Cancer therapeutic agent comprising naltrexone and cannabinoid

Abstract

The present invention is based on the discovery that inhibition of cancerous cell proliferation by cannabinoids is more effectively achieved by treatment in combination with low doses of naltrexone (LDN) or 6-beta-naltrexone (6BN) (a metabolite of naltrexone). There is provided a pharmaceutical composition comprising naltrexone or a metabolite thereof or an analog thereof for use in the treatment of cancer in a subject wherein a therapeutically effective amount of naltrexone or a metabolite thereof or an analog of any one thereof is administered to the subject in a first treatment stage wherein after the first treatment stage, a therapeutically effective amount of naltrexone or a metabolite thereof or an analog of any one thereof is administered to the subject. A therapeutically effective amount of cannabinoid is administered to the subject in the second treatment phase, and wherein a chemotherapeutic agent is administered to the subject before, during or after treatment.

Description

[0001] This application is a divisional application of the PCT international application PCT / GB2020 / 050638 filed on March 13, 2020, which entered the Chinese national phase on September 14, 2021, and whose Chinese patent application number is 202080021221.0 and invention name is “Cancer therapeutic agent containing naltrexone and cannabinoids”. Technical Field

[0002] The present invention relates to drug dosing regimens and pharmaceutical compositions for cancer treatment. Background Art

[0003] Cannabinoids are a class of phenolic compounds produced in large quantities in the cannabis plant.

[0004] However, recent studies have shown that certain cannabinoids have therapeutic value for a wide variety of conditions, such as inflammatory diseases, neurodegenerative and psychiatric disorders, chronic pain, anxiety, and PTSD. While cannabinoids are currently used to combat wasting, vomiting, and nausea associated with cancer treatment, there is evidence that certain 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.

[0005] Endocannabinoid-mediated signaling systems operate in mammals and, on the basis of evidence, probably 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.

[0006] Pharmacological evidence to date suggests that the psychoactive effects of cannabinoid ingestion are primarily caused by activation of CB1, whereas therapeutic effects are primarily mediated by CB2. Therefore, an ideal therapeutic molecule would activate CB2 preferentially over CB1, mitigating unwanted psychoactive side effects. Cannabidiol (CBD), a component of cannabis extract, is a cannabinoid with this binding property that has recently attracted considerable attention, but its complete mechanism of action is still being elucidated.

[0007] However, in general, natural cannabinoids are not designed to show high specificity for one or other receptor, 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 a variety of tumor implantation mouse models.

[0008] Sometimes, receptors in one signaling pathway, in response to a specific ligand, can have their expression levels or downstream effects altered by changes in the signaling output of another, seemingly independent signaling pathway. Such cross-regulation has been demonstrated, for example, between signaling pathways following activation of the growth hormone and insulin receptors. Summary of the invention

[0009] The present inventors have discovered that treatment with cannabinoids and chemotherapeutic agents to inhibit the proliferation of cancer cells is more effectively achieved by combining treatment with low-dose naltrexone (LDN) or 6-β-naltrexol (6BN), a metabolite of naltrexone. They have also discovered that the effectiveness of this treatment surprisingly depends on the order in which LDN or 6BN and the cannabinoid are administered, with the most effective regimen being a phase of treatment with LDN or 6BN followed by a phase of treatment with the cannabinoid.

[0010] The inventors have also discovered that treatment with LDN or 6BN significantly increases the levels of CB2 receptors in cancer cells, making cannabinoids more effective.

[0011] According to a first aspect of the present invention, there is provided a pharmaceutical composition comprising naltrexone or a metabolite thereof or an analog selected from methylnaltrexone, naloxone, nalmefene and nalorfene for use in treating cancer in a subject, wherein a chemotherapeutic agent is administered to the subject before, during or after the treatment, and wherein in a first treatment phase a therapeutically effective amount of naltrexone or a metabolite or analog thereof is administered to the subject, and wherein after the first treatment phase, in a second treatment phase a therapeutically effective amount of a cannabinoid is administered to the subject.

[0012] According to a second aspect of the present invention, there is provided a pharmaceutical composition comprising a cannabinoid for use in treating cancer in a subject, wherein the subject is characterized by having undergone a first treatment phase during which the subject has been administered a therapeutically effective amount of naltrexone or a metabolite thereof or an analog selected from methylnaltrexone, naloxone, nalmefene and nalorfene, and wherein after the first treatment phase, the subject will be administered a therapeutically effective amount of the cannabinoid, and wherein the subject is administered a chemotherapeutic agent before, during or after treatment.

[0013] According to a third aspect of the present invention, there is provided a formulation comprising naltrexone or a metabolite thereof or an analog selected from methylnaltrexone, naloxone, nalmefene and nalorfene and a cannabinoid and a chemotherapeutic agent for treating cancer in a subject, wherein the naltrexone or a metabolite or analog thereof is provided in a therapeutically effective amount to be administered in a first treatment phase, wherein the cannabinoid is provided in a therapeutically effective amount to be administered in a second treatment phase following the first treatment phase, and wherein the chemotherapeutic agent is provided in a therapeutically effective amount before, during or after the treatment.

[0014] According to a fourth aspect of the present invention, there is provided a method for determining the suitability of a subject with cancer for treatment with a cannabinoid in a second treatment phase, the subject being characterized by having undergone a first treatment phase as defined above, the method comprising the steps of:

[0015] i. contacting a sample obtained from the subject with a CB2-specific probe after or during the recovery phase;

[0016] ii. determining the concentration of CB2 in the sample; and

[0017] iii. comparing the concentration of CB2 in the sample with the concentration of CB2 determined in a sample obtained from the subject prior to the first treatment period,

[0018] wherein the subject is suitable for a second treatment phase if the concentration of CB2 increases at least two-fold after the first treatment phase, and wherein the subject is or has been administered a chemotherapeutic agent.

[0019] According to a fifth aspect of the present invention, there is provided the use of naltrexone or a metabolite thereof or an analog selected from methylnaltrexone, naloxone, nalmefene and nalorfene 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 combined treatment regimen, the treatment regimen comprising a first treatment phase followed by a second treatment phase, wherein a cannabinoid is administered to the subject in the second treatment phase, and wherein a chemotherapeutic agent is administered to the subject before, during or after the treatment regimen.

[0020] According to a sixth aspect of the present invention, there is provided the use of a cannabinoid in the manufacture of a medicament for treating cancer in a subject, wherein the medicament is to be administered in a second treatment phase of a combined treatment regimen, the treatment regimen comprising a first treatment phase followed by a second treatment phase, wherein in the first treatment phase naltrexone or a metabolite thereof or an analogue selected from methylnaltrexone, naloxone, nalmefene and nalorfene is to be administered to the subject, and wherein a chemotherapeutic agent is administered to the subject before, during or after the treatment regimen.

[0021] According to a seventh aspect of the present invention, there is provided the use of naltrexone or a metabolite thereof or an analogue selected from 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 of which are used to treat cancer, wherein the first and second medicaments are to be administered to a subject suffering from cancer in a combined treatment regimen, the treatment regimen comprising a first treatment phase followed by a second treatment phase, wherein the first medicament is to be administered to the subject during the first treatment phase and the second medicament is to be administered to the subject in the second treatment phase, and wherein a chemotherapeutic agent is administered to the subject before, during or after the treatment regimen.

[0022] According to an eighth aspect of the present invention, there is provided a method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of naltrexone or a metabolite thereof or an analog selected from methylnaltrexone, naloxone, nalmefene and nalorfene in a first treatment phase, and then administering to the subject a therapeutically effective amount of a cannabinoid in a second treatment phase, and wherein a chemotherapeutic agent is administered to the subject before, during or after the treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention is described with reference to the accompanying drawings, in which:

[0024] Figure 1 The effects of two-day treatment of MCF7 breast cancer cell line cultures with 10 nM naltrexone (LDN), 1 μM 6-β-naltrexol (6-1), or 10 μM 6-β-naltrexol (6-2) are shown. Levels of Bcl2-associated death promoter (BAD), p21 protein, opioid receptors kappa 1 (OPRK1) and mu 1 (OPRM1), and cannabinoid receptors CBR1 (CB1) and CBR2 (CB2) were visualized by western blot (left) using GAPDH as a loading control and quantified by densitometry analysis relative to GAPDH (right).

[0025] Figure 2 The effects of various two-stage treatments on MCF7 cell proliferation and survival are shown. Each treatment lasted four days and consisted of two days of treatment with the first agent (UN = control, LDN = low-dose naltrexone at 10 nM, CBD = cannabidiol, 6BN = 6-β-naltrexol), followed by the introduction of the second agent for another two days.

[0026] Figure 3 shows the effects of various two-stage treatments on A549 cell proliferation (3A) and survival (3B) according to Example 3 (0 = no treatment; LC = LDN and CBD; G = gemcitabine; P = oxaliplatin).

[0027] Figure 4 shows the effects of various two-stage treatments on HCT116 cell proliferation (4A) and survival (4B) according to Example 3 (0 = no treatment; LC = LDN and CBD; G = gemcitabine; P = oxaliplatin). DETAILED DESCRIPTION

[0028] The present invention provides a specific therapeutic regimen for treating cancer in a subject, wherein a cannabinoid is administered after administration of a low dose of naltrexone (LDN), a metabolite of naltrexone, or an analog selected from methylnaltrexone, naloxone, nalmefene, and nalorfene, and wherein a chemotherapeutic agent is administered to the subject before, during, or after the therapeutic regimen.

[0029] The present inventors have discovered that staged administration of LDN followed by cannabinoids has a priming effect on human immune cells and cancer cells, and thus its combination with a chemotherapeutic agent is more effective in inhibiting cancer cell proliferation than either single or simultaneous administration or staged administration of cannabinoids followed by LDN or continuous administration of a chemotherapeutic agent alone.

[0030] It was also found that CB2 receptor levels in MCF7 cells increased significantly after treatment with LDN. Without wishing to be bound by theory, this increase in CB2, which is a therapeutic target of cannabinoids such as CBD, is a plausible contributing factor to the increased effectiveness of treatment with CBD.

[0031] Those skilled in the art will be able to perform the phased administration of therapeutic agents as described. The present invention may be further understood with reference to the following definitions:

[0032] 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]isoquinolin-7-one), and pharmaceutically acceptable salts, solvates, hydrates, racemates, stereoisomers, clathrates, polymorphs and prodrugs thereof. Analogs thereof are also contemplated for use according to the present invention. Suitable analogs include methylnaltrexone, naloxone, nalmefene and nalorfene.

[0033] Naltrexone is usually in the form of its hydrochloride salt.

[0034] "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 low dose is up to a total of 3 mg per patient per day.

[0035] Naltrexone metabolites include 6-β-naltrexol, 2-hydroxy-3-methoxy-6β-naltrexol, and 2-hydroxy-3-methoxy-naltrexone.

[0036] A 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-methylenebenzofurano[3,2-e]isoquinoline-4a,7,9-triol), and pharmaceutically acceptable salts, solvates, hydrates, racemates, stereoisomers, clathrates, polymorphs and prodrugs thereof.

[0037] 6BN or the like can also be administered in a "low dose." In this context, a "low dose" can be the same as the low dose listed above for naltrexone.

[0038] Cannabinoids are a class of compounds known to those skilled in the art and include those produced in large quantities by plants of the genus Cannabis, as well as endogenous cannabinoids synthesized in animals. Synthetic active compounds directed against CB receptors are also contemplated. As used herein, the term may refer to any cannabinoid, but is preferably selected from the group consisting of cannabidiol, cannabidiolic acid, cannabinol, cannabigerol, cannabivarin, tetrahydrocannabivarin, cannabidiol, cannabicyclol, anandamide, 2-arachidonic acid glycerol, 2-arachidonic acid glycerol ether, N-arachidonic dopamine, virodhamine, dronabinol, nabilone, rimonabant, R-(+)-methylarachidonic acid aminoethanol (R-(+)-Met-anandamide), WIN-55, 212-2, HU-210, JWH-133, SR141716, SR144528 or a combination thereof, or a pharmaceutically acceptable salt, solvate, hydrate, racemate, stereoisomer, clathrate, polymorph, prodrug, and analogs thereof that produce equivalent effects.

[0039] As used herein, "chemotherapy" and "chemotherapeutic" have their conventional meanings in the art. The term "anti-cancer agent" is used synonymously with "chemotherapeutic agent." For the avoidance of doubt, in this context, cannabinoids are not anti-cancer agents.

[0040] In certain embodiments, chemotherapy involves the administration of an anticancer agent selected from the group consisting of a PI3-kinase inhibitor, an AKT inhibitor, a taxane, an antimetabolite, an alkylating agent, a cell cycle inhibitor, a topoisomerase inhibitor, and a cytotoxic antibody.

[0041] When the chemotherapeutic agent is a PI3-kinase inhibitor, suitable examples include, but are not limited to, wortmannin, LY294002, demethoxychloroquine, IC87114, NVP-BEZ235, BAY 80-6946, BKM120, GDC-0941, GDC-9080; including combinations thereof; and pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, inclusion compounds and prodrugs of any of the foregoing.

[0042] When the anticancer agent is an AKT inhibitor, suitable examples include, but are not limited to, MK-2206, GSK690693, perifosine, PHT-427, AT7867, honokiol, PF-04691502; including combinations thereof; and pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, inclusion compounds and prodrugs of any of the foregoing.

[0043] When the anticancer agent is a taxane, suitable examples include, but are not limited to, paclitaxel and docetaxel; including combinations thereof; and pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates and prodrugs of any of the foregoing.

[0044] When the anticancer agent is an antimetabolite, suitable examples include, but are not limited to, methotrexate, 5-fluorouracil, capecitabine, cytosine arabinoside (Ara-C), gemcitabine, 6-thioguanosine, pentostatin, azathioprine, 6-mercaptopurine, fludarabine and cladribine; including combinations thereof; and pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, inclusion compounds and prodrugs of any of the foregoing.

[0045] When the anticancer agent is an alkylating agent, suitable examples include, but are not limited to, chloroethylamine, cyclophosphamide, ifosfamide, trofosfamide, melphalan (L-myosin), chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine (BCNU), streptozocin (streptozotocin), dacarbazine (DTIC; dimethyltriazene imidazole carboxamide) temozolomide and oxaliplatin; including combinations thereof; and pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, inclusion compounds and prodrugs of any of the foregoing.

[0046] When the anticancer agent is a cell cycle inhibitor, suitable examples include, but are not limited to, epothilone, vincristine, vinblastine, UCN-01, 17AAG, XL844, CHIR-124, PF-00477736, CEP-3891, flavopiridol, berberine, P276-00, terameprocol, isoflavone daidzein, BI2536, BI6727, GSK461364, cyclosporine, ON-01910, NMS-P937, TAK-960, Ispins, Monastrol, AZD485073, LY2523355, ARRY-520, MK-0731, SB743921, GSK923295, lonafarnib, proTAME, bortezomib, MLN9708, ONX0912, CEP-18770; including combinations thereof; and any of the above drugs pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates and prodrugs of any of the foregoing; particularly suitable examples of cell cycle inhibitors include, but are not limited to, Hespaeradin, ZM447439, VX680, MLN-8054, PHA-739358, AT-9283, AZD1152, MLN8237, ENMD2076, SU6668; including combinations thereof; and other Aurora kinase inhibitors; and pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, clathrates and prodrugs of any of the foregoing.

[0047] The anticancer agent can be a checkpoint inhibitor. Checkpoint inhibitors are a type of cancer immunotherapy, and currently approved inhibitors target the molecules CTLA4, PD-1, and PD-L1.

[0048] As defined herein, "simultaneous" administration includes administration of the substances within about 2 hours or about 1 hour or less of each other, even more preferably simultaneously.

[0049] As defined herein, "separate" administration includes administration of the substances more than about 12 hours, or about 8 hours, or about 6 hours, or about 4 hours, or about 2 hours apart.

[0050] As defined herein, "sequentially" administration includes administration of the substances each time in multiple aliquots and / or doses and / or at different times.

[0051] The anticancer agent may be administered to the subject before, during or after the treatment regimen. The anticancer agent may be administered to the subject at any time during the treatment regimen, such as during the first treatment phase or during the second treatment phase.

[0052] During the first treatment phase, the anticancer agent may be administered to the patient before and / or after the administration of naltrexone, its metabolite or its analogue. The anticancer agent and naltrexone, its metabolite or its analogue may be administered simultaneously, sequentially, separately, preferably simultaneously. The anticancer agent may also be continued to be administered to the patient after cessation of treatment with naltrexone, thus administering the anticancer agent in both the first treatment phase and the second treatment phase.

[0053] During the second treatment phase, the anticancer agent may be administered to the patient before and / or after the administration of the cannabinoid. The anticancer agent and the cannabinoid may be administered simultaneously, sequentially, separately, preferably simultaneously. The anticancer agent may also continue to be administered to the patient after the cessation of treatment with the cannabinoid, thus also administering the anticancer agent after the treatment regimen.

[0054] In another embodiment, the treatment may further include administering vitamin D to the subject. Vitamin D may be administered to the subject before, during, or after the treatment regimen. Vitamin D may be administered to the subject at any time during the treatment regimen, such as during the first treatment phase or during the second treatment phase. Preferably, vitamin D should be administered to the patient before the start of the disclosed treatment regimen.

[0055] During the first treatment phase, vitamin D may be administered to the patient before and / or after administration of naltrexone or its metabolite or analog thereof. Vitamin D and naltrexone or its metabolite or analog thereof may be administered simultaneously, sequentially, separately, preferably simultaneously. Vitamin D may also be continued to be administered to the patient after cessation of treatment with naltrexone, thus administering vitamin D during both the first treatment phase and the second treatment phase.

[0056] During the second treatment phase, vitamin D may be administered to the patient before and / or after administration of the cannabinoid. Vitamin D and cannabinoids may be administered simultaneously, sequentially, separately, preferably simultaneously. Vitamin D may also continue to be administered to the patient after cessation of treatment with cannabinoids, and thus may also be administered after the treatment regimen.

[0057] Preferably, during the treatment regimen, the subject should be administered vitamin D daily to maintain correct vitamin D levels during all phases of treatment.

[0058] As used herein, "vitamin D" refers to any intermediate or product of vitamin D and vitamin D metabolic pathways that produces active metabolites that can enhance cell growth inhibition. Metabolites can refer to vitamin D precursors that can be incorporated into the naturally occurring vitamin D synthetic pathway in a subject for treatment of the present invention. Alternatively, metabolites can refer to molecules obtained by anabolic or catabolic methods utilizing vitamin D. Non-limiting examples of vitamin D metabolites include ergocalciferol, cholecalciferol, calcidiol and calcitriol, 1a-hydroxycholecalciferol, 25-hydroxycholecalciferol, 1a, 25-hydroxycholecalciferol, 24, 25-hydroxycholecalciferol. "Active" metabolites are metabolites that can be used in the context of the present invention. The dosage regimen of vitamin D or its active metabolites is well known to those skilled in the art. The term vitamin D also includes pharmaceutically acceptable salts of any of the above. A vitamin D metabolite particularly suitable for use in the present invention is calcitriol.

[0059] In certain embodiments, where the agent is 6-β-naltrexol, 6-β-naltrexol will be 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 certain embodiments, 6-β-naltrexol will be administered in an amount effective to increase the plasma concentration of 6-β-naltrexol to within the range of 0.3 ng / ml to 3,400 ng / ml, preferably within the range of 34 ng / ml to 3,400 ng / ml, more preferably within the range of 340 ng / ml to 3,400 ng / ml. An effective amount to achieve this amount can be determined using any number of routine techniques known to those skilled in the art. For example, one skilled in the art can perform mass spectrometry analysis on a plasma sample obtained from a subject to determine the increase in 6-β-naltrexol concentration in the sample after administration of 6-β-naltrexol. An effective amount is an amount determined to achieve the desired increase in plasma concentration. Typically, naltrexol will be administered in an amount up to 3 mg per patient per day.

[0060] Cannabinoids can be administered in conventional amounts based on the specific cannabinoid and patient details. In certain embodiments, cannabinoids will be administered daily in a dosage of between 10 mg and 1000 mg, preferably between 200 and 800 mg, more preferably between 300 and 500 mg.

[0061] The chemotherapeutic agent can be administered in conventional amounts depending on the particular chemotherapeutic agent and the particular circumstances of the patient. In certain embodiments, the chemotherapeutic agent is administered daily at a dose used in standard practice.

[0062] In certain embodiments, a vitamin D product is administered to a patient in an amount sufficient to increase the subject's blood concentration of vitamin D to at least 40 ng / ml, more preferably at least 50 ng / ml. Preferably, the vitamin D solution concentration is increased to 40 to 220 ng / ml, more preferably, the vitamin D solution concentration is increased to 40 to 90 ng / ml.

[0063] The technician can determine the sufficient amount for administration by routine assessment of certain parameters of the patient, such as but not limited to age, weight, sex, medical history and / or other lifestyle factors, including smoking, drinking and exercise level. In addition, the technician can determine whether the dose is sufficient to increase the blood concentration of vitamin D to a sufficient concentration by performing routine biochemical and analytical measurements on biological samples obtained from the subject. Preferably, the sample subjected to the analysis is blood. Such well-known measurement examples include, but are not limited to, mass spectrometry, in which the level of vitamin D or its active metabolite can be quantitatively measured. Therefore, a sufficient amount refers to an amount that achieves the desired blood concentration of vitamin D. The desired concentration can be achieved after a single or repeated administration of a certain dose of vitamin D or its active metabolite. When a vitamin D product and a naltrexone product are administered simultaneously, it is not important whether the blood concentration of vitamin D is within the desired range before the naltrexone product is administered, provided that the amount of the vitamin D product administered is sufficient to increase the blood concentration of vitamin D to the desired concentration range. Other methods for determining the concentration of vitamin D or its active metabolite in a biological sample obtained from a patient are well known to the technician. In certain embodiments, the amount of vitamin D sufficient to increase the blood concentration of vitamin D to more than a certain level is referred to as the "therapeutically effective amount" of the vitamin D product.

[0064] As used herein, "preparation" may refer to a substance or a collection of substances in the form of one or more compositions, intended for simultaneous or non-simultaneous use.

[0065] For any therapeutic agent, the method of administration is not particularly limited, but in various embodiments of the present invention, LDN, cannabinoids and chemotherapeutic agents are administered by oral, buccal, sublingual, nasal, pulmonary, intravenous, rectal, topical and transdermal routes. Oral administration is preferred for LDN, sublingual administration is preferred for cannabinoids, and oral or intravenous administration is preferred for chemotherapeutic agents.

[0066] 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 thereof is administered. In the second treatment phase, an effective amount of one or more cannabinoids is administered. A chemotherapeutic agent may be administered before, during, or after the treatment regimen.

[0067] In a preferred embodiment, the chemotherapeutic agent is administered after the treatment regimen. This can be advantageous because it enables the immune cells and cancer cells to be "primed" (activated) and thus more effectively reduce the cancer when the chemotherapeutic agent is subsequently administered.

[0068] Preferably, the second treatment phase begins 1 to 7 days after the start of the first treatment phase, more preferably 1 to 4 days, most preferably 1 to 2 days, where "day" is a continuous period of 24 hours.

[0069] In another preferred embodiment of the present invention, there is a "recovery phase" between the end of the first treatment phase and the start of the second treatment phase. During the recovery phase, no LDN, cannabinoids or chemotherapeutic agents are administered. In one embodiment, the duration of the recovery phase is at least two days, preferably no more than 1 week apart, and most preferably two days in duration.

[0070] As used herein, the terms "treating" and "treatment" and "to treat" refer to therapeutic measures that cure, slow down and / or stop the progression of a diagnosed pathological condition or disease, and also refer to prophylactic measures or preventative measures that prevent and / or slow down the development of a target pathological condition or disease. Thus, those subjects in need of treatment include those already suffering from the condition, those susceptible to the condition, and those for which the condition is to be prevented. In some cases, a subject's tumor / cancer is successfully "treated" according to the present invention if the subject exhibits one or more of the following: a reduction in the number of cancer cells or a complete absence; a reduction in tumor size; inhibition or absence of infiltration of cancer cells into peripheral organs (including, for example, spread of cancer to soft tissue and bone); inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; reduced morbidity and mortality; reduced tumorigenicity, frequency of tumorigenesis, or ability to develop tumors; a reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells into a non-tumorigenic state; or some combination of the effects.

[0071] As used herein, the term "subject" refers to any animal that will be the 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.

[0072] As used herein, the term "tumor / cancer" refers to any mass of tissue resulting from excessive cell growth, proliferation and / or survival, benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0073] The types of cancers that can be treated by the present invention are not limited in any way and include, for example, carcinoma, sarcoma, adenocarcinoma, melanoma, neurocarcinoma (blastoma, glioma), mesothelioma and reticuloendothelial, lymphoid 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 cancer type is preferably breast cancer.

[0074] In a preferred embodiment, the cancer to be treated is selected from brain cancer, breast cancer, colon cancer, lung cancer, prostate cancer and pancreatic cancer and leukemia. The cancer to be treated is preferably breast cancer, lung cancer or colon cancer. The cancer to be treated is most preferably breast cancer.

[0075] 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 male or female breast tissue, including, for example, carcinomas and sarcomas. Specific embodiments of breast cancer include ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), or mucinous carcinoma. Breast cancer also refers to invasive ductal (IDC), lobular neoplasia, or invasive lobular carcinoma (ILC).

[0076] Similarly, the definitions of "lung cancer" and "colon cancer" are well known in the medical sciences. The skilled artisan will understand that lung cancer and colon cancer refer to any malignant tumor of the lung and colon tissues of males or females, respectively, including, for example, carcinomas and sarcomas.

[0077] As used herein, the term "cancer cell" refers to a cell or immortalized cell line derived from a tumor or cancer.

[0078] In one aspect of the invention, a method for determining whether a subject having cancer and having been treated with LDN is suitable for treatment with a cannabinoid is provided. The subject may have been treated with a chemotherapeutic agent. In this regard, a sample is obtained from the subject and contacted with a CB2-specific probe. The concentration of CB2 in the sample is thereby determined and compared with a reference measurement prior to a first treatment phase.

[0079] As used herein, "suitability" refers to the property of having a high likelihood of treatment success as defined above, compared to those subjects tested as unsuitable. A subset of subjects tested by the method contemplated by an aspect of the present invention will be considered unsuitable for the second phase of treatment. No part of the present invention is intended to be used for these subjects. The method of determining suitability is intended to be considered by those skilled in the art in conjunction with other tests known to them and using their intuition and judgment.

[0080] 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. However, preferably, the sample is a tumor biopsy obtained from a subject.

[0081] As used herein, a "probe" is any moiety that, when contacted with a sample obtained from a subject, allows the concentration of CB2 in the sample to be measured in some manner. In one embodiment, the probe is an antibody or other CB2 binding molecule that is 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.

[0082] In another aspect, there is provided the use of naltrexone, its metabolite or an analogue selected from methylnaltrexone, naloxone, nalmefene and nalorfene, and a cannabinoid in the manufacture of a medicament to be administered as part of a "combination therapy regimen", wherein "combination therapy regimen" as used herein refers to a regimen consisting of a first treatment phase as defined above, preferably a recovery phase, and a second treatment phase, and wherein the combination therapy regimen comprises administering a chemotherapeutic agent in the manner described above.

[0083] The invention will now be described with reference to the following non-limiting examples.

[0084] Example

[0085] Example 1

[0086] An illustrative and non-limiting experiment was performed on cultures of MCF7 (breast cancer) cells. Briefly, 10 nM LDN or 1 μM or 10 μM 6BN was given to the cultures in vitro for two days, after which the expression levels of BAD, p21, opioid receptors kappa and mu, CB1, CB2, and GAPDH (as a loading control) were analyzed by Western blotting, followed by quantification by density measurement of the bands.

[0087] Completely, MCF7 cells were seeded into 6-well plates at a density of 1x 105 / well and left 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 western blot analysis. Primary probing was performed with specific antibodies generated against BAD, p21, OPRK1, OPRK2, CBR-1, and CBR-2. BAD is the Blc2-associated death promoter, a pro-apoptotic protein that, if upregulated in cancer cells, increases their propensity to be killed by various therapies. Anti-GAPDH was used as a loading control. All antibodies were used at a dilution of 1:1000, and then the appropriate HRP-conjugated secondary antibodies were also used at a dilution of 1:1000. Bands were visualized using the SuperSignal Chemiluminescent Detection System, and densitometry of band intensities was determined using Adobe Photoshop CS3, v10.0 and normalized to the loading control.

[0088] In response to all three treatments, CB2 and BAD levels increased relative to untreated controls. For CB2, the increase was greatest in response to 10 μM 6BN. This can be seen in Figure 1 It can be seen in.

[0089] Example 2

[0090] Another illustrative and non-limiting experiment was performed on cultures of MCF7 (breast cancer) cells. Briefly, cultures were treated with LDN or 6BN for two days and then treated for another two days after cannabinoid administration. Cell number and viability were assessed on the fourth day. The order of administration of each treatment was reversed and the experiment was repeated.

[0091] Completely, MCF7 cells were seeded into 6-well plates at a density of 1.5x104 / well and left to adhere. Cells were then cultured with naltrexone (10nM), 6-β-naltrexol (10μM) or cannabidiol (10μM). The drug-containing medium was removed after 48h and the cells were gently rinsed with drug-free medium. Fresh medium was then added to cells supplemented with naltrexone (10nM), 6-β-naltrexol (10μM) or cannabidiol (10μM), as shown. Cell number and viability were assessed 48 hours later, and the percentage of live and dead cells was distinguished by trypan blue dye exclusion. The data were then merged to compare the effects of the order on the overall effect.

[0092] As from Figure 2 As can be seen in the Figure 3, the timing of LDN or 6BN administration before cannabinoids was found to have a greater effect on inhibiting cancer proliferation.

[0093] Example 3

[0094] A549 (lung cancer) and HCT116 (colon cancer) cells were incubated with drugs according to the schedule in Table 1. The schedule lasted for a total of 96 hours.

[0095] Treatment was divided into two blocks, each lasting 48 hours. Due to the complexity of conducting cell-based tests with three different drugs in three different treatment phases, CBD and LDN were administered together in one treatment phase, while the chemotherapy agent was administered in the other treatment phase.

[0096] • Between blocks there is a washout phase where the spent medium containing the drug is removed, followed by washing the cells and then adding fresh medium containing the next treatment block.

[0097] • Chemotherapeutic agents were gemcitabine (GEM) or oxaliplatin (OXP) and were used at ~IC20 concentrations (1 μM). Low-dose naltrexone (LDN) was used at 10 nM, and cannabinoids (CBD) were used at 1 μM. "0" means no drug administration and was used as a control.

[0098] Cell number and percentage of cell viability were assessed at 96 hours. Data were then combined to compare the effect of the sequence on the overall effect.

[0099] Block 1 (0-48 hours) Block 2 (48-96 hours) 0 0 LDN + CBD LDN + CBD 0 GEM LDN + CBD 0 LDN + CBD GEM GEM 0 0 LDN + CBD GEM LDN + CBD GEM GEM 0 OXP(P) LDN + CBD OXP(P) OXP(P) 0 OXP(P) LDN + CBD OXP(P) OXP(P)

[0100] Table 1: Shows the timeline of the test.

[0101] As shown in Figures 3 and 4, it is effective to use CBD+LDN before chemotherapy.In 96 hours, using chemotherapeutics after LDN+CBD produces improvement or similar effect than only continuous use chemotherapy (it thus uses chemotherapeutics total amount higher than combined treatment).These results have been observed for two cell types (A549 and HCT116) and two chemotherapeutics (GEM and OXP) tested.Compared with the cells not receiving pre-treatment, using LDN+CBD treatment cells before chemotherapy reduces cell number and cell survival rate % reduction.

[0102] Thus, Example 3 demonstrates that pretreatment produces an effect similar to 96 hours of chemotherapy. This allows for an effective treatment regimen that maintains better or comparable overall efficacy compared to continuous chemotherapy, but uses significantly reduced chemotherapy doses, which is beneficial because it can reduce chemotherapy-related toxicity experienced by patients.

[0103] References

[0104] 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. Use of a pharmaceutical composition in the preparation of a medicament for improving the effect of cannabidiol and / or a chemotherapeutic agent in treating cancer in a subject, the pharmaceutical composition include: (a) naltrexone, or (b) its metabolites, or (c) an analog selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorfene, wherein the subject is administered a chemotherapeutic agent before, during or after treatment, and wherein in a first treatment phase, a therapeutically effective amount of naltrexone or a metabolite or any analog thereof is administered to the subject, and wherein after the first treatment phase, in a second treatment phase, a therapeutically effective amount of a cannabinoid is administered to the subject, The low-dose naltrexone refers to naltrexone administered at a dose of less than 0.5 mg / kg.

2. Use of a pharmaceutical composition comprising a cannabinoid in the preparation of a medicament for enhancing the effect of a chemotherapeutic agent in treating cancer in a subject, wherein the subject is characterized by having undergone a first treatment phase during which the subject has been administered a therapeutically effective amount of: (a) naltrexone, or (b) its metabolites, or (c) an analog selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorfene, and wherein after the first treatment period, a therapeutic amount of said cannabinoid is administered to the subject, and wherein a chemotherapeutic agent is administered to the subject before, during or after treatment, The low-dose naltrexone refers to naltrexone administered at a dose of less than 0.5 mg / kg.

3. A preparation for treating cancer in a subject, wherein include: (a) naltrexone, or a metabolite thereof, or an analog selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorfene, (b) cannabinoids, and (c) chemotherapeutic agents, wherein in a first treatment phase the subject is administered a therapeutically effective amount of naltrexone or a metabolite or any analog thereof, wherein after the first treatment phase, in a second treatment phase the subject is administered a therapeutically effective amount of a cannabinoid, and wherein the subject is administered a chemotherapeutic agent before, during or after treatment, The low-dose naltrexone refers to naltrexone administered at a dose of less than 0.5 mg / kg.

4. The use or formulation according to any one of the preceding claims, wherein the first treatment period is administration for at least two days.

5. The use or formulation according to any one of the preceding claims, wherein the first treatment phase and the second treatment phase are separated by a recovery phase characterized by the absence of administration of naltrexone or metabolites or analogs, cannabinoids and chemotherapeutic agents.

6. Use or formulation according to claim 5, wherein the recovery phase is at least one day, preferably in the range of 1 to 7 days.

7. The use or formulation according to any one of the preceding claims, wherein the second treatment phase is administration for at least one day.

8. The use or formulation according to any one of the preceding claims, wherein the composition or formulation comprises naltrexone.

9. The use or formulation according to any one of claims 1 to 8, wherein the composition or formulation comprises 6-β-naltrexol.

10. The use or formulation of any one of the preceding claims, wherein the cannabinoid is selected from cannabidiol, cannabidiolic acid, cannabinol, cannabigerol, cannabinol, tetrahydrocannabinol, cannabidiol, cannabicyclol, anandamide, 2-arachidonic acid, 2-arachidonic acid ether, N-arachidonic acid dopamine, arachidonic acid (2-aminoethyl) ester, dronabinol, nabilone, rimonabant or a combination thereof.

11. The use or formulation according to any one of the preceding claims, wherein the cannabinoid is cannabidiol.

12. The use or formulation according to any one of the preceding claims, wherein the chemotherapeutic agent is selected from PI3-kinase inhibitors, AKT inhibitors, taxanes, antimetabolites, alkylating agents, cell cycle inhibitors, topoisomerase inhibitors and cytotoxic antibodies.

13. The use or formulation according to any one of the preceding claims, wherein the chemotherapeutic agent is administered during the first treatment phase.

14. The use or formulation according to any one of the preceding claims, wherein a chemotherapeutic agent is administered during the second treatment phase.

15. The use or formulation according to any one of the preceding claims, wherein the chemotherapeutic agent is administered after the second treatment period.

16. The use or formulation according to any one of the preceding claims, wherein the cancer is breast cancer.

17. The use or formulation according to any one of claims 1 to 15, wherein the cancer is lung cancer or colon cancer.

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