Cancer therapy

Abstract

The present invention is based on the discovery that the inhibition of cancer cell proliferation by cannabinoids is more effectively achieved by combination therapy with low dose naltrexone (LDN) or 6-beta-naltrexone (6BN), a metabolite of naltrexone. A pharmaceutical composition comprising naltrexone or a metabolite thereof or an analog of any of them for use in the treatment of cancer in a subject is provided, wherein the subject will be administered a therapeutically effective amount of naltrexone or a metabolite thereof or an analog of any of them in a first treatment phase, wherein after the first treatment phase, the subject will be administered a therapeutically effective amount of a cannabinoid in a second treatment phase.

Description

Technical Field

[0001] This invention relates to drug administration regimens and drug combinations for cancer treatment. Background Technology

[0002] Cannabinoids are a class of phenolic compounds produced in large quantities in plants of the genus *Cannabis*. Various cannabinoid compounds have long been believed to have psychoactive effects in humans and have been widely used for recreational purposes, although such use is illegal in many jurisdictions.

[0003] However, recent research has shown that certain cannabinoids have therapeutic value for a wide range of conditions, such as inflammatory diseases, neurodegenerative diseases and mental illnesses, chronic pain, anxiety, and PTSD. While cannabinoids are currently used to combat weight loss, vomiting, and nausea associated with cancer treatment, there is evidence that some cannabinoid compounds may be effective in treating the underlying pathology of cancer. Currently available evidence suggests that they do this by disrupting cancer cell migration, adhesion, and tumor vascularization.

[0004] Endocannabinoid-mediated signaling systems function in mammals and, based on evidence, are likely to do so in most other vertebrates as well. In humans, two cannabinoid receptors have been identified and named CB1 and CB2, both of which are part of the G protein-coupled receptor superfamily.

[0005] Pharmacological evidence to date suggests that the psychoactive effects of cannabinoid intake are primarily mediated by CB1 activation, while the therapeutic effects are mainly mediated by CB2. Therefore, the ideal therapeutic molecule would activate CB2 preferentially over CB1, mitigating the unwanted psychoactive side effects. Cannabidiol (CBD), a component of cannabis extracts, is a cannabinoid exhibiting this binding property and has recently attracted considerable attention, but its complete mechanism of action remains to be elucidated.

[0006] However, naturally occurring cannabinoids are generally not designed to exhibit high specificity for one or more receptors, but newly developed synthetic cannabinoids are available that bind more specifically, such as JWH-133 and SR141716 (Cridge & Rosengren 2013). These compounds have been shown to inhibit tumor growth and cancer cell viability in vitro and in various tumor-implanted mouse models.

[0007] Sometimes, the expression levels or downstream effects of receptors in one signaling pathway, in response to specific ligands, can be altered by changes in the signal output of another seemingly independent signaling pathway. This cross-regulation has been demonstrated, for example, between signaling pathways following the activation of growth hormone and insulin receptors. Summary of the Invention

[0008] The inventors have discovered that the inhibition of cancer cell proliferation by cannabinoids is more effectively achieved through combination therapy with low-dose naltrexone (LDN) or 6-β-naltrexol (6BN) (a metabolite of naltrexone). They have also found that the effectiveness of this treatment is surprisingly dependent on the order of administration of the two agents, with the most effective regimen being a phase of treatment with LDN or 6BN followed by a phase of treatment with cannabinoids.

[0009] The inventors also made a previously unknown discovery that treatment with LDN or 6BN significantly increases the level of CB2 receptors in cancer cells, thereby making cannabinoids more effective.

[0010] According to a first aspect of the invention, a pharmaceutical composition is provided comprising naltrexone or a metabolite thereof or an analogue selected from methylnaltrexone, naloxone, nalmefen and nalprofen, for treating cancer in a subject, wherein the subject is given a therapeutically effective amount of naltrexone or a metabolite thereof or an analogue thereof during a first treatment phase, and wherein, after the first treatment phase, the subject is given a therapeutically effective amount of cannabinoids during a second treatment phase.

[0011] According to a second aspect of the invention, a pharmaceutical composition comprising cannabinoids is provided for treating cancer in a subject, wherein the subject is characterized by having undergone a first treatment phase during which the subject is given a therapeutically effective amount of naltrexone or a metabolite thereof or an analogue selected from methylnaltrexone, naloxone, nalmefen, and nalprofen, wherein after the first treatment phase, the subject is given a therapeutically effective amount of the cannabinoids.

[0012] According to a third aspect of the invention, there is provided a formulation comprising naltrexone or a metabolite thereof or an analogue selected from methylnaltrexone, naloxone, nalmefen and nalprofen, and cannabinoids, for treating cancer in a subject, wherein naltrexone or a metabolite thereof or an analogue thereof is provided in a therapeutically effective amount to be administered in a first treatment phase, and wherein cannabinoids are provided in a therapeutically effective amount to be administered in a second treatment phase following the first treatment phase.

[0013] According to a fourth aspect of the invention, a method is provided for determining the suitability of a subject suffering from cancer for treatment with cannabinoids in a second treatment phase, said subject being characterized in that they have undergone a first treatment phase as defined above, the method comprising the steps of:

[0014] i. Contact samples obtained from the subject with a CB2-specific probe after or during the recovery phase:

[0015] ii. Determine the concentration of CB2 in the sample; and

[0016] iii. Compare the concentration of CB2 in the sample with the concentration of CB2 determined in samples obtained from the subject prior to the first treatment phase.

[0017] If the concentration of CB2 increases at least twice after the first treatment phase, the subject is suitable for the second treatment phase.

[0018] According to a fifth aspect of the invention, there is provided the use of naltrexone or a metabolite thereof or an analogue selected from methylnaltrexone, naloxone, nalguanfen and nalprofen in the manufacture of a medicament for treating cancer in a subject, wherein the medicament is administered to the subject in a first treatment phase of a combination therapy regimen comprising the first treatment phase followed by a second treatment phase, wherein the subject is administered cannabinoids in the second treatment phase.

[0019] According to a sixth aspect of the invention, the use of cannabinoids in the manufacture of a medicament for treating cancer in a subject is provided, wherein the medicament is administered in a second treatment phase of a combination treatment regimen comprising a first treatment phase followed by a second treatment phase, wherein in the first treatment phase the subject is given naltrexone or a metabolite thereof or an analogue selected from methylnaltrexone, naloxone, nalmefen, and nalprofen.

[0020] According to a seventh aspect of the invention, there is provided the use of naltrexone or its metabolites or analogs selected from methylnaltrexone, naloxone, nalmefen, and nalopefen in the manufacture of a first drug and the use of cannabinoids in the manufacture of a second drug, both of which are used to treat cancer, wherein in a combined treatment regimen the first and second drugs are administered to a subject suffering from cancer, the treatment regimen comprising a first treatment phase followed by a second treatment phase, wherein the first drug is administered to the subject during the first treatment phase and the second drug is administered to the subject during the second treatment phase.

[0021] According to an eighth aspect of the invention, a method for treating cancer in a subject is provided, comprising administering a therapeutically effective amount of naltrexone or a metabolite thereof or an analogue selected from methylnaltrexone, naloxone, nalmefen, and nalprofen to the subject in a first treatment phase, and then administering a therapeutically effective amount of cannabinoid to the subject in a second treatment phase. Attached Figure Description

[0022] Figure 1The effects of treating MCF7 breast cancer cell line cultures for two days with 10 nM naltrexone (LDN), 1 μM 6-β-naltrexol (6-1), or 10 μM 6-β-naltrexol (6-2) are shown. With GAPDH as a loading control, the levels of Bcl2-associated death promoter (BAD), p21 protein, opioid receptors κ1 (OPRK1) and mu1 (OPRM1), and cannabinoid receptors CBR1 (CBI) and CBR2 (CB2) were visualized by Western blotting (left) and quantified by density analysis relative to GAPDH (right).

[0023] Figure 2 The effects of various two-stage treatments on the proliferation and viability of MCF7 cells were demonstrated. Each treatment lasted four days and consisted of two days of treatment with the first agent (UN = control, LDN = 10 nM low-dose naltrexone, CBD = cannabidiol, 6BN = 6-β-naltrexol), followed by two more days of treatment with the second agent. Detailed Implementation

[0024] This invention provides a specific treatment regimen for treating cancer in subjects, wherein cannabinoids are administered after administration of a low dose of naltrexone (LDN), a metabolite of naltrexone, or an analogue selected from methylnaltrexone, naloxone, nalmefen, and nalprofen.

[0025] The inventors discovered that administering LDN followed by cannabinoids in stages was more effective in inhibiting cancer cell proliferation than administering cannabinoids alone or simultaneously or in stages followed by LDN.

[0026] Furthermore, it was found that CB2 receptor levels significantly increased in MCF7 cells after LDN treatment. Without being bound by theory, this increase in CB2—a therapeutic target for cannabinoids such as CBD—is a feasible contributing factor to increasing the effectiveness of CBD treatment.

[0027] Those skilled in the art will be able to administer the therapeutic agents as described in stages. The invention can be further understood with reference to the following definitions:

[0028] As used herein, “naltrexone” refers to the compound 17-cyclopropylmethyl-4,5α-epoxy-3,14-dihydroxymorphinan-6-one (IUPAC name (4R,4aS,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methylenebenzofuran[3,2-e]isoquinoline-7-one), and its pharmaceutically acceptable salts, solvates, hydrates, racemates, stereoisomers, cage-like inclusion compounds, polymorphs, and prodrugs. Analogs thereof are also contemplated for use according to the invention. Suitable analogs include methylnaltrexone, naloxone, nalmefen, and nalprofen.

[0029] Naltrexone is usually in its hydrochloride form.

[0030] "Low-dose naltrexone" (LDN) refers to naltrexone administered at a "low" dose of less than 0.5 mg / kg, preferably less than 0.2 mg / kg, more preferably between 0.01 mg / kg and 0.08 mg / kg, even more preferably between 0.03 mg / kg and 0.06 mg / kg, and most preferably between 0.04 mg / kg and 0.05 mg / kg. Typically, the total low dose per patient is up to 3 mg per day.

[0031] The metabolites of naltrexone include 6-β-naltrexol, 2-hydroxy-3-methoxy-6β-naltrexol, and 2-hydroxy-3-methoxy-naltrexone.

[0032] The preferred metabolite of naltrexone is 6-β-naltrexol (6BN), which, as used herein, refers to the compound N-cyclopropylmethyl-7,8-dihydro-14-hydroxynorisomorphine (IUPAC name (4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-1,2,4,5,6,7,7a,13-octahydro-4,12-methylenebenzofuran[3,2-e]isoquinoline-4a,7,9-triol), and its pharmaceutically acceptable salts, solvates, hydrates, racemates, stereoisomers, cage inclusion complexes, polymorphs, and prodrugs.

[0033] 6BN or similar substances can also be administered at “low doses.” In this context, “low dose” can be the same as the low dose listed for naltrexone above.

[0034] Cannabinoids are a class of compounds known to those skilled in the art, including those produced in large quantities from cannabis plants and endogenous cannabinoids synthesized in animals. Synthetic active compounds targeting CB receptors are also envisioned. As used herein, the term may refer to any cannabinoid, but is preferably selected from the following: cannabidiol, cannabidiol acid, cannabinol, cannabivarin, cannabivarin, tetrahydrocannabivarin, cannabicycloterpene, arachidonic acid, 2-arachidonic glycerol, 2-arachidonic glycerol ether, N-arachidonic dopamine, arachidonic acid (2-aminoethyl) ester (virodhamine), dronabinol, cannabinone, rimonaban, R-(+)-methylarachidonic acid aminoethanol (R-(+)-Met-anandamide), WIN-55, 212-2, HU-210, JWH-133, SR141716, SR144528 or combinations thereof, or pharmaceutically acceptable salts, solvates, hydrates, racemates, stereoisomers, cage inclusion compounds, polymorphs, prodrugs, and analogues that produce equivalent effects.

[0035] In some embodiments, the agent is 6-β-naltrexol, and the 6-β-naltrexol is administered in an effective amount to increase the plasma concentration of 6-β-naltrexol to at least 0.34 ng / ml, preferably at least 3.4 ng / ml, more preferably at least 34 ng / ml, or most preferably at least 340 ng / ml. In some embodiments, the 6-β-naltrexol is administered in an effective amount to increase the plasma concentration of 6-β-naltrexol to the range of 0.3 ng / ml to 3,400 ng / ml, preferably in the range of 34 ng / ml to 3,400 ng / ml, more preferably in the range of 340 ng / ml to 3,400 ng / ml. The effective amount to achieve this amount can be determined using any number of conventional techniques known to those skilled in the art. For example, those skilled in the art can perform mass spectrometry analysis on a plasma sample obtained from a subject to determine the increase in the concentration of 6-β-naltrexol in the sample after administration of 6-β-naltrexol. The effective amount is the amount determined to achieve the desired increase in plasma concentration. Normally, naltrexol will be administered at a dose of up to 3 mg per patient per day.

[0036] Cannabinoids can be administered in conventional doses based on the specific cannabinoid and the patient's details. In some embodiments, the cannabinoid will be administered daily at a dose between 10 mg and 1000 mg, preferably between 200 and 800 mg, and more preferably between 300 and 500 mg.

[0037] As used herein, “preparation” can refer to a substance or a collection of substances in the form of one or more compositions intended for simultaneous or separate use.

[0038] For any therapeutic agent, the method of administration is not particularly limited, but in various embodiments of the invention, LDN and cannabinoids are administered via oral, buccal, sublingual, nasal, pulmonary, intravenous, rectal, topical, and transdermal routes. Oral administration is preferred for LDN, and sublingual administration is preferred for cannabinoids.

[0039] The proposed treatment regimen includes a "first treatment phase" and a "second treatment phase." In the first treatment phase, a therapeutically effective amount of LDN, its metabolites, or any analogue is administered. In the second treatment phase, an effective amount of one or more cannabinoids is administered.

[0040] Preferably, the second treatment phase begins 1 to 7 days after the start of the first treatment phase, more preferably 1 to 4 days, and most preferably 1 to 2 days, where "day" refers to a continuous 24-hour period.

[0041] In another preferred embodiment of the invention, there is a "recovery phase" between the end of the first treatment phase and the beginning of the second treatment phase. During the recovery phase, no LDN or cannabinoids are administered. In one embodiment, the recovery phase lasts for at least two days, preferably with an interval of no more than one week, and most preferably for two days.

[0042] As used herein, the terms “treating,” “treatment,” and “totreat” refer to therapeutic measures that cure, slow, and / or stop the progression of a diagnosed pathological condition or disease, and also to prophylactic or preventative measures that prevent and / or slow the development of a target pathological condition or disease. Therefore, those subjects requiring treatment include those who already have the condition, those who are susceptible to the condition, and those who wish to prevent the condition. In some cases, according to the invention, a subject's tumor / cancer is successfully “treated” if the subject exhibits one or more of the following: a reduction or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs (including, for example, cancer spread to soft tissue and bone); inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; a reduction in morbidity and mortality; a reduction in tumor neoplasia, frequency of tumorigenesis, or ability to produce tumors; a reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenetic cells into a non-tumorigenic state; or some combination of the aforementioned effects.

[0043] As used herein, the term “subject” means any animal that will become a recipient of cancer treatment, including, but not limited to, humans, non-human primates, horses, canines, felines, rodents, and other vertebrates. The terms “subject” and “patient” are used interchangeably herein.

[0044] As used herein, the term “tumor / cancer” refers to any mass of tissue resulting from excessive cell growth, proliferation and / or survival, which may be benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0045] The types of cancers treatable by this invention are not limited in any way and include, for example, carcinoma, sarcoma, adenocarcinoma, melanoma, neurocarcinoma (germ cell tumor, glioma), mesothelioma and reticuloendothelial, lymphoblastic or hematopoietic neoplastic diseases (e.g., myeloma, lymphoma or leukemia). In specific embodiments, the tumor may include lung adenocarcinoma, lung cancer, diffuse or interstitial gastric cancer, colon cancer, prostate adenocarcinoma, esophageal cancer, breast cancer, pancreatic adenocarcinoma, ovarian adenocarcinoma, adrenal adenocarcinoma, endometrial adenocarcinoma or uterine adenocarcinoma, but the preferred type of cancer is breast cancer.

[0046] In a preferred embodiment, the cancer to be treated is selected from brain cancer, breast cancer, colon cancer, lung cancer, prostate cancer, pancreatic cancer, and leukemia. Breast cancer is preferred.

[0047] The definition of “breast cancer” is well-known in medical science. Those skilled in the art will understand that breast cancer refers to any malignant tumor of the breast tissue in men or women, including, for example, carcinomas and sarcomas. Specific manifestations of breast cancer include ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), or mucinous carcinoma. Breast cancer also refers to invasive ductal carcinoma (IDC), lobular neoplasia, or invasive lobular carcinoma (ILC).

[0048] As used in this article, the term "cancer cell" refers to cells or immortalized cell lines derived from tumors or cancers.

[0049] In one aspect of the invention, a method is provided for determining whether a subject suffering from cancer and treated with LDN is suitable for treatment with cannabinoids. In this method, a sample is obtained from the subject and contacted with a CB2-specific probe. The concentration of CB2 in the sample is then determined and compared with a reference measurement prior to a first treatment phase.

[0050] As used herein, “fitness” refers to a characteristic that indicates a higher probability of treatment success compared to those subjects deemed unfit by testing. A subset of subjects tested using methods conceived by any aspect of this invention would be considered unfit for the second phase of treatment. No part of this invention is permitted for use with these subjects. Methods for determining fit are intended to be considered by those skilled in the art, along with other tests known to them, and using their intuition and judgment.

[0051] In some embodiments, the biological "sample" obtained from the subject for use in the method is blood, plasma, serum, lymph, tissue, or cells from a tissue sample. However, preferably, the sample is a tumor biopsy obtained from the subject.

[0052] As used herein, a “probe” is any part that, upon contact with a sample obtained from a subject, allows for the measurement of the concentration of CB2 in the sample in a certain manner. In one embodiment, the probe is an antibody or other CB2-binding molecule used to provide a purer CB2 solution that can be analyzed spectrophotometrically and calibrated using methods known to those skilled in the art to determine the concentration of CB2 in the original sample.

[0053] In another aspect, the use of naltrexone, its metabolites, or analogues selected from methylnaltrexone, naloxone, nalmefen, and nalprofen, as well as cannabinoids, in the manufacture of a medicine to be administered as part of a “combination therapy regimen,” which, as used herein, means a regimen consisting of a first treatment phase—preferably a recovery phase—and a second treatment phase as defined above.

[0054] The invention will now be described with reference to the following non-limiting embodiments.

[0055] Example

[0056] Example 1

[0057] An illustrative and non-restrictive experiment was performed on MCF7 cell cultures. Briefly, cultures were injected with 10 nM LDN or 1 μM or 10 μM 6BN in vitro for two days. The expression levels of BAD, p21, opioid receptors κ and mu, CB1, CB2, and GAPDH (as loading controls) were analyzed by protein immunoblotting, followed by quantification by band density measurement.

[0058] MCF7 cells were seeded completely into 6-well plates at a density of 1 x 10⁵ / well and allowed to adhere overnight. Naltrexone (10 nM) or 6-β-naltrexol (1 μM or 10 μM) was added to the cells, and then the cells were collected for protein immunoblotting analysis. Primary probing was performed using generated specific antibodies against BAD, p21, OPRK1, OPRK2, CBR-1, and CBR-2. BAD is a Blc2-associated death promoter, a pro-apoptotic protein that, if upregulated in cancer cells, increases their tendency to be killed by various therapies. Anti-GAPDH was used as a loading control. All antibodies were used at a 1:1000 dilution, followed by appropriate HRP-conjugated secondary antibodies at a 1:1000 dilution. Bands were visualized using an ultrasignal chemiluminescence detection system, and density measurements of band intensity were determined using Adobe Photoshop CS3, v10.0 and normalized to the loading controls.

[0059] In the responses to all three treatments, CB2 and BAD levels increased relative to the untreated control group. For CB2, this increase was highest in the response to administration of 10 μM 6BN. This can be seen in... Figure 1 This can be seen from the text.

[0060] Example 2

[0061] Another illustrative and non-limiting experiment was performed on MCF7 cell cultures. Briefly, the cultures were treated with LDN or 6BN for two days, followed by treatment with cannabinoids for another two days. Cell number and viability were assessed on day four. The order of administration of each treatment was reversed, and the experiment was repeated.

[0062] MCF7 cells were seeded completely into 6-well plates at a density of 1.5 x 10⁴ cells / well and allowed to adhere. Cells were then cultured with naltrexone (10 nM), 6-β-naltrexol (10 μM), or cannabidiol (10 μM). The drug-containing medium was removed after 48 h, and the cells were gently rinsed with drug-free medium. Fresh medium was then added to the cells supplemented with naltrexone (10 nM), 6-β-naltrexol (10 μM), or cannabidiol (10 μM), as shown in the figure. Cell number and viability were assessed after another 48 h, and the percentage of live and dead cells was distinguished using trypan blue exclusion. The data were then pooled to compare the effect of the order on the overall results.

[0063] As from Figure 2 As can be seen, the timing of LDN or 6BN administration before cannabinoids has a greater impact on inhibiting cancer proliferation.

[0064] refer to

[0065] Cridge,B.&Rosengren,R(2013)Critical appraisal of the potential use ofcannabinoids in cancer management.Cancer Management and Research 2013:5 301-313

Claims

1. The use of a formulation for preparing a pharmaceutical agent for treating cancer in a subject, wherein the formulation comprises naltrexone or 6-β-naltrexol, and cannabinoid, wherein naltrexone or 6-β-naltrexol is provided in an amount between 0.01 mg / kg and 0.08 mg / kg for administration in a first treatment phase, and wherein the cannabinoid is provided in a therapeutically effective amount for administration in a second treatment phase following the first treatment phase. The cannabinoid mentioned therein is cannabidiol, and The cancer mentioned is breast cancer.

2. The use according to claim 1, wherein the first treatment phase is administration for at least two days.

3. The use according to claim 1 or 2, wherein the first treatment phase and the second treatment phase are separated by a recovery phase, wherein the recovery phase is characterized by the absence of administration of naltrexone or 6-β-naltrexol, and cannabinoids.

4. The use according to claim 3, wherein the recovery phase is at least one day.

5. The use according to claim 3, wherein the recovery phase is in the range of 1 to 7 days.

6. The use according to claim 1 or 2, wherein the second treatment phase is administration for at least one day.

7. The use according to claim 1 or 2, wherein the formulation comprises naltrexone.

8. The use according to claim 1 or 2, wherein the formulation comprises 6-β-naltrexol.

Images