Cancer Treatment

Abstract

SOLUTION: There is provided a pharmaceutical composition comprising naltrexone or a metabolite thereof or an analogue thereof, for use in treatment of cancer within a subject, where a therapeutically effective amount of the naltrexone or metabolite thereof or either of analogue is to be administered to the subject in a first treatment phase, where after the first treatment phase a therapeutically effective amount of a cannabinoid is to be administered to the subject in a second treatment phase.EFFECT: Inhibition of proliferation of cancer cells by cannabinoids can be brought about more effectively by combined treatment with low dose naltrexone (LDN) or 6-β-naltrexone (6BN), which is a metabolite of naltrexone.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to drug administration regimens and drug combinations for use in the treatment of cancer. [Background technology]

[0002] Cannabinoids are a class of phenolic compounds that are abundantly produced in the Cannabis plant. Various cannabinoid compounds have long been known to have psychotropic effects in humans and are widely used recreationally, even though such use is illegal in many jurisdictions.

[0003] However, recent studies have demonstrated that some cannabinoids may be therapeutically useful for a wide range of conditions, including inflammatory diseases, neurodegenerative and psychiatric disorders, chronic pain, anxiety, and PTSD. Cannabinoids are currently used to combat the exhaustion, vomiting, and nausea associated with cancer treatment, but there is evidence to suggest that some cannabinoid compounds may be effective in treating the underlying pathology of cancer. Currently available evidence suggests that they do so by interfering with the migration, adhesion, and angiogenesis of cancer cells.

[0004] An endogenous cannabinoid-mediated signaling system is operative in mammals and, based on evidence, may also function in many other vertebrates. Two cannabinoid receptors have been identified in humans, termed CB1 and CB2. Both are part of the G protein-coupled receptor superfamily.

[0005] Current pharmacological evidence indicates that CB1 activation is responsible for most of the psychotropic effects of cannabinoid intake, whereas the majority of therapeutic effects are mediated by CB2. An ideal therapeutic molecule would therefore preferentially activate CB2 over CB1, thus reducing the undesirable psychotropic effects. Cannabidiol (CBD), a component of cannabis extract, is a cannabinoid with such a binding profile and has attracted considerable interest in recent years, although its mechanism of action remains to be fully elucidated.

[0006] In general, however, natural cannabinoids do not tend to be highly specific for one receptor or the other, and newly developed synthetic cannabinoids, such as JWH-133 and SR141716 (Cridge & Rosengren 2013), are available that exhibit more specific binding. These compounds have been shown to inhibit tumor growth and cancer cell viability in vitro and in various tumor-implanted mouse models.

[0007] In response to a particular ligand, the expression level or downstream effect of a receptor in one signaling pathway may be altered, seemingly by altering the signal output of another signaling pathway. Such cross-modulation has been shown, for example, between the signaling pathways that follow activation of growth hormone and insulin receptors. Summary of the Invention

[0008] The inventors of the present invention have discovered that the inhibition of cancer cell proliferation by cannabinoids can be more effectively induced by combined treatment with low-dose naltrexone (LDN) or 6-β-naltrexol (6BN), a metabolite of naltrexone. The inventors have found that the effectiveness of such treatment is surprisingly dependent on the order in which the two drugs are administered, with the most effective regimen being a treatment phase with LDN or 6BN followed by a treatment phase with cannabinoids.

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

[0010] According to a first aspect of the present invention there is provided a pharmaceutical composition comprising naltrexone, a metabolite thereof, or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine for use in treating cancer in a subject, wherein a therapeutically effective amount of the naltrexone, metabolite thereof, or analogue is administered to the subject in a first treatment phase, and following the first treatment phase a therapeutically effective amount of a cannabinoid is administered to the subject in a second treatment phase.

[0011] According to a second aspect of the present invention there is provided a pharmaceutical composition comprising a cannabinoid for use in the treatment of cancer in a subject, characterized in that the subject undergoes a first treatment phase in which a therapeutically effective amount of naltrexone, a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine is administered, and after the first treatment phase a therapeutically effective amount of the cannabinoid is administered to the subject.

[0012] According to a third aspect of the present invention, there is provided a formulation comprising naltrexone, a metabolite thereof, or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene, and nalorphine; and a cannabinoid; for use in treating cancer in a subject, wherein the naltrexone, metabolite thereof, or analogue is provided in a therapeutically effective amount for administration in a first treatment phase, and the cannabinoid is provided in a therapeutically effective amount for administration in a second treatment phase following the first treatment phase.

[0013] According to a fourth aspect of the present invention, there is provided a method for determining the suitability of a subject having cancer for treatment with a cannabinoid in a second treatment phase, the subject being characterised by having undergone a first treatment phase as defined above, the method comprising: i. contacting a sample obtained from the subject after or during a recovery phase with a probe specific for CB2; ii. determining the concentration of CB2 in the sample; iii. comparing the concentration of CB2 in the sample to the concentration of CB2 determined from a sample obtained from the subject prior to the first treatment phase; wherein the subject is suitable for the second treatment phase if the CB2 concentration increases at least 2-fold after the first treatment phase.

[0014] According to a fifth aspect of the present invention there is provided the use of naltrexone, a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine in the manufacture of a medicament for the treatment of cancer in a subject, said medicament being administered to the subject in a first treatment phase of a combination treatment regimen comprising said first treatment phase followed by a second treatment phase, said subject being administered a cannabinoid in said second treatment phase.

[0015] According to a sixth aspect of the present invention there is provided the use of a cannabinoid in the manufacture of a medicament for the treatment of cancer in a subject, said medicament being administered in a second treatment phase of a combination treatment regimen comprising a first treatment phase followed by said second treatment phase, said subject being administered in said first treatment phase naltrexone, a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine.

[0016] According to a seventh aspect of the present invention there is provided the use of naltrexone, a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine in the manufacture of a first medicament and the use of a cannabinoid in the manufacture of a second medicament, both medicaments being for the treatment of cancer, said first and second medicaments being administered to a subject having cancer in a combined treatment regimen, said treatment regimen comprising a first treatment phase followed by a second treatment phase, said first drug being administered to said subject in the first treatment phase and said second drug being administered to said subject in the second treatment phase.

[0017] According to an eighth aspect of the present invention there is provided a method for the treatment of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of naltrexone, a metabolite thereof, or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene, and nalorphine in a first treatment step, and thereafter administering to the subject a therapeutically effective amount of a cannabinoid in a second treatment step. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows the effect of 2 days of treatment with 10 nM naltrexone (LDN), 1 μM 6-β-naltrexol (6-1), or 10 μM 6-β-naltrexol (6-2) on cultures of the MCF7 breast cancer cell line. The amounts of Bcl2-associated death promoter (BAD), p21 protein, opioid receptors kappa 1 (OPRK1) and μ1 (OPRM1), and cannabinoid receptors CBR1 (CB1) and CBR2 (CB2) were visualized by Western blotting using GAPDH as a loading control (left) and quantified by analysis of relative concentration to GAPDH (right). [Diagram 2]Figure 2 shows the effect of various two-step treatments on the proliferation and viability of MCF7 cells, each lasting 4 days, where treatment with the first drug (UN=control, LDN=10 nM low dose naltrexone, CBD=cannabidiol, 6BN=6-β-naltrexol) was performed for 2 days, followed by introduction of the second drug named for 2 more days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention provides certain therapeutic regimens for treating cancer in a subject, in which a cannabinoid is administered following administration of low dose naltrexone (LDN), a metabolite of naltrexone, or an analog selected from the group consisting of methylnaltrexone, naloxone, nalmefene, and nalorphine.

[0020] The inventors have found that administering LDN followed by a cannabinoid in a stepwise manner inhibits cancer cell proliferation more effectively than administering them separately or simultaneously, and more effectively than administering a cannabinoid followed by LDN in a stepwise manner.

[0021] It was also shown that the amount of CB2 receptors was significantly increased in MCF7 cells after treatment with LDN. Without being bound by theory, this increase in CB2, the therapeutic target of cannabinoids such as CBD, is a favorable factor that contributes to the improved efficacy of CBD treatment.

[0022] One skilled in the art can perform stepwise administration of said therapeutic agents as described above. The present invention can be further understood with reference to the following definitions.

[0023] The term "naltrexone" as used herein 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-methanobenzofuro[3,2-e]isoquinolin-7-one) and its pharmacologically acceptable salts, solvates, hydrates, racemates, stereoisomers, clathrates, polymorphs, and prodrugs. The use of analogs thereof according to the present invention is also contemplated. Examples of suitable analogs include methylnaltrexone, naloxone, nalmefene, and nalorphine.

[0024] The naltrexone is typically in the form of its hydrochloride salt.

[0025] "Low dose naltrexone" (LDN) refers to naltrexone administered at "low" doses of less than 0.5 mg / kg, preferably less than 0.2 mg / kg, more preferably 0.01 mg / kg to 0.08 mg / kg, even more preferably 0.03 mg / kg to 0.06 mg / kg, and most preferably 0.04 mg / kg to 0.05 mg / kg. Typically, a low dose is up to 3 mg / day total per patient.

[0026] Examples of metabolites of naltrexone include 6-β-naltrexol, 2-hydroxy-3-methoxy-6β-naltrexol, and 2-hydroxy-3 methoxy-naltrexone.

[0027] The preferred metabolite of naltrexone is 6-β-naltrexol (6BN), which is referred to herein as 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-methanobenzofuro[3,2-e]isoquinoline-4a,7,9-triol) and its pharmacologically acceptable salts, solvates, hydrates, racemates, stereoisomers, clathrates, polymorphs, and prodrugs.

[0028] The 6BN or analogue may also be administered in a "low dose." In this context, a "low dose" may be as outlined above for naltrexone.

[0029] Cannabinoids are understood by those skilled in the art to be a class of compounds that includes endocannabinoids synthesized in animals as well as those abundantly produced by plants of the Cannabis genus. Synthetic compounds active at the CB receptor are also contemplated. The term as used herein may refer to any cannabinoid, but is preferably selected from the list comprising cannabidiol, cannabidiolic acid, cannabinol, cannabigerol, cannabivarin, tetrahydrocannabivarin, cannabidivarin, cannabichromene, arachidonoylethanolamine, 2-arachidonoylglycerol, 2-arachidonoylglyceryl ether, N-arachidonoyldopamine, virodamine, dronabinol, nabilone, rimonabant, R-(+)-Met-anandamide, WIN-55,212-2, HU-210, JWH-133, SR141716, SR144528, or combinations thereof, or pharmacologically acceptable salts, solvates, hydrates, racemates, stereoisomers, clathrates, polymorphs, prodrugs, and analogues thereof that produce equivalent effects.

[0030] In one embodiment, when the agent is 6-β-naltrexol, the 6-β-naltrexol is administered in an amount effective 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 one embodiment, the 6-β-naltrexol is administered in an amount effective to increase the plasma concentration of 6-β-naltrexol to within the range of 0.3 ng / ml to 3400 ng / ml, preferably within the range of 34 ng / ml to 3400 ng / ml, more preferably within the range of 340 ng / ml to 3400 ng / ml. An amount effective to achieve such an amount can be determined using any number of conventional methods known to those of skill in the art. For example, one of skill in the art can perform mass spectrometry on a plasma sample obtained from a subject to determine the increase in 6-β-naltrexol concentration in the sample after administration of an amount of 6-β-naltrexol. An effective amount is an amount determined to result in a desired increase in plasma concentration. Typically, naltrexol is administered in amounts up to 3 mg per patient per day.

[0031] The cannabinoids may be administered in conventional amounts based on the particular cannabinoid and patient details, in one embodiment the cannabinoids are administered in a dosage of 10 mg to 1000 mg, preferably 200 to 800 mg, more preferably 300 to 500 mg per day.

[0032] As used herein, the term "formulation" can refer to a substance or collection of substances in the form of one or more compositions intended for simultaneous or non-simultaneous use.

[0033] Although the method of administration is not limited to any of the therapeutic agents, in various embodiments of the invention, LDN and cannabinoids are administered orally, bucally, sublingually, nasally, pulmonary, intravenously, rectally, topically, and transdermally. LDN is preferably administered orally, and cannabinoids are preferably administered sublingually.

[0034] The contemplated 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 analogs is administered. In the second treatment phase, an effective amount of one or more cannabinoids is administered.

[0035] The second treatment phase is preferably initiated 1 to 7 days, more preferably 1 to 4 days, and most preferably 1 to 2 days after the initiation of the first treatment phase, where "day" refers to any consecutive 24-hour period.

[0036] In a further preferred embodiment of the present 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, LDN or cannabinoids are not administered. In one embodiment, the recovery phase has a duration of at least 2 days, preferably no more than 1 week, most preferably 2 days.

[0037] As used herein, the terms "treating" and "treatment" and "to treat" refer to both therapeutic measures that cure, slow, and / or halt the progression of a diagnosed condition or disease, and prophylactic or preventative measures that prevent and / or delay the onset of the targeted condition or disease. Thus, subjects in need of treatment include those already with the disease, those prone to having the disease, and those in which the disease is to be prevented. In some instances, a subject's tumor / cancer will be successfully "treated" in accordance with the present invention if the subject exhibits one or more of the following: a reduction in the number of cancer cells or the complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell invasion into surrounding organs, such as the spread of cancer 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 tumorigenicity, tumor frequency, or tumorigenic potential of the tumor; a reduction in the number or frequency of cancer stem cells within the tumor; differentiation of tumor-forming cells into non-tumorigenic cells; or a combination of multiple effects.

[0038] The term "subject" as used herein refers to any animal, including but not limited to humans, non-human primates, horses, dogs, cats, rodents, and other vertebrates, to be treated for cancer. The terms "subject" and "patient" are used interchangeably herein.

[0039] As used herein, the term "tumor / cancer" refers to any mass of tissue that results from excessive growth, proliferation, and / or survival of cells, and can be either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0040] The types of cancer that can be treated by the present invention are not limited in any way, but include, for example, carcinoma, sarcoma, adenocarcinoma, melanoma, neuro(blastoma, glioma), mesothelioma, and neoplastic diseases of the reticuloendothelial, lymphatic, or hematopoietic systems (myeloma, lymphoma, or leukemia). In a specific embodiment, the tumor may include lung adenocarcinoma, lung cancer, diffuse or interstitial gastric cancer, colon adenocarcinoma, prostate adenocarcinoma, esophageal cancer, breast cancer, pancreatic adenocarcinoma, ovarian adenocarcinoma, adrenal adenocarcinoma, and endometrial or uterine adenocarcinoma, but the type of cancer is preferably breast cancer.

[0041] In a preferred embodiment, the cancer to be treated is selected from brain, breast, colon, lung, prostate, and pancreatic cancer, and leukemia. The cancer to be treated is preferably breast cancer.

[0042] The definition of "breast cancer" is well known in medicine. Those skilled in the art recognize that breast cancer refers to any malignant tumor of male or female breast tissue, such as carcinoma or sarcoma. Specific forms 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 neoplasm, or invasive lobular carcinoma (ILC).

[0043] The term "cancer cell" as used herein refers to a cell or an immortalized cell line derived from a tumor or cancer.

[0044] In one aspect of the present invention, a method is provided for determining the suitability of a subject with cancer and already treated with LDN for treatment with cannabinoids.In this aspect, a sample is obtained from the subject and contacted with a CB2 specific probe.The CB2 concentration in the sample is thereby determined and compared with a baseline measurement made before the first treatment stage.

[0045] The term "suitability" as used herein refers to the property of having a higher probability of success in the treatment as defined above compared to subjects deemed unsuitable by the test. A subset of subjects who are tested by the method envisaged in one aspect of the present invention are deemed unsuitable for the second stage of treatment. Nothing in the present invention in any way prohibits the use of such subjects. The method of determining suitability is intended to be considered by those skilled in the art using intuition and judgment in conjunction with other tests known to those skilled in the art.

[0046] In certain embodiments, the biological "sample" obtained from a subject for use in the method is blood, plasma, serum, lymph, tissue, or cells from a tissue sample, but preferably the sample is a tumor biopsy obtained from the subject.

[0047] As used herein, a "probe" refers to any moiety that, when contacted with a sample obtained from a subject, allows for the measurement of the concentration of CB2 in the sample in some 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 by methods known to those of skill in the art, thereby allowing the concentration of CB2 in the original sample to be determined. can.

[0048] In another aspect, there is provided the use of naltrexone, a metabolite thereof, or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene, and nalorphine; and a cannabinoid; in the manufacture of a medicament for administration as part of a "combination therapeutic regimen", wherein said "combination therapeutic regimen" refers to a regimen consisting of a first therapeutic phase, and preferably a recovery phase, and a second therapeutic phase, as defined above.

[0049] The invention will now be described with reference to the following non-limiting examples. EXAMPLES

[0050] Example 1 One illustrative, non-limiting experiment was performed on cultures of MCF7 cells. Briefly, either 10 nM LDN or 1 μM or 10 μM 6BN were administered in Cultures were treated for 2 days in vitro, after which the expression levels of BAD, p21, opioid receptors kappa and μ, CB1, CB2, and GAPDH (as a loading control) were analyzed by Western blotting followed by quantification by band densitometry.

[0051] In detail, MCF7 cells were seeded at a density of 1 × 105 / well in 6-well plates and allowed to attach overnight. Naltrexone (10 nM) or 6-β-naltrexol (1 μM or 10 μM) was added to the cells, after which the cells were harvested and subjected to Western blot analysis. As primary probes, specific antibodies raised against BAD, p21, OPRK1, OPRK2, CBR-1, and CBR-2 were used. BAD is a Bcl2-associated death promoter. It is a proapoptotic protein whose upregulation in cancer cells increases the propensity of cells to be killed by various therapies. Anti-GAPDH was used as a loading control. All antibodies were used at a dilution of 1:1000, followed by the appropriate HRP-conjugated secondary antibody at a dilution of 1:1000. Bands were visualized using the SuperSignal chemiluminescent detection system, and densitometry of bands was performed using Adobe Photoshop CS3, v10.0 and normalized to loading controls.

[0052] All three treatments increased the levels of CB2 and BAD compared to no drug controls. For CB2, this increase was greatest with 10 μM 6BN treatment. See FIG. 1.

[0053] Example 2 Another non-limiting experiment for illustrative purposes was carried out on MCF7 cell cultures. Briefly, cultures were treated with LDN or 6BN for 2 days, and then administered with cannabinoids for another 2 days. Cell count and viability were assessed on the fourth day. The order of administration of each treatment was reversed, and the experiment was repeated.

[0054] In detail, MCF7 cells were seeded in 6-well plates at a density of 1.5 x 104 / well and allowed to attach. The cells were then cultured with naltrexone (10 nM), 6-β-naltrexol (10 μM), or cannabidiol (10 μM). After 48 hours, the drug-containing medium was removed and the cells were gently rinsed with drug-free medium. Fresh medium containing either naltrexone (10 nM), 6-β-naltrexol (10 μM), or cannabidiol (10 μM) was then added to the cells as indicated in the graph. After an additional 4 After 8 hours, cell number and viability were assessed by the percentage of live and dead cells as identified by trypan blue dye exclusion, and data were then pooled to compare the effect of sequence on overall efficacy.

[0055] As can be seen in Figure 2, schedules in which LDN or 6BN was used prior to the cannabinoid were more effective at inhibiting cancer growth.

[0056] literature Cridge, B. & Rosengren, R (2013) Critical appraisal of the potential use of cannabinoids in cancer management. Cancer Management and Research 2013:5 301-313

Claims

1. 1. A method for determining the suitability of a subject having cancer for treatment with a cannabinoid in a second treatment phase, the subject having been administered in a first treatment phase a therapeutically effective amount of naltrexone; a metabolite of naltrexone selected from the group consisting of 6-β-naltrexol, 2-hydroxy-3-methoxy-6β-naltrexol, and 2-hydroxy-3 methoxy-naltrexone; or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene, and nalorphine; the method comprising: i. contacting a sample obtained from said subject after or during a recovery phase with a probe specific for CB2; ii. determining the concentration of CB2 in the sample; iii. comparing the concentration of CB2 in the sample to a concentration of CB2 determined from a sample obtained from the subject prior to the first treatment phase; wherein if the CB2 concentration increases at least 2-fold after the first treatment phase, the subject is suitable for the second treatment phase.

2. The method of claim 1 , wherein the sample is a tumor biopsy sample.

3. The method of claim 1 or 2, wherein the subject has breast cancer.

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