Here are some of the examples. Example 5 caught m
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Posted On: 03/10/2017 5:39:39 AM
Here are some of the examples. Example 5 caught my eye. To get a quick idea of what each example does, just read the first few lines and the last few lines of the example. Read all of it if you want to get very technical.
EXAMPLE 1
Reference is now made to Figure 1 which demonstrates a graph of the relative viability of MM cells vs. different concentrations of CBD and THC, during different time periods (i.e 0, 24 and 48 hours). The effect of different concentrations of CBD and THC on the viability of different multiple myeloma cell lines and primary cells isolated from bone marrow of myeloma patients in the presence and absence of bone marrow stroma cells, was tested. Several MM cell lines were plated at 2 xlO<4>cells per well in 96-wells and reacted with different concentrations of CBD and THC. Samples were taken from bone marrow aspirates from MM patients. Mononuclear cells were separated by Ficoll density gradient centrifugation and myeloma cells selected using CD138 microbeads (Miltenyi Biotec). Purified CD 138+ patient cells were be plated at a density of 2x10<4>cells per well and treated for 48 hours with different concentrations of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%). Cell viability was measured using XTT cell proliferation Kit (Biological Industries) according to manufacture instructions. It can be seen from figure 1, that in comparison to the control sample (in which only buffer was added) all combinations of CBD and THC showed an effect upon the viability of the cells.
EXAMPLE 2
Combinations of novel and/or conventional anti-MM agents can achieve higher clinical response rates than single agent(s). In addition, many patients experience significant dose-limiting side effects requiring dose reductions or cessation of therapy.
The anti-MM activity induced by the combination of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2% with other MM chemotherapeutic drugs in vitro was assessed. The response of MM cells to treatment with CBD and THC in combination with currently in use anti-MM agents (bortezomib (BTZ), lenalidomide (LEN), dexamethasone (DEX), melphalan (MEL) and doxorubicin (DOXO) were evaluated. The anti-MM activity of combined treatment was analyzed by XTT assays as previously described in example 1, and the presence of synergistic cytotoxic effects are be evaluated using the Chou-Talalay method based on the median-effect equation and the classic isobologram equation and compusyn computer software. It appears that in comparison to the control (in which only buffer was added) or to currently in use anti-MM agents, all combinations of CBD and THC affected the viability of the cells.
EXAMPLE 3
This example presents the mode of action of cannabis as an anti-myeloma agent, the effect of cannabis on MM cell lines is evaluated regarding apoptosis, angiogenesis, cell cycle, mitochondrial transmembrane potential, ROS production, and cell signaling.
Apoptosis analysis: MM cells were treated with different concentrations of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) during 0, 24 and 48 h. For evaluation of apoptosis, cells were processed using an Annexin V/propidium iodide (PI) kit (Becton Dickinson Biosciences) according to manufacture instructions. Cell-cycle analysis: MM cells were exposed to different concentrations of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) for different intervals of time. The cells were then permeabilized by 70% ethanol at -20 °C overnight and incubated with 50 μg/ml PI and 20 units/ml RNase-A (Roche Diagnostics). DNA content was analyzed by flow cytometry. Data was collected using FACS Calibur (Becton Dickinson) and analyzed with the CellQuest software.
Cell signaling: MM cell lines were plated in RPMI 1640 with 10% FBS, penicillin, and streptomycin. CBD and THC (THC 2% CBD 20%; THC 10% CBD 10% and; THC 20% CBD 2%) was added after 0, 30 minutes, 2, 6, 24 and 48 h. Cells were then lysed in RIPA-lysis buffer (lOmM sodium pyrophosphate, 2mM sodium orthovanadate, 5mM sodium fluoride, 5 g/mL aprotinin, 5 g/mL leupeptin, and ImM phenylmethylsulfonyl fluoride). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes and immunoblotted with cell signaling antibodies. Immunoreactive bands were detected by Western Blot chemiluminescence reagents (Thermo Scientific) and exposed on Kodak-XAR film.
Cell signaling arrest was achieved with the THC and CBD extract combinations, with different THC and CBD ratios providing different levels of arrest.
Mitochondrial transmembrane potential: Mitochondrial transmembrane potential (Dwm) was evaluated by 5,5',6,6'-tetrachloro-l, ,3,3'-tetraehylbenzimidazolylcarbocyanineiodide (JC-1) staining. Briefly, 2 x 104 cells were treated with of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) for different times and then incubated for 10 mm at room temperature with 10 μg/ml of JC-1. JC-1 was excited by an argon laser (488 nm), and the green (530 nm)/red (570 nm) emission fluorescence was collected simultaneously. Carbonyl cyanide chlorophenylhydrazone protonophore, a mitochondrial uncoupler that collapses (Dwm), was be used as a positive control. Samples were analyzed using a FACScan cytofluorimeter with CellQuest software. Different levels of reduction arrest in mitochondrial transmembrane potential were achieved with the various THC and CBD extracts of the present invention.
ROS production: The fluorescent probe dichlorodihydrofluorescein diacetate (DCFDA) was used to assess oxidative stress levels. Briefly, 2 x 104 cells treated with the appropriate compounds were incubated with 20 μΜ DCFDA (Life Technologies Italia, Italy) 20 min prior to the harvest time point. The cells were then washed, and the intensity of the fluorescence was assayed using flow cytometry and CellQuest software. Different levels of reduction arrest ROS production was obtained with the THC and CBD extracts herein described.
EXAMPLE 4
This example presents the effect of cannabis on osteoblasts (OB) function, MC3T3-E1 pre- osteoblastic cells (ATCC) and bone marrow-derived stromal cells were cultured in osteoblastic differentiation media, with or without MM cells, in the presence of different concentrations of CBD and THC; (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) for different periods of time. At the end of the culture period, cells will be evaluated for OB differentiation. To evaluate the effect of cannabis on OC function, mononuclear cells from MM patients were differentiated to osteoclasts and treated with cannabis and their activity evaluated in the presence and absence of stroma cells.
EXAMPLE 5
This example examines anti-tumor efficacy of cannabis in murine xenograft MM model SCID mice (6-8 week old) were maintained in accordance with Institutional Animal Care Use Committee guidelines. Mice were gamma- irradiated (150 rads) using Csl37 γ-irradiator source and (24 hours post-irradiation) injected subcutaneously with MM cells (7xl06/mouse) suspended in PBS. 2-3 weeks later, when palpable tumors were developed, mice were be randomly divided into different groups (15 mice/group), and the following treatment protocol will be implemented: Group la-b*: vehicle control administered ip, every day, 5 days a week throughout the duration of the experiment; Group 2-4 a-b*: the best combination of THC 2% CBD 20%, CBD 10% THC 10% or THC 20% CBD 2% according to in vitro results at different doses (1, 10 and 20 mg/kg) administered ip, every day, 5 days a week throughout the duration of experiment; Group 5-6 a-b*: THC and CBD at 20 mg/kg administered ip, every day, 5 days a week throughout the duration of experiment; * Subgroup a: mice treated during 3 weeks (n=5) and tumor removed and analyzed *Subgroup b: mice treated until the end of the experiment (n=15). Evaluation of efficacy will include inhibition of tumor growth, survival, blood tests, animals' vital signs and gross pathology. Tumor size will be measured by caliper. Caliper measurements of the longest perpendicular tumor diameters will be performed every other day to estimate tumor volume. All compositions showed decrease in tumor size but the highest reduction was shown in the solution having THC 2% CBD 20%.
OWCP
EXAMPLE 1
Reference is now made to Figure 1 which demonstrates a graph of the relative viability of MM cells vs. different concentrations of CBD and THC, during different time periods (i.e 0, 24 and 48 hours). The effect of different concentrations of CBD and THC on the viability of different multiple myeloma cell lines and primary cells isolated from bone marrow of myeloma patients in the presence and absence of bone marrow stroma cells, was tested. Several MM cell lines were plated at 2 xlO<4>cells per well in 96-wells and reacted with different concentrations of CBD and THC. Samples were taken from bone marrow aspirates from MM patients. Mononuclear cells were separated by Ficoll density gradient centrifugation and myeloma cells selected using CD138 microbeads (Miltenyi Biotec). Purified CD 138+ patient cells were be plated at a density of 2x10<4>cells per well and treated for 48 hours with different concentrations of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%). Cell viability was measured using XTT cell proliferation Kit (Biological Industries) according to manufacture instructions. It can be seen from figure 1, that in comparison to the control sample (in which only buffer was added) all combinations of CBD and THC showed an effect upon the viability of the cells.
EXAMPLE 2
Combinations of novel and/or conventional anti-MM agents can achieve higher clinical response rates than single agent(s). In addition, many patients experience significant dose-limiting side effects requiring dose reductions or cessation of therapy.
The anti-MM activity induced by the combination of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2% with other MM chemotherapeutic drugs in vitro was assessed. The response of MM cells to treatment with CBD and THC in combination with currently in use anti-MM agents (bortezomib (BTZ), lenalidomide (LEN), dexamethasone (DEX), melphalan (MEL) and doxorubicin (DOXO) were evaluated. The anti-MM activity of combined treatment was analyzed by XTT assays as previously described in example 1, and the presence of synergistic cytotoxic effects are be evaluated using the Chou-Talalay method based on the median-effect equation and the classic isobologram equation and compusyn computer software. It appears that in comparison to the control (in which only buffer was added) or to currently in use anti-MM agents, all combinations of CBD and THC affected the viability of the cells.
EXAMPLE 3
This example presents the mode of action of cannabis as an anti-myeloma agent, the effect of cannabis on MM cell lines is evaluated regarding apoptosis, angiogenesis, cell cycle, mitochondrial transmembrane potential, ROS production, and cell signaling.
Apoptosis analysis: MM cells were treated with different concentrations of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) during 0, 24 and 48 h. For evaluation of apoptosis, cells were processed using an Annexin V/propidium iodide (PI) kit (Becton Dickinson Biosciences) according to manufacture instructions. Cell-cycle analysis: MM cells were exposed to different concentrations of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) for different intervals of time. The cells were then permeabilized by 70% ethanol at -20 °C overnight and incubated with 50 μg/ml PI and 20 units/ml RNase-A (Roche Diagnostics). DNA content was analyzed by flow cytometry. Data was collected using FACS Calibur (Becton Dickinson) and analyzed with the CellQuest software.
Cell signaling: MM cell lines were plated in RPMI 1640 with 10% FBS, penicillin, and streptomycin. CBD and THC (THC 2% CBD 20%; THC 10% CBD 10% and; THC 20% CBD 2%) was added after 0, 30 minutes, 2, 6, 24 and 48 h. Cells were then lysed in RIPA-lysis buffer (lOmM sodium pyrophosphate, 2mM sodium orthovanadate, 5mM sodium fluoride, 5 g/mL aprotinin, 5 g/mL leupeptin, and ImM phenylmethylsulfonyl fluoride). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes and immunoblotted with cell signaling antibodies. Immunoreactive bands were detected by Western Blot chemiluminescence reagents (Thermo Scientific) and exposed on Kodak-XAR film.
Cell signaling arrest was achieved with the THC and CBD extract combinations, with different THC and CBD ratios providing different levels of arrest.
Mitochondrial transmembrane potential: Mitochondrial transmembrane potential (Dwm) was evaluated by 5,5',6,6'-tetrachloro-l, ,3,3'-tetraehylbenzimidazolylcarbocyanineiodide (JC-1) staining. Briefly, 2 x 104 cells were treated with of CBD and THC (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) for different times and then incubated for 10 mm at room temperature with 10 μg/ml of JC-1. JC-1 was excited by an argon laser (488 nm), and the green (530 nm)/red (570 nm) emission fluorescence was collected simultaneously. Carbonyl cyanide chlorophenylhydrazone protonophore, a mitochondrial uncoupler that collapses (Dwm), was be used as a positive control. Samples were analyzed using a FACScan cytofluorimeter with CellQuest software. Different levels of reduction arrest in mitochondrial transmembrane potential were achieved with the various THC and CBD extracts of the present invention.
ROS production: The fluorescent probe dichlorodihydrofluorescein diacetate (DCFDA) was used to assess oxidative stress levels. Briefly, 2 x 104 cells treated with the appropriate compounds were incubated with 20 μΜ DCFDA (Life Technologies Italia, Italy) 20 min prior to the harvest time point. The cells were then washed, and the intensity of the fluorescence was assayed using flow cytometry and CellQuest software. Different levels of reduction arrest ROS production was obtained with the THC and CBD extracts herein described.
EXAMPLE 4
This example presents the effect of cannabis on osteoblasts (OB) function, MC3T3-E1 pre- osteoblastic cells (ATCC) and bone marrow-derived stromal cells were cultured in osteoblastic differentiation media, with or without MM cells, in the presence of different concentrations of CBD and THC; (THC 2% CBD 20%; THC 10% CBD 10%; and THC 20% CBD 2%) for different periods of time. At the end of the culture period, cells will be evaluated for OB differentiation. To evaluate the effect of cannabis on OC function, mononuclear cells from MM patients were differentiated to osteoclasts and treated with cannabis and their activity evaluated in the presence and absence of stroma cells.
EXAMPLE 5
This example examines anti-tumor efficacy of cannabis in murine xenograft MM model SCID mice (6-8 week old) were maintained in accordance with Institutional Animal Care Use Committee guidelines. Mice were gamma- irradiated (150 rads) using Csl37 γ-irradiator source and (24 hours post-irradiation) injected subcutaneously with MM cells (7xl06/mouse) suspended in PBS. 2-3 weeks later, when palpable tumors were developed, mice were be randomly divided into different groups (15 mice/group), and the following treatment protocol will be implemented: Group la-b*: vehicle control administered ip, every day, 5 days a week throughout the duration of the experiment; Group 2-4 a-b*: the best combination of THC 2% CBD 20%, CBD 10% THC 10% or THC 20% CBD 2% according to in vitro results at different doses (1, 10 and 20 mg/kg) administered ip, every day, 5 days a week throughout the duration of experiment; Group 5-6 a-b*: THC and CBD at 20 mg/kg administered ip, every day, 5 days a week throughout the duration of experiment; * Subgroup a: mice treated during 3 weeks (n=5) and tumor removed and analyzed *Subgroup b: mice treated until the end of the experiment (n=15). Evaluation of efficacy will include inhibition of tumor growth, survival, blood tests, animals' vital signs and gross pathology. Tumor size will be measured by caliper. Caliper measurements of the longest perpendicular tumor diameters will be performed every other day to estimate tumor volume. All compositions showed decrease in tumor size but the highest reduction was shown in the solution having THC 2% CBD 20%.
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