemistry, post-mortem gross examination, and histopathological examination of major organs were performed at Comparative Pathology Department at the University of Miami, Fla.
During the treatment period, changes in body weights of mice were checked weekly and no significant variation in the body weight was observed (
Complete blood count (CBC) was performed to measure levels of hemoglobin (HB), hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood counts (RBC), and white blood cells counts (WBC). The levels of HB, MCH, MCHC, and MCV were not significantly altered in various treatment groups (
Total protein level was analyzed to evaluate the impact of treatment regimens on protein metabolism. No significant changes were observed in total protein levels in all treatment groups (
Assessments of liver function were accomplished by measuring the level of ALT. Significant elevation of ALT level was observed in TMZ treated group (
To further investigate the in vivo tumor growth inhibitory effects of F16 and to confirm the earlier study with the subcutaneous model, an intracranial glioblastoma xenograft model using U87MG cells was established as described earlier in the materials and methods section. U87MG-luc cells were implanted into the mice brains and tumor growth was monitored with BLI. One week after cell implantation, animals were randomly divided into five groups (control-PBS, control-KP, F16, TMZ, F16+TMZ). Tumor growth was monitored with BLI every week and representative mice from the five groups are shown in
The survival of mice with glioma xenografts after vehicle-PBS, vehicle-KP, F16, TMZ and combination treatments was examined. Tumor bearing mice treated with F16 showed a significant increase in the survival time with a median survival of 39 days compare to mice treated with vehicles-PBS and vehicles-KP with a median survival of 34 days and 36 days respectively (
Changes in body weight of the experimental mice were examined weakly from the day of implantation until the end of the experiment (
The xenograft brains and tumors were excised and subjected to IHC analysis. The expression of glioblastoma marker CD31 in F16, TMZ and combination of F16 and TMZ treated tumor section were compared to the tumors extracted from the control groups (FIGS. 20A-F). In the control-PBS and control-KP tumor sections, CD31 was expressed in high levels, which indicated that the exponential growth of GBM is associated with angiogenesis (
As noted above, Glioblastoma multiforme (GBM) treatment is very challenging as evidenced by the low survival rate of GBM patients, who generally do not live more than one year [22]. The current standard treatment for patients with GBM is multi-modal, which begins with extensive surgical resection of the tumor mass. Thereafter, patients are subjected to radiotherapy (RT) and concomitantly chemotherapy with Temozolomide (TMZ). Indeed, TMZ plus RT treatment regimen is considered to be the most effective as it increases the median overall survival by 2.6 months to be 14.6 months compared to RT alone 12 months, and the percentage of patients who live 2 years increases from 10.4% to 26.5% [4]. Unfortunately, 60-75% of TMZ treated patients do not respond to TMZ treatment and more than 50% of patients fail the treatment after 6 months of tumor progression [23, 24]. This lack of response is due to the over-expression of O6-methylguanine methyltransferase (MGMT) and/or DNA damage repair systems in GBM cells [25]. Moreover, 15-20% of TMZ treated patients develop significant toxicity, which can lead to disconsolation of treatment [23]. All these shortfalls associated with TMZ have promoted scientists to develop more effective therapeutic options. In this context, novel therapeutic strategies targeting vascular endothelial growth factor (VEGF) or its downstream signaling pathways have been yielding promising results as an addendum to standard therapy [26].
The dependence of tumor growth and metastasis on angiogenesis has supported the notion of using anti-angiogenic approaches in treating cancer. Moreover, angiogenesis inhibitors are clinically proven to improve patients' quality of life, extend progression free survival (PFS) and/or overall survival (OS) of several advanced stage cancers, which has prompted scientist to study using angiogenesis inhibitors for GBM treatment. In 2009, BVZ was approved by FDA for recurrent GBM treatment [27]. In fact, using BVZ for recurrent GBM treatment failed to improve the OS, but did improve the PFS [17, 28]. Moreover, angiogenesis inhibitors are proposed to be useful in alleviating the intracranial pressure associated with brain cancer by reducing the vessel permeability through normalization of the existing vasculature [29]. Unluckily, using angiogenesis inhibitors for GBM treatment is faced with two hurdles which are a few angiogenesis inhibitors can cross the blood brain barrier (BBB) [30], and some angiogenesis inhibitors associated with severe toxicities that limit their clinical benefits [31]. Therefore, there is a crucial need to develop novel angiogenesis inhibitors that can cross the BBB with little or no toxicity.
In 2011, F16, a novel antiangiogenic agent, was disclosed in U.S. Pat. No. 7,939,557 B2. F16 not only showed strong binding and inhibition of vascular endothelial growth factor receptor-2 (VEGFR2) phosphorylation in human umbilical vein endothelial cells (HUVEC) but also exhibited a significant in vivo tumor growth inhibition in mice implanted with GI-IOTA (breast cancer) xenograft and Colo-320 DM (colon cancer) xenograft [19]. In addition, the preclinical pharmacokinetic studies revealed substantial disposition of F16 in major organs of mice after a single i.p. administration [20]. It was an unexpected finding that F16 concentration at 12 h post injection was the highest in the brain compared to liver and kidneys. The concentration of F16 in the brain was close to the concentration that observed in the plasma, which was over 1.3 and 6.1 folds than liver and kidneys respectively. This result indicates that F16 is easily transported across the BBB and slowly accumulated into the brain regions without evidence of clinical behavioral toxicities. In fact, two important factors play a significant role in facilitating the BBB penetration of any drug, which are lipophilicity and molecular weight [32]. In consistent with these criteria, F16 is highly lipophilic an has a small molecular weight (301.2 g/mol), which may explain the penetration of the BBB. All these results inspired the inventors to test the effectiveness of F16 in the treatment of GBM.
Generally, treatment-related toxicity is one of the most common limitations of clinically available agents for cancer treatment. Hepatotoxicity and nephrotoxicity are the common toxicities associated with chemotherapeutic agents including angiogenesis inhibitors. In this toxicity study, mice treated with TMZ showed signs of liver toxicities as evidenced by the increase in the ALT (
Xenograft models using human cancer cells have provided tremendous benefits to oncology field. Initially, the subcutaneous xenograft model, which is called heterotopic, has been the most commonly used preclinical procedure to establish tumor xenografts because it is fast, inexpensive and easily reproducible [34, 35]. However, it has been consistently noticed that some drug regimens that are curative in heterotopic models do not have a significant effect on human disease. Therefore, the emphasis has been shifted towards orthotopic xenograft establishment such as intracranial brain tumor xenograft. In the orthotopic model, the tumor xenograft is implanted into the same anatomical location or organ from which the cancer is initiated, which will provide an appropriate location for tumor-host interactions, the ability to study the site-specific dependence of therapy and organ-specific expression of genes, and a sufficient preclinical test for anti-cancer drugs [35, 36]. Moreover, it is well known that tumor progression and metastasis are dependent on the formation of new blood vessels in most situations [37]. Also, the biochemical imbalance in the tumor microenvironment contributes to pathological angiogenesis and tumor growth progressions through continuous secretion of growth factors [38].
In order to mimic tumor growth with appropriate tumor microenvironment, the intracranial GBM xenograft model was established, which provides a better representation of the clinical features of tumor angiogenesis and be more relevant to the real situation inside the human brain. Results show that F16 significantly inhibited xenograft tumor growth (
F16 is effectively mediated anti-tumor activity through inhibition of angiogenesis [19]. The IHC results confirmed the in vivo anti-angiogenic activity of F16 using CD31 expression as a biomarker to demonstrate the presence of endothelial cells in tumor tissues [42]. As expected, F16 treatment was associated with a low level of CD31 expression, representing a significant reduction of tumor micro-vessel density (
In conclusion to Example 2, the in vivo results clearly proved high potency of F16 treatment in inhibiting tumor growth and prolonging the median survival of mice implanting intracranially with U87MG-luc cells. In comparison to TMZ, F16 was well tolerated in mice without evidence of significant pre-clinical or laboratory toxicities. Though using KP formulation has improved the brain delivery of F16 by 40% compare to PBS formulation [data not shown], the KP formulation caused some hypersensitivity reactions which may lead to more serious side effect when it used for longer time [43]. Finally, these findings provide a new avenue for GBM treatment, which might benefit a significant number of patients by extending their overall survival or improve their quality of life.
The findings disclosed herein provide a new avenue for treatment of solid cancers having angiogenic ability, particularly for treatments of brain cancers such as glioblastoma multiforme (GBM). Such novel treatments might benefit a significant number of patients by extending their overall survival and/or improve their quality of life.
All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. It is to be understood that while a certain form of the invention is illustrated, it is not intended to be limited to the specific form or arrangement herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification. One skilled in the art will readily appreciate that the present invention is adapted to carry out the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods using F16 described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention. 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 which are obvious to those skilled in the art are intended to be within the scope of the invention.
1. Mohammad Algahtanil, 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-Apr. 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 Fla., USA.
3. Sivanesan Dhandayuthapani, Thanigaivelan Kanagasabai, Khadija Cheema and Appu Rathinavelu (2017), Bioavailability, pharmacokinetics and safety profile of a novel anti-50 angiogenic compound JFD in pre-clinical models. Presented at the AACR Annual Meeting 2017, April 1-5 Washington, D.C.
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, D.C., 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, Presented at the Annual Conference of the American Association for Cancer Research (AACR) April 1-5, Washington, D.C., USA
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, Presented at the Annual Conference of the American Association for Cancer Research (AACR) April 1-5, Washington, D.C., USA
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 D.C., 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, Fla. on Nov., 12th-15th 2016.
14. Thanigaivelan Kanagasabai, Rohin Chand, Amy Aman Kaur, Sivanesan Dhandayuthapani, Olena Bracho, Appu Rathinavelu. MDM2 stabilizes and induces HIF-lu 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: Feb. 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, November; 39 (11):1010428317726841. https://doi: 10.1177/1010428317726841.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/060549 | 11/13/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62935448 | Nov 2019 | US |
