Patent Application
20230049415
The present disclosure relates to medical uses of cannabinoids.
References considered to be relevant as background to the presently disclosed subject matter are listed below:
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
The Gi protein associated cell surface, the A3 adenosine receptor (A3AR), has been demonstrated as an effective target for treatment of a variety of diseases or disorders. Furthermore, it has also been described that the A3AR is over-expressed in cancer cells as well as in inflammatory cells and in peripheral blood mononuclear cells
(PBMCs) derived from patients with various auto-immune inflammatory diseases, such as rheumatoid arthritis psoriasis and Crohn's Disease.
Activation of the A3AR with highly specific ligands, such as the A3AR agonists 3-iodobenzyl-5′-N-methylc arboxamido adeno sine (piclidenososn) and 2-Chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (namodenoson) was found to be effective in the treatment of cancer (U.S. Pat. No. 6,790,839 and WO 2013/111132), inflammatory diseases (U.S. Pat. Nos. 7,141,553, 8,987,228, WO 2007/063538), inhibiting viral replication (U.S. Pat. No. 7,589,075), inducing hepatocyte proliferation (WO 2009/050707), reducing ectopic fat accumulation (WO 2017/090036) and others.
Cannabinoids have also been described for their potential pharmaceutical use in the treatment of a variety of human diseases and disorders. An examples is the treatment of non-alcoholic fatty liver disease (NAFLD) by the use of 7-Hydroxy cannabidiol (7-OH-CBD), a metabolite of CBD (WO 2009/093018.
The present disclosure provides, in accordance with a first of its aspects a formulation for treating a disease or disorder, that is treatable by an activator of the A3 adenosine receptor (A3AR) and that comprises at least one cannabinoid at an amount effective to treat or ameliorate the disease or disorder.
Provided by another aspect of this disclosure is a cannabinoid for treating a disease or disorder treatable by an A3AR activator.
Also provided by a further aspect of this disclosure is a method of treating a disease or disorder, treatable by and A3AR activator, comprising administering to a subject having said disease or disorder at least one cannabinoid in an amount effective to improve said disease or disorder or a condition exhibited by the subject as a result of said disease or disorder.
Also provided by an additional aspect of this disclosure is a method of treating a disease or disorder in a subject, comprising obtaining data on level of expression A3AR in tissue or cells of the subject; and when said data is indicative that expression level is elevated as compared to level of expression of the A3AR on cells or tissue of same lineage in subjects not suffering from said disease or condition, administering to the subject at least one cannabinoid in an amount effective to achieve improvement in said disease or condition.
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Among the findings of the present disclosure is that the proliferation of Stellate cells is significantly inhibited when exposed to nM or even pM concentrations of a cannabinoid extract comprising Cannabidiol (CBD) and Tetrahydrocannabinol (THC). A similar inhibitory effect was exhibited when hepatocellular carcinoma cells were exposed to the same cannabinoid extract, an effect that was reversed when the same cells were exposed to an A3 adenosine receptor (A3AR) antagonist. These surprising findings suggest that the cannabinoid extract exerts this effect, at least partially, through the A3AR.
Accordingly, cannabinoids, be it a single cannabinoid or a mixture of cannabinoids, may be used, according to this disclosure, for treating diseases, disorders or conditions that are treatable by an A3AR activator (an A3AR agonist or allosteric modulator). The term “clinical condition” will be used hereinafter to collectively denote said disease, disorder, or condition. The terms “treatment”, “treating” or “treat” is being used herein to refer to the therapeutic administration of cannabinoids to subjects having said clinical condition, in accordance with this disclosure. A specific subset of such treatment is of subjects in which the clinical condition is associated with an elevated expression the A3AR as compared to the expression of the A3AR in healthy subjects (i.e., that are not diagnosed as having said clinical condition).
The term treatable denotes, among others, that: (i) said clinical condition, the physiological manifestation thereof, or symptoms associated therewith may be controlled (typically reduced) by the administration of an A3AR activator, such as an agonist of A3AR such as piclidenoson or namodenoson (also known in the scientific literature as IB-MECA and Cl-IB-MECA, respectively); (ii) following such administration an improvement in general health scores (by a physician assessment or by a patient's own assessment, e.g., an improvement in the ACR score in rheumatoid arthritis patients, PASI or PGA scores in psoriasis and many others); or (iii) that such administration yields an improvement in the treated subject's quality of life. The cannabinoid is used, in accordance with this disclosure, for the treatment of a clinical condition otherwise treatable with an A3AR activator, the treatment by the cannabinoid being in the alternative or in addition to the A3AR activator.
Accordingly, the at least one cannabinoid acts, in accordance with this disclosure, as an A3AR activator.
Specifically, in accordance with the present disclosure there is provided a formulation comprising at least one cannabinoid for use in treating said clinical condition; a cannabinoid for use in such treatment; and a method for said treatment.
In the following, when referring to elements of the formulation, it is to be understood as equally defining elements of the use or method disclosed herein, and vice versa, the description relating to the method disclosed herein should be regarded as equally, and mutatis mutandis, defining the formulation disclosed herein.
The formulation used in the context of the present disclosure comprises at least one cannabinoid. The formulation may comprise a cannabis oil, cannabis concentrate, cannabis extract, a natural isolated cannabinoid, any chemical derivative of a natural cannabinoid or a synthetic cannabinoid. Phytocannabinoids, namely cannabinoids derived from a plant such as cannabis plant are a specific example of cannabinoids used in accordance with this disclosure. There are at least 113 different known phyotcannabinoids, including THC (tetrahydrocannabinol) or CBD (cannabidiol) and their derivatives.
The cannabinoids can be chemically classified into distinct chemical classes: the classical cannabinoids that are structurally related to THC or CBD; the nonclassical cannabinoids (cannabimimetics) including the aminoalkylindoles, 1,5-diarylpyrazoles, quinolines, and arylsulfonamides; and others.
Non-limiting examples of phytocannabinoids that may be potentially used in accordance with the present disclosure includes selected from the group consisting of Tetrahydrocannabinolic Acid (THCA), Tetrahydrocannabinol (THC), Cannabidiolic Acid (CBDA), Cannabidiol (CBD), Cannabinol (CBN), Cannabigerol (CBG), Cannabichromene (CBC), Cannabicyclol (CBL), Cannabivarin (CBV), Cannabichromevarin (CBCV), Cannabigerovarin (CBGV), Cannabigerol monomethyl ether (CBGM), Cannabielsoin (CBE), Cannabicitran (CBT), Tetrahydrocannabivarin (THCV) and Cannabidivarin (CBDV). Also contemplated for use according to this disclosure are synthetic derivatives of these phytocannabinoids. The cannabinoids may be selected on the basis of their A3AR agonistic activity, as explained below.
In accordance with the present disclosure, the formulation can include a single or any combinations of n cannabinoids (n being an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) of the above non-limiting examples of cannabinoids.
In the context of the present disclosure, when referring to a phytocannabinoid, or a cannabinoid (one or more) derived from a plant, it is to be understood as being any one of a plant-derived material including plant extract, a plant concentrate, a plant isolate, a plant-derived oil and/or one or more cannabinoid compounds isolated from the plant material; the plant being typically (although not exclusively) a cannabis plant. CBD and THC, whether purified, synthetic or whether provided in the form of a plant-derived material, are examples of phytocannabinoids.
The formulation of this disclosure is used for the treatment of a clinical condition treatable by an A3AR activator, for example by an A3AR agonists such as piclidenoson or namodenoson. The selection of the cannabinoid to be used for treatment may be through studies conducted in the appropriate in vitro or animal disease models or through the conduct of the appropriate human clinical study. Cannabinoids may be screened in such studies for those having an effect that is similar to that achieved with the A3AR activator used as the comparable in such studies. To ensure that the selected cannabinoid acts through the A3AR, an antagonist of that receptor may be used to examine whether the effect exerted by the cannabinoid is reduced or even eliminated by said antagonist. By comparing effects with or without an antagonist to the A3AR, a cannabinoid with an effect exerted specifically or a least primarily through the A3AR may be selected.
By an embodiment of this disclosure the cannabinoid that is used is one that exerts it effect primarily through the A3AR; particularly, have an A3AR activator effect, meaning that it can bind to and activate this receptor. It should be noted, however, that while the at least one cannabinoid used in accordance with this embodiment exerts its effect primarily through an A3AR activator effect, it is possible that the cannabinoid has a parallel effect, e.g., exerted through other receptors on the same or other cells. For example, the cannabinoid used in accordance with this embodiment may have an effect of reducing disease symptoms of the clinical condition, e.g., an anti-inflammatory or anti-cancer effect, that is exerted through the A3AR receptor and have in parallel also an effect of reducing pain, an effect on the general well-being of the subject, or another general effect.
An A3AR activator or A3AR activator effect denotes an effect exerted directly on the A3AR (e.g., via the adenosine binding site) or indirectly (e.g., via an allosteric binding site) to thereby activate the A3AR, including full or partial activation of this receptor. The A3AR activator effect is thus an enhancement of the activity of the A3AR by (i) agonistic activation via the receptor's adenosine binding site to thereby induce a direct activation of the receptor, or (ii) allosteric modulation of the receptor via an allosteric binding site.
An A3AR activator effect can, as noted above, be reduced or, at times, totally eliminated by an antagonist of this receptor. This is one of the typical characteristics of said effect and it may be tested in vitro with cells that express the A3AR.
A subset of clinical conditions treatable by cannabinoids in accordance with this disclosure is one in which there is an elevated level of expression of an A3AR in cells or tissue, as compared to cells or tissue of the same lineage in subjects not suffering from said or disorder. Such increased level may be an average expression level of the A3AR in said cells or tissue in subjects suffering from said clinical condition, which is at least 1.5 times the average expression level of the A3AR in cells or tissue of a same lineage in subjects not suffering from said clinical condition. While a clinical condition may be selected as a therapeutic target by such average increase in A3AR expression level, the patients may also be selected individually as recipients of the cannabinoid-based treatment according to this disclosure, based on a pre-treatment testing of the A3AR level and selecting only those subjects with an elevated expression level of this receptor versus that in subjects not suffering from said clinical condition. The elevated expression level of the A3AR may be at least 1.5, 1.6, 1.7, 1.8, 1.9, or at least 2 times that of subjects not suffering from said clinical condition; but may also be, at times, at least 2.5 or at least 3 times that of subjects not suffering from said clinical condition.
By one embodiment the cells or tissue exhibiting the elevated A3AR expression are cells or tissue of a diseased organ or tissue having a disease-related abnormality; e.g., cancer cells, inflammatory cells, cells of the immune system, adipose cells, liver cells. In some clinical conditions the elevated A3AR expression may be on other cells, for example on circulating while blood cells, e.g., mononuclear cells.
Examples of the clinical condition treatable in accordance with this disclosure are cancer, inflammatory disease, liver disease such as NAFLD or obesity.
It has been found that the formulation comprising at least one cannabinoid is effective, in in vitro assays, at very low concentrations, even at the pM range. Thus, irrespective of the form of administration, and in accordance with some examples, the concentration of the at least one cannabinoid in the formulation is sufficient to induce, after administration, a peak blood concentration of the least one cannabinoid within the range of 0.01-100 nM, at times, within the range of 0.01-50 nM, at times, below 50 nM, at times, between 0.1 μM and 10 nM, at times, between 10 μM and 1 nM, at times between 1 pM and 20 nM or any range between 1 pM and 100 nM.
By an embodiment of this disclosure, the at least one cannabinoid is administered to the subject in combination with an A3AR agonist, such as piclidenoson or namodenoson. Such combination means that the A3AR agonist may be co-administered with the at least one cannabinoid, for example, within the same formulation, or may be administered to the subject within the same therapeutic regiment, for example one given once, twice or thrice daily and the other given once, twice or thrice daily at different times.
Oral administration of the at least one cannabinoid is one exemplary administration form. Parenteral is another. Accordingly, the at least one cannabinoid may be formulated in a dosage form suitable for, respective, oral and parenteral delivery. Other examples of delivery forms are by inhalation, oro-mucosal or sublingual administration, topical administration or rectal administration.
The therapeutic treatment according to this disclosure may be for a short time period of 1 day, several days or several weeks, or may be a chronic treatment over prolonged time periods of months to years. Chronic treatement it is to be understood as involving routine administration of the at least one cannabinoid for a prolonged time period (as opposed to a single time administration).
The clinical conditions treatable according to this disclosure include, for examples, inflammation, cancer, e.g., liver cancer, non-alcoholic fatty liver disease (NAFLD) or other liver conditions, obesity, fibrosis, e.g., liver fibrosis, neuropathic pain.
The A3AR activator effect encompasses A3AR agonistic effect or A3AR allosteric modulator effect.
An A3AR agonistic effect denotes an effect that is exerted through binding to the adenosine binding site of this receptor, thereby fully or partially activating the A3 adenosine receptor.
When referring to A3AR allosteric modulator effect denotes an effect that is exerted through binding at the receptor's allosteric site, which may be different from the binding site of the endogenous ligand or agonist thereof, to thereby impart a positive regulation on the receptor's activity. Such modulation may be (i) an increased affinity to binding of adenosine or an A3AR agonists to the receptor's adenosine binding site (the orthosteric binding site) and/or (ii) a decrease in dissociation rate of adenosine or an A3AR ligand to the orthosteric binding site.
The method disclosed herein may also involve, in accordance with the invention the step of obtaining information with respect to the expression level of the A3AR in the tissue or cells indicative of the existence of the disease or condition to be treated, so as to determined that the subject is suitable for the treatment. Thus, prior to the administration of the at least one cannabinoid, the subject or the physician responsible for the subject's treatment is of knowledge/or in possession of data indicative of the level of expression of the A3AR in tissue or cells of the subject and the subject is administered with the at least one cannabinoid only if the level of expression is above a predefined reference level, i.e., is considered by predetermined parameters to be elevated levels that require the treatment.
As used herein, the forms “a”, “an” and “the” include singular as well as plural references unless the context clearly dictates otherwise. For example, the term “a cannabinoid” includes one or more cannabinoids.
Further, as used herein, the term “comprising” is intended to mean that the composition include the recited active agent, i.e. a cannabinoid, but not excluding other elements, such as physiologically acceptable carriers and excipients as well as other active agents.
Further, all numerical values, e.g. when referring the amounts or ranges of the elements constituting the formulations comprising the at least one cannabinoid as an active ingredient, are approximations which are varied (+) or (−) by up to 20%, at times by up to 10% of from the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term “about”.
Defined in the following numbered paragraphs are embodiments of the disclosure herein. These embodiments are intended to add and not limit the above disclosure.
The disclosure will now be exemplified in the following description of experiments that were carried out in accordance with the teaching herein. It is to be understood that these examples are intended to be in the nature of illustration rather than of limitation. Obviously, many modifications and variations of these examples are possible in light of the above teaching.
Inhibition of Human Hepatocellular Carcinoma and Human Hepatic Stellate Cell Growth by CBD/THC extract
Cell culture. Hep-3B hepatocellular carcinoma cells and LX-2 hepatic stellate cells were grown in MeM-Eagle medium. All media contained penicillin (10 units/ml), streptomycin (10 μg/ml), L-glutamine (2 mM) and 10% fetal bovine serum (FBS). The cells were maintained in T-75 flasks at 37° C. in a 5% CO2 incubator and transferred to a freshly prepared medium twice weekly. For all studies serum starved cells were used. FBS was omitted from the cultures for 18 hours and the experiment was carried out on monolayers of cells in DMEM medium supplemented with 1% FBS in a 37° C., 5% in a CO2 incubator. CBD/THC in different ratios (C15/T3 and C3/T15) was introduced to the culture system at concentrations of 100 pM, 1, 10 and 100 nM (stock solution of 100 mM at DMSO was prepared from the original oil and further diluted in culture medium to reach the nM concentration in the experimental system.
3H-thymidine incorporation assay. 3H-thymidine incorporation assay was used to evaluate cell growth. cells (5,000 cells/well) were incubated with different concentration of T3/C15, T15/C3 in 96-well plate for 48/72 hours. Each well was pulsed with 1 μCi 3H-thymidine for the last 24 hours. Cells were harvested and the 3H-thymidine uptake was determined in an LKB liquid scintillation counter (LKB, Piscataway, N.J., USA). These experiments were repeated at least 4 times.
Western Blot Analysis. Protein extract from cell line were utilized. The samples were homogenized with an ice-cold RIPA lysis buffer (Thermo scientific; 89900) with protease phosphatase inhibitor cocktail (Thermo scientific; 1861281). Cell debris were removed by centrifugation for 10 min, at 7500 g. The supernatant was utilized for Western Blot (WB) analysis. Protein concentrations were determined using the NanoDrop (ThermoFisher Scientific, Mass., US). Equal amounts of the sample (50 μg) were separated by SDS -PAGE, using 4-12% polyacrylamide gels. The resolved proteins were then electro blotted onto nitrocellulose membranes (Pall Corporation, Fla., US). Membranes were blocked with 5% bovine serum albumin and incubated with the desired primary antibody (Santa cruz; A3AR sc-13938, PI3K sc-1637,GSK-3β sc-9166, β-catenin sc-7963, cyclin D1 sc-8396, NF-κB sc-372, LEF-1 sc-374522, α-SMA sc-32251 and β-actin sc-47778 dilution 1:1000) for 24 h hour at 4° C. Blots were then washed and incubated with a secondary antibody (Abcam; Mouse ab97020, Rabbit ab97048) for 1 h at room temperature. Bands were recorded using BCIP/NBT color development kit (Promega, Madison, Wis., USA). Densitometry of protein expression was normalized against β-actin and expressed as % of control.
In addition,
B and β-catenin, showing the involvement of both the NF-
B and the Wnt/β-catenin pathways in mediating the anti-cancer and the liver anti-fibrotic effects of the cannabinoids.
| Number | Date | Country | Kind |
|---|---|---|---|
| 272078 | Jan 2020 | IL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2021/050046 | 1/14/2021 | WO |
