The present invention relates to phytocannabinoids for use in the treatment of a breast cancer. In a first embodiment the invention relates to an oral presentation of tetrahydrocannabinol (THC) for use in the treatment of aggressive breast cancer, characterised by overexpression of the Her2 gene. In a second embodiment the invention relates to the phytocannabinoid cannabidiol (CBD) for use in the treatment of aggressive breast cancer, characterised by overexpression of the Her2 gene. In a third embodiment the invention relates to the combination of the phytocannabinoids tetrahydrocannabinol (THC) and cannabidiol (CBD) for use in the treatment of breast cancer or to treat, prevent or to reduce the risk of a cancer metastasising.
Breast cancer occurs due to mutations in the genes responsible for regulating the growth of breast cells which causes the cells to grow in an unregulated manner. Usually breast cancer either begins in the cells of the milk producing glands, known as the lobules, or in the ducts. Less commonly breast cancer can begin in the stromal tissues. These include the fatty and fibrous connective tissues of the breast.
Over time the breast cancer cells can invade nearby tissues such the underarm lymph nodes or the lungs in a process known as metastasis.
The stage of the breast cancer, the size of the tumour and it's rate of growth are all factors which determine the type of treatment that is offered. Treatment options include surgery to remove the tumour, drug treatment which includes chemotherapy and hormonal therapy, radiation therapy and immunotherapy.
The prognosis and survival rate varies widely; the five year relative survival rates vary from 98% to 23% depending on the type of breast cancer that occurs. Worldwide breast cancer constitutes 23% of all cancers with the majority of breast cancers occurring in women.
A particularly aggressive form of breast cancer is characterised by amplification of the Her2 gene which results in overexpression of its protein product. Her2 stands for “human epidermal growth factor receptor 2” and is also known as ErbB-2.
Approximately 15 to 20% of breast cancers have amplification of the Her2 gene. This receptor is stimulated by a growth factor which causes the cell to divide; in the absence of the growth factor the cell will normally stop growing. Overexpression of this receptor in breast cancer is associated with increased resistance of the breast tumour to treatment and recurrence of the disease.
The compound trastuzumab is a monoclonal antibody to Her2 and has improved the 5 year relative survival rates of stage 1 to 3 Her2-positive breast cancers to approximately 87%, however trastuzumab is very expensive, a full course costs approximately $70,000, in addition approximately 2% of patients suffer significant heart damage from its use.
Although the use of this therapy has clearly improved the outcome of patients with Her2-positive tumours, innate and acquired resistance to trastuzumab is still a clinical challenge. Indeed only 25% of Her2-positive tumours respond to trastuzumab and most of the responders eventually relapse.
Cannabinoids have been shown to have an anti-proliferative effect on different cancer cell lines. The cannabinoids THC, THCA, CBD, CBDA, CBG and CBC and the cannabinoid BDS THC and CBD were tested on eight different cell lines including DU-145 (hormone-sensitive prostate cancer), MDA-MB-231 (breast cancer), CaCo-2 (colorectal cancer) and C6 (glioma cells). Furthermore as well as pure cannabinoids a THC botanical drug substance (BDS) and a CBD BDS containing about 95% (w/w) of the respective primary cannabinoid were used, (Ligresti, 2006).
CBD was also shown to inhibit id-1 gene expression in some aggressive forms of breast cancer. The cell lines used were MDA-MB231 and MDA-MB436, (McAllister et al. 2007).
In the application WO 2008/144475, McAllister also describes the use of a combination of the cannabinoids THC and CBD in the treatment glioma. Furthermore CBD is described for use in the treatment of breast cancer as exemplified by the cell lines MDA-MB231 and MDA-MB436, but not cell lines characterized by overexpression of the Her2 gene. In glioma the combination consisted of a high ratio of THC to a low ratio of CBD (4:1 THC:CBD). The problem with such a composition is that the high ratio of THC leads to side effects such as psychosis and anxiety. This work is further described in McAllister et al. 2010.
The present applicants in application WO 2009/147439 themselves describe the use of a combination of cannabinoids, particularly tetrahydrocannabinol (THC) and cannabidiol (CBD), in the treatment of cancer. In particular the cancer to be treated is a brain tumour, more particularly a glioma; more particularly still a glioblastoma multiforme (GBM).
Caffarel et al. (2006, 2008) describes the mechanism whereby THC inhibits cell cycle proliferation and describes the use of THC in an animal model of breast cancer.
More recently Caffarel et al. 2010 documented the anti-tumour action of THC in different models of breast cancer, including cell cultures, xenografted mice, and more recently a genetically engineered mouse model of ErbB2-positive breast cancer, the THC was injected peritumourally.
Shrivastava (2011) describes the how cannabidiol induces programmed cell death in breast cancer cells by co-ordinating cross-talk between apoptosis and autophagy.
Because breast cancer is such a major problem, and existing treatments have limited long-term success, it is a primary object of the present invention to identify alternative treatments which might improve a patient's prognosis.
In one aspect it is a further object to provide treatments of aggressive breast cancer characterised by overexpression of the Her2 gene since only about 25% of patients with this subset of breast cancer respond to the existing drug of choice trastuzumab and of those treated many relapse and die from secondary tumours which have metastasised from the primary cancer forming secondary tumours in the lung and lymph nodes.
In accordance with a first aspect of the present invention there is provided an oral presentation of tetrahydrocannabinol (THC) for use in the treatment of aggressive breast cancer characterised by overexpression of the Her2 gene.
This aspect also extends to the manufacture of a medicament for use in the treatment of aggressive breast cancer characterised by overexpression of the Her2 gene and to the method of treatment per se.
In accordance with a second aspect of the present invention there is provided cannabidiol (CBD) for use in the treatment of aggressive breast cancer characterised by overexpression of the Her2 gene.
This aspect also extends to the manufacture of a medicament for use in the treatment of aggressive breast cancer characterised by overexpression of the Her2 gene and to the method of treatment per se
In accordance with a third aspect of the present invention there is provided a combination of tetrahydrocannabinol (THC) and cannabidiol (CBD) for use in the treatment of breast cancer or to treat, prevent or to reduce the risk of a cancer metastasising.
This aspect also extends to the manufacture of a medicament for use in the treatment of breast cancer or to treat, prevent or to reduce the risk of a cancer metastasising and to the method of treatment per se.
The combination of THC and CBD may also be used to treat or prevent cancer of the lymph nodes or lungs.
Preferably the combination of THC and CBD is in a ratio of between 15:1 to 1:15 (THC:CBD), more preferably between 3:1 to 1:10 (THC:CBD), and most preferably still between 1:4 to 1:10 (THC:CBD).
Preferably the THC, CBD or a combination thereof are in the form of a botanical drug substance (BDS).
The THC, CBD or a combination thereof are present in a therapeutically acceptable amount, which may, for example, be between 1 mg and 2000 mg.
The human dose equivalent (HED) can be estimated using the following formula:
The Km for a mouse is 3 and the Km for a human is 37.
The THC, CBD or a combination thereof may further comprise a non-cannabinoid chemotherapeutic agent.
The non-cannabinoid chemotherapeutic agent may be a monoclonal antibody, such as trastuzumab.
In a fourth embodiment of the present invention there is provided a composition comprising a combination of tetrahydrocannabinol (THC) and cannabidiol (CBD) for use in the treatment of breast cancer or to treat, prevent or to reduce the risk of a cancer metastasising, wherein the ratio of THC to CBD is between 3:1 to 1:10 (THC:CBD).
In this specification the following terms are used and are intended to have the following meanings/definitions:
“Cannabinoids” are a group of compounds including the endocannabinoids, the phytocannabinoids and those which are neither endocannabinoids or phytocannabinoids, hereafter “syntho-cannabinoids”.
“Endocannabinoids” are endogenous cannabinoids, which are high affinity ligands of CB1 and CB2 receptors.
“Phytocannabinoids” are cannabinoids that originate in nature and can be found in the cannabis plant. The phytocannabinoids can be present in an extract including a botanical drug substance, isolated, or reproduced synthetically.
“Syntho-cannabinoids” are those compounds capable of interacting with the cannabinoid receptors (CB1 and/or CB2) but are not found endogenously or in the cannabis plant. Examples include WIN 55212 and rimonabant.
An “isolated phytocannabinoid” is one which has been extracted from the cannabis plant and purified to such an extent that all the additional components such as secondary and minor cannabinoids and the non-cannabinoid fraction have been removed.
A “synthetic cannabinoid” is one which has been produced by chemical synthesis this term includes modifying an isolated phytocannabinoid, by for example forming a pharmaceutically acceptable salt thereof.
A “botanical drug substance” or “BDS” is defined in the Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research as: “A drug derived from one or more plants, algae, or microscopic fungi. It is prepared from botanical raw materials by one or more of the following processes: pulverisation, decoction, expression, aqueous extraction, ethanolic extraction or other similar processes.” A botanical drug substance does not include a highly purified or chemically modified substance derived from natural sources. Thus, in the case of cannabis, BDS derived from cannabis plants do not include highly purified Pharmacopoeial grade cannabinoids.
In the present invention a BDS is considered to have two components: the phytocannabinoid-containing component and the non-phytocannabinoid containing component. Preferably the phytocannabinoid-containing component is the larger component comprising greater than 50% (w/w) of the total BDS and the non-phytocannabinoid containing component is the smaller component comprising less than 50% (w/w) of the total BDS.
The amount of phytocannabinoid-containing component in the BDS may be greater than 55%, through 60%, 65%, 70%, 75%, 80% to 85% or more of the total extract. The actual amount is likely to depend on the starting material used and the method of extraction used.
The “principle phytocannabinoid” in a BDS is the phytocannabinoid that is present in an amount that is higher than that of the other phytocannabinoids. Preferably the principle phytocannabinoid is present in an amount greater than 40% (w/w) of the total extract. More preferably the principle phytocannabinoid is present in an amount greater than 50% (w/w) of the total extract. More preferably still the principle phytocannabinoid is present in an amount greater than 60% (w/w) of the total extract.
The amount of the principle phytocannabinoid in the BDS is preferably greater than 50% of the phytocannabinoid-containing fraction, more preferably still greater than 55% of the phytocannabinoid-containing fraction, and more preferably still greater than 60% through 65%, 70%, 75%, 80%, 85%, 90% and 95% of the phytocannabinoid-containing fraction.
The “secondary phytocannabinoid/s” in a BDS is the phytocannabinoid/s that is/are present in significant proportions. Preferably the secondary phytocannabinoid is present in an amount greater than 5% (w/w) of the total extract, more preferably greater than 10% (w/w) of the total extract, more preferably still greater than 15% (w/w) of the total extract. Some BDS's will have two or more secondary phytocannabinoids that are present in significant amounts. However not all BDS's will have a secondary phytocannabinoid.
The “minor phytocannabinoid/s” in a BDS can be described as the remainder of all the phytocannabinoid components once the principle and secondary phytocannabinoids are accounted for. Preferably the minor phytocannabinoids are present in total in an amount of less than 5% (w/w) of the total extract, and most preferably the minor phytocannabinoid is present in an amount less than 2% (w/w) of the total extract.
The term “consisting essentially of” is limited to the phytocannabinoids which are specified, it does not exclude non-cannabinoid components that may also be present.
Typically the non-phytocannabinoid containing component of the BDS comprises terpenes, sterols, triglycerides, alkanes, squalenes, tocopherols and carotenoids.
These compounds may play an important role in the pharmacology of the BDS either alone or in combination with the phytocannabinoid.
The “terpene fraction” may be of significance and can be broken down by the type of terpene: monoterpene or sesquiterpene. These terpene components can be further defined in a similar manner to the cannabinoids.
The amount of non-phytocannabinoid containing component in the BDS may be less than 45%, through 40%, 35%, 30%, 25%, 20% to 15% or less of the total extract. The actual amount is likely to depend on the starting material used and the method of extraction used.
The “principle monoterpene/s” in a BDS is the monoterpene that is present in an amount that is higher than that of the other monoterpenes. Preferably the principle monoterpene/s is present in an amount greater than 20% (w/w) of the total terpene content. More preferably the principle monoterpene is present in an amount greater than 30% (w/w) of the total terpene content, more preferably still greater than 40% (w/w) of the total terpene content, and more preferably still greater than 50% (w/w) of the total terpene content. The principle monoterpene is preferably a myrcene or pinene. In some cases there may be two principle monoterpenes. Where this is the case the principle monoterpenes are preferably a pinene and/or a myrcene.
The “principle sesquiterpene” in a BDS is the sesquiterpene that is present in an amount that is higher than all the other sesquiterpenes. Preferably the principle sesquiterpene is present in an amount greater than 20% (w/w) of the total terpene content, more preferably still greater than 30% (w/w) of the total terpene content. The principle sesquiterpene is preferably a caryophyllene and/or a humulene.
The sesquiterpene components may have a “secondary sesquiterpene”. The secondary sesquiterpene is preferably a pinene, which is preferably present at an amount greater than 5% (w/w) of the total terpene content, more preferably the secondary sesquiterpene is present at an amount greater than 10% (w/w) of the total terpene content.
The secondary sesquiterpene is preferably a humulene which is preferably present at an amount greater than 5% (w/w) of the total terpene content, more preferably the secondary sesquiterpene is present at an amount greater than 10% (w/w) of the total terpene content.
Alternatively botanical extracts may be prepared by introducing isolated phytocannabinoids or their synthetic equivalent into a non-cannabinoid plant fraction as can be obtained from a zero cannabinoid plant or one or more non-cannabinoid components found in the cannabis plant such as terpenes.
The structures of the phytocannabinoids CBD and THC are as shown below:
Phytocannabinoids can be found as either the neutral (decarboxylated form) or the carboxylic acid form depending on the method used to extract the cannabinoids. For example it is known that heating the carboxylic acid form will cause most of the carboxylic acid form to decarboxylate into the neutral form.
Where a synthetic phytocannabinoid is used the term is intended to include compounds, metabolites or derivatives thereof, and pharmaceutically acceptable salts of such compounds.
The term “pharmaceutically acceptable salts” refers to salts or esters prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids, as would be well known to persons skilled in the art. Many suitable inorganic and organic bases are known in the art.
For the purpose of this invention the term ‘treatment’ is intended to encompass decreasing the viability of cancer cells and their ability to metastasise, and a therapeutically effective amount is an amount that achieves this aim.
Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which
The Examples below demonstrate the effectiveness of the cannabinoids THC and CBD in Her2-positive breast cancer. This type of breast cancer represents a subset of breast tumours characterized by very aggressive clinical courses. These tumours are currently treated with trastuzumab, a humanized monoclonal antibody against Her2.
The effect of THC and CBD, alone and in combination, on the viability of two different Her2-positive human breast cancer cell lines: SkBr3 and BT474 were tested.
Briefly, cells were incubated in RPMI (SkBr3) or DMEM (BT474) supplemented with 10% foetal bovine serum. After 12 hours of serum starvation, the cells were challenged with different concentrations of THC or CBD (or the corresponding vehicle, DMSO) for 72 h. Cell viability was then determined by the colorimetric MTT test.
The analysis of the combined drug effect was performed with the CalcuSyn v2.0 software. To determine whether the combination of THC and CBD was additive or synergistic, we calculated different combination indexes (Cis) using the algorithm by Chou and Talalay with the CalcuSyn v2.0 software.
The cell lines BT474 and SkBr3 were firstly demonstrated to express Her2 (
The two cell lines were then challenged with different concentrations of THC and CBD. As shown in
The BT474 cells were then exposed to different concentrations of a combination of THC and CBD in a 1:1 ratio. Interestingly, we observed that the combination of low concentrations (<1 μM) of the cannabinoids (concentrations with no effect on cell viability when administered alone) significantly diminished the viability of the cell cultures (
We obtained the CI values shown in Table 1.1 below, all of the CI values were less than 1, indicating that these compounds were producing a strong synergistic effect.
This synergism can be also observed in the isobologram shown in
The results demonstrate that a low dose of a combination of THC and CBD have significant antiproliferative effects in human Her2-positive breast cancer cells.
Importantly a synergic effect was achieved with half the dose of THC that was required when administered alone. This observation may have important implications for the development of cannabinoid-based therapies for breast tumours, as the dose of THC could be reduced, in consequence diminishing or removing any potential side effects, without affecting its potency.
These data provide strong evidence for the use of cannabinoid-based therapies for the management of Her2-positive breast cancer, which represents a subset of breast tumours characterized by very aggressive clinical courses, reduced responsiveness to conventional and targeted therapies and high relapsing frequencies.
Ectopic xenografts were generated by subcutaneous injection of 5×106 human BT474 cells (Her2-positive human breast cancer cells). Animals were divided into 11 experimental groups (8 animals/group) and when tumours reached 200 mm3, they were treated as detailed in Table 2.1 below for a period of 4 weeks.
Different routes of administration were tested along with different combinations of THC, CBD and trastuzumab.
The cannabinoids THC and CBD were in the form of botanical drug substances (BDS) whereby the major cannabinoid is present along with other cannabinoid components and a non-cannabinoid fraction.
The relative amounts of the different cannabinoids in the THC and CBD BDSs are shown in Table 2.2 below. It will be appreciated that variations of plus or minus 25% (w/w) can occur with BDS components depending on the method of extraction used.
Tumours were routinely measured during this period with an external calliper, and their volume was calculated as (4π/3)×(width/2)2×(length/2).
Oral administration of THC significantly reduced tumour growth and interestingly this route of administration is more effective than IP administration (
The results also clearly show an important anti-tumour effect of CBD in this model (
Both THC and CBD were able to improve the anti-tumour action of trastuzumab, especially when administered in combination (
These data demonstrate that human Her2-positive cancer cells are sensitive to phytocannabinoids in vivo. Indeed the combination of THC and CBD were more effective than the standard treatment trastuzumab which leads to a positive indication for these cannabinoids in the treatment of aggressive types of breast cancer.
CBD has been shown to have significant anti-tumour properties in breast cancer. Since this compound has a very safe profile, experiments were performed to determine whether the proportion of the psychoactive compound THC can be decreased in the combination treatments
The effect of THC and CBD, alone and in combination at different ratios, on the viability of BT474 cells was examined.
Cells were incubated in DMEM supplemented with 10% foetal bovine serum. After 12 hours of serum starvation, cells were challenged for 72 h as described in Table 3.1. Cell viability was then determined by the colorimetric MTT test.
These data indicate that combinations of THC and CBD in which THC proportion was as low as 1:10 were as effective as the 1:1 ratios in decreasing cell viability (
Human Her2-positive cancer cells are sensitive to phytocannabinoids both in cell cultures and in vivo. It is noteworthy that decreasing the ratio of THC to as low as 1:10 (THC:CBD) did not make any difference in the effectiveness of the combination of the cannabinoids.
As such ratioed combinations of THC and CBD could be used to treat aggressive forms of breast cancer with very few, if any side effects.
The effect of THC and CBD on the viability of a series of Her2-positive human breast cancer cell lines: MDA-MB-231 cells that ectopically overexpress Her2 (231-Her2), a highly metastatic version of 231-Her2 (231-Met), and three different trastuzumab-resistant 231-Her2-derived cell lines (TrR1, TrR2 and TrR3).
Cells were incubated in DMEM supplemented with 10% foetal bovine serum. After 12 hours of serum starvation, cells were challenged with different concentrations of THC or CBD (or the corresponding vehicle, DMSO) for 72 h. Cell viability was then determined by the colorimetric MTT test.
As shown in
Similar studies were performed with trastuzumab-resistant cells and the results were analogous: these cell lines decreased their viability in response to phytocannabinoids in a concentration dependent manner (
Together, these results suggest that, whatever molecular changes these cells have suffered to become extremely aggressive (highly metastatic or resistant to trastuzumab), phytocannabinoids are still effect agents in decreasing these cells viability and ability to metastasize.
The previous Examples demonstrate that 1:9 THC:CBD is as effective as the 1:1 ratio in reducing BT474 cell viability in vitro. This data demonstrating the effect of 1:9 THC:CBD combinations in BT474 cells in vivo is provided in this example. In addition the effect of such a combination on trastuzumab-resistant and in highly metastatic Her2-overexpressing MDA-MB-231 cells in vitro is also demonstrated.
The effect of a 1:9 combination of THC:CBD on Her2-positive cancer cells in vitro, was demonstrated by challenging the following cell lines with the cannabinoid concentrations indicated in the corresponding figure legends:
Cells were incubated in the presence of cannabinoids for 48 h and cell viability was assessed by the MTT colorimetric test.
To study the effect of different THC:CBD ratios on human Her2-positive breast cancer in vivo, we generated ectopic xenografts in immune-deficient female mice by subcutaneous injection of 5×106 BT474 cells. Animals were divided into 7 experimental groups (8 animals/group) and when tumours reached 200 mm3 (typically 40 days after cancer cell injection), they started to be treated as indicated in the table below.
Tumours were routinely measured with external calliper, and volume was calculated as (4π/3)×(width/2)2×(length/2).
Previous examples demonstrated that THC:CBD combinations in which THC represents just one tenth of the total cannabinoid concentration were as effective as 1:1 ratios in reducing the viability of human breast cancer cells naturally overexpressing the oncogene Her2 (BT474 cells).
This example demonstrates similar experiments with MDA-MB-231-derived cell lines. MDA-MB-231 cells are widely used in breast cancer studies due to their ability to form tumours and metastasis in vivo. However, this cell line does not express Her2. To convert MDA-MB-231 cells into a Her2-positive breast cancer model, we used 231 cells stably transfected with Her2 (231-Her2) and two clones derived from them: one showing resistance to trastuzumab treatment (231-Her2-TrR) and another with an increased metastatic potential (231-Her2-Met).
Overexpression of the Her2 oncogene in MDA-MB-231 cells did not alter responsiveness to phytocannabinoids (
Trastuzumab resistant (
The effect of phytocannabinoid combinations on Her2-positive cells in vivo was also examined. BT474 subcutaneous xenografts decreased their growth in response to THC-BDS and CBD-BDS alone (
These data demonstrate that MDA-MB-231 and 231-Her2 cells are equally sensitive to THC and CBD alone; that 1:1 THC:CBD combinations have additive effects on 231 and 231-Her2 cells in vitro; and that 1:9 THC:CBD combinations are as effective as 1:1 combinations in decreasing cell viability.
Despite the molecular changes suffered to become highly aggressive, trastuzumab-resistant and highly metastatic Her2-positive cells retain their capacity to respond to phytocannabinoids as described in the example above.
It is also demonstrated that a 1:9 THC:CBD combination is more effective than 1:1 combinations in decreasing BT474 xenograft growth.
Caffarel, M. M., Andradas, C., Mira, E., Perez-Gomez, E., Cerutti, C., Moreno-Bueno, G., Flores, J. M., Garcia-Real, I., Palacios, J., Manes, S., et al. 2010. Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition. Mol Cancer 9:196.
Number | Date | Country | Kind |
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1117956.1 | Oct 2011 | GB | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/GB2012/052565 | 10/17/2012 | WO | 00 | 4/17/2014 |
Number | Date | Country | |
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61550069 | Oct 2011 | US |