WO2001058445A1 - Therapy with cannabinoids in the treatment of cerebral tumor - Google Patents

Therapy with cannabinoids in the treatment of cerebral tumor Download PDF

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WO2001058445A1
WO2001058445A1 PCT/ES2000/000450 ES0000450W WO0158445A1 WO 2001058445 A1 WO2001058445 A1 WO 2001058445A1 ES 0000450 W ES0000450 W ES 0000450W WO 0158445 A1 WO0158445 A1 WO 0158445A1
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cannabinoids
cannabinoid
tumors
malignant
natural
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PCT/ES2000/000450
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Spanish (es)
French (fr)
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Manuel Guzman Pastor
Cristina Sanchez Garcia
Ismael Galve Roperh
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Universidad Complutense De Madrid
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/335Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. cannabinols, methantheline
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/045Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05Phenols
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47Quinolines; Isoquinolines
    • A61K31/473Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/33Heterocyclic compounds
    • A61K31/395Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/53751,4-Oxazines, e.g. morpholine
    • A61K31/53831,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Abstract

The therapy with cannabinoids in the treatment of cerebral tumors involves (intracranial or systematic) administration of (natural or synthetic) cannabinoids to (human or non-human) mammals having cerebral tumors. Activation of the specific receptors of the cannabinoids leads to selective death of the transformed cells. Regression or eradication of the cerebral tumors is achieved without any significant side-effects.

Description

Title

CANNABINOID THERAPY FOR THE TREATMENT OF BRAIN TUMORS

Technical Field of the Invention

The present invention falls within the field of brain tumor therapy.

Object of the invention

The present invention as set forth in this specification refers to the therapeutic use of cannabinoids for the treatment of brain tumors. The therapies used today for the treatment of these tumors (surgery, radiotherapy, chemotherapy, immunotherapy, gene therapy) usually be ineffective or at most palliative The invention involves a technically simple approach, lacking noticeable side effects and highly effective for the treatment of brain tumors including the most malignant (glioblastomas)

Background

Among the brain tumors that affect humans, glioblastomas are the most frequent (1 per 50 000 people and year) malignant (mortality close to 100%) and of more rapid evolution (life expectancy of weeks / months after diagnosis ) Today, the treatment of glioblastomas is usually ineffective or merely palliative, and involves techniques such as surgery, radiotherapy, chemotherapy and immunotherapy (Louis, DN & Gusella, JF, Trenas Genet. 11, 412-415 1995 Avgeropoulos, NG & Batchelor, TT, Oncologist 4, 209-224, 1999) In addition, gene therapy has begun to be used as an experimental treatment of glioblastomas, although so far it has provided few positive results (Martuza, RL, Nature Med 3, 1323, 1997) The already unlikely success of these therapeutic approaches usually It is also complicated by factors such as rapid growth, notorious heterogeneity, high level of infiltration and extreme resistance to chemotherapy exhibited by glioblastomas (Maintz, D et al, J Neuropathol Exp Neurol 56, 1098-1 104, 1997, Masón , W, Louis, DN & Cairncross, JG, J Clin Oncol 15, 3423-3426, 1997, Martuza, op cit, Avgeropoulos & Batchelor, op cit) It seems clear therefore that the development of alternative therapies for treatment is highly recommended of brain tumors Cannabinoids are compounds that owe their name to the fact that they are synthesized by the Cannabis sativa L plant. These compounds, among which Δ 9 -tetrahιdrocannabιnol (THC) stands out for its high potency and abundance, are responsible for the central effects and peripherals of marijuana use (Pertwee, RG, Pharmacol Ther 74, 129-180,1997, Felder, CC & Glass, M Annu Rev Pharmacol Toxicol 38, 179-200, 1998) C sativa cannabinoids (Fig 1) carry ac Above its effects because they are similar to certain molecules produced by animals (including humans) that probably play important roles in the nervous system. These molecules are therefore called endogenous cannabmoids or endocannabinoids, and anandamide (= araquιdonoιletanolamιda) is their main Representative (Di Marzo, V, Melck, D, Bisogno, T & De Petrocellis, L, Trends Neurosa 21, 521-528 1998, Martin, BR, Mechoulam, R & Razdan, RK, Ufe S 65, 573-595, 1999 ) Moreover, it has been possible to obtain in the laboratory compounds that mimic the action of natural cannabinoids but with a much higher potency. They are called synthetic cannabinoids, one of whose representatives is WIN-55,212-2 (Fig 2) (Pertwee, op cit Barth, F, Expert Opm Ther Patents 8, 301-313, 1998) Cannabinoids, both natural and synthetic, act by binding to specific membrane receptors (cannabinoid receptors or CB type), of which two different subtypes CB Í and CB 2 are known today (Pertwee, op cit, Howlett, A er al, in The lUPHAR Compendium of Receptor Characteπzation and Classification, eds Godframd, T, Humphrey , P Ruffolo, R & Vanhoutte, P, lUPHAR Media, pp 97-104, 1998) Not all The body's tissues possess these receptors are located mainly in the nervous system, and that is why cannabinoids may exert their effects on the brain (Pertwee, op at, Childers, SR & Breivogel, CS, Drug Alcohol Depen 51, 173-187, 1998 ) There are today a large number of studies about the possible therapeutic applications of cannabinoids. Moreover, doctors are now allowed in the United Kingdom and in various states of the United States to prescribe THC or certain synthetic cannabinoids such as appetite stimulants and vomit inhibitors in patients with AIDS or cancer chronically treated with chemotherapy (Gπnspoon, L & Bakalar, JB, JAMA 273, 1875-1876,1995, Voth, E & Schwartz, R, Ann Intem Med 126, 791- 798, 1997) Among the potential therapeutic uses of cannabinoids, the following (a) stand out as analgesic agents have been shown to act highly effectively in the attenuation of acute pain o and chronic, (b) as agents that reduce motor activity are being tested today in the treatment of disorders associated with Parkinson's disease, Huntington's chorea and multiple sclerosis, (c) as anticonvulsant agents its study is studied application in the treatment of epilepsy, (d) as agents that decrease infraocular pressure could be used in the treatment of glaucoma (Voth and Schwartz, op cit, Manzanares, J er a /., Trends Pharmacol Sci 20, 287-294, 1999, Pop, E, Curr Opm CPNS Invest Drugs 1, 587-596, 1999, Sanudo-Pena, MC, Tsou, K & Walker, JM, Ufe Sci 65, 703-713, 1999) Some of these potential therapeutic uses of the cannabinoids have already been patented (see for example US4189491, US5939429, WO9711668, WO9832441 and WO9957106)

One of the most intriguing and unexplored effects of cannabinoids is their ability to inhibit the growth of transformed cells in vitro. Thus, it has been shown that various cannabinoids inhibit the proliferation of MCF-7 breast tumor cells (De Petrocellis, L et al. , Proc Nati Acad. Sci. USA 95, 8375-8380, 1998), of C6 glioblastoma cells (Sánchez, C, Galve-Roperh, I, Canova, C, Brachet, P & Guzmán, M, FEBS Lett 436, 6-10, 1998) and PC-3 prostate tumor cells (Ruiz, L, Miguel, A & Díaz-Laviada, I, FEBS Lett 458, 400-404, 1999) Without However, these findings in cell culture systems have never been observed in vivo so far, their biomedical significance is unknown.

Description of the invention

The present invention makes use of cannabinoids for the treatment of brain tumors for the first time, and is based on our original observations that cannabinoids induce a marked regression (which leads to a lengthening of life) and even eradication (which entails Cure) of ghoblastomas in laboratory animals This invention involves the use of a technically simple therapy, devoid of noticeable side effects and, more importantly, very effective for the treatment of brain tumors, which as mentioned above cannot be treated satisfactorily today with other techniques or compounds The experiments that have led to the present invention are detailed below.

Antitumor action of cannabinoids in rats

Injection of C6 glioblastoma cells in the rat brain is widely used as an experimental model of malignant brain tumor (Barth, RF, J Neur∞ncol 36, 91-102, 1998) C6 glioblastoma cells were inoculated directly into the brain of Wistar rats, and tumors were visualized by magnetic resonance imaging All animals that were left untreated died uniformly 12-18 days after cell inoculation (Fig 3a). To assess the antitumor potential of cannabinoids, 12 days after cell inoculation, a group of animals was administered for 7 days THC or WIN-55,212-2 through a cannula located at the site of inoculation. Animals treated with Cannabinoids had a significantly longer life than control animals (Fig 3a). Thus, the administration of cannabinoids managed to increase survival to 19-35 days in 9/15 animals (treatment with THC) or 19-43 days in 4/15 animals (treatment with WIN-55,212-2) Moreover, cannabinoids completely eradicated the tumor in 3 / 15 animals (treatment with THC) or in 5/15 animals (treatment with WIN-55,212-2) An image of magnetic resonance imaging of one of the animals cured with THC is shown in Fig 3b, after administration of the cannabinoid tumor mass disappeared completely, and instead a residual hypomteπsa area was observed that is interpreted as a fibrous scar at the site of inoculation No recurrence was observed in the 8 animals cured with caπnabmoides

Antitumor action of cannabinoids in immunodeficient mice

To discern whether the antiproliferative action of cannabinoids is due to a direct effect on tumor cells or an indirect effect mediated by an immune response, C6 glioblastoma cells were inoculated subcutaneously in mice deficient in RAG-2 recombinase (RAG-2 ' 1 ' ), lacking mature T and B lymphocytes (Shinkai, Y et al, Ce // 68, 855-867 1992) As shown in Fig 4a, the tumor size was extraordinarily smaller in animals treated with THC or WIN-55,212-2 than in control animals Examples of tumor-bearing mice and dissected tumors after treatment with or without cannabinoids for 7 days are shown in Fig 4b

Secondary treatment with cannabinoids in vivo The possible side effects of cannabinoid treatment were examined below Rats without tumor and treated with cannabinoids were not affected at all their survival (Fig 3a) As in the 8 animals mentioned above whose tumors were eradicated with cannabinoids, thorough magnetic resonance analysis of all animals without tumor revealed that treatment with cannabinoids produced no sign of damage due to necrosis, edema, infection, inflammation or trauma to rule out the possibility that Cannabinoids were toxic to dividing nerve cells, TUNNEL stains were performed in the subventπcular area of the rat's brain, which continues to proliferate in the adult animal. Cannabinoid administration not only did not produce any significant apoptotic effect on the brain in vivo. , but also the slight dizziness observed in the caudate putamen of the control animals was not evident in the animals treated with cannabinoids

In both animals without tumor and in tumor carriers, cannabinoids did not induce any significant alteration of behavioral parameters such as motor coordination and physical activity. Water and food intake, as well as weight gain, were also not affected by cannabinoids. Likewise, in the blood tests the biochemical parameters (glucose, urea, uric acid, creatinine, cholesterol, bilirubin) and the markers of tissue damage (alanine and aspartate ammotransferases, γ-glutamyltransferase, creatine kinase, lactate dehydrogenase) were not affected neither over the period of 7 days of administration nor up to 2 months after the end of cannabinoid treatment Data from other authors support the idea that cannabinoids are not only not toxic compounds for nerve cells, but even protect them of toxic stimuli such as glutamatergic agonists (Skaper, SD et al, Pr oc Nati Acad Sα USA 93, 3984-3989, 1996, Shen, M & Thayer, SA, Mol Pharmacol 54, 459-462, 1998), oxidizing agents (Hampson, AJ, Gπmaldi, M Axelrod, J & Wink, D , Proc Nati Acad Sci USA 95, 8268-8273, 1998) and ischemia (Nagayama, T er a /, J Neurosa 19, 2987-2995, 1999)

Pharmacological characterization of the antitumor action of cannabinoids

Experiments aimed at pharmacologically characterizing the cannabinoid-induced death of C6 glioblastoma cells in culture High potency synthetic agonists such as

WIN-55,212-2, CP-55,940 and HU-210 induced the death of these cells at doses lower than THC, as expected from its higher affinity for cannabinoid receptors (Pertwee, op at) Thus, after 5 days of exposure to cannabinoids, the viability of glioblastoma C6 was reduced by 50% at concentrations of 20 nM WIN-55, 212-2, 45 nM CP- 55,940, 10 nM HU-210 and 480 nM THC (n = 4) Neither SR141716 (a selective CB antagonist nor SR144528 (a selective CB 2 antagonist)) Shire, D et al, Life Sci 65, 627-635, 1999) were able separately to prevent THC-induced cell death. However, when the two antagonists were added together to the incubations an effective prevention of death was observed. THC-induced cell (Fig 5a) Accordingly, Western blot analysis showed that C6 glioblastoma cells expressed both the CBi and CB 2 receptors (Fig 5b)

Application of the invention in other cases The experiments that have led to the present invention have been carried out with rats and mice as tumor-bearing animals. However, both for the experimental design employed and for the similarity that brain tumors exhibit in different mammals (RF Barth, op cit), the invention can be applied to the treatment of brain tumors in other mammals, including humans

The experiments that have led to the present invention have been carried out with glioblastomas as a brain tumor model. However, because of the experimental design that has been used for the induction and treatment of tumors, the invention can be applied to the treatment of other tumors. brain diseases, for example medulloepitheliomas, medulloblastomas, neuroblastomas, germinomas, embryonic carcinomas, astrocytomas, astroblastomas, ependymomas, oligodendrog omas, plexal carcinomas, neuroepitehomas, pineoblastomas, ependyloblastomas, neuroectodermal tumors, malignant melanomas, malignant melanomas The experiments that have led to the present invention have been carried out with two paradigmatic cannabinoids, one natural (THC) and one synthetic (WIN-55,212-2) In a preferred embodiment of the invention, the cannabinoid with more potent antiproliferative effect will be used for a given tumor However, since the antiproliferative effect of these compounds is mediated by cannabinoid receptors (CB type receptors, Howlett et al, op αt), the invention is applicable to any other agonist of these receptors, both cannabmoids. of C sativa (for example Δ 8 -tetrahιdrocannabιnol, cannabinol, cannabidiol) (Fig 1) as synthetic cannabinoids (for example HU-210, CP-55,940, CP-50,556) (Fig 2) (Pertwee, op αt, F Barth, op cit) Also included in this section are medications that contain any cannabinoid in their composition

The experiments that have led to the present invention have been carried out by intratumorally administering the cannabinoid. In a preferred embodiment of the invention this will be the chosen route of administration, since it allows a high accessibility of the cannabinoid to the tumor. However, since the action of the cannabinoid is direct on the tumor and does not seem to substantially affect peripheral systems, the route of administration can also be systemic, for example intrapeπtoneal, intravenous or oral

The experiments that have led to the present invention have been carried out by the continuous administration of a dose of cannabinoid for a certain time. In a preferred embodiment of the invention these parameters will be optimized depending on the specific requirements of the treatment patient condition, size and tumor location, number of tumors, etc. Thus, for example, the mode of application may be continuous (preferred mode) or sequential in one or more dose per day This will obviously vary the doses of compound administered and the total duration of treatment.

Brief description of the figures

Figure 1

Chemical formula of the main C sativa cannabmoids

Figure 2

Chemical formula of the main synthetic cannabinoids

Figure 3

Antitumor action of cannabinoids in rats (a) Survival curves of rats with brain tumors Glioblastomas were induced in 45 rats (day 0), 15 animals were not treated with cannabinoids (---), while another 15 were treated with THC (-) and another 15 with WIN-55,212-2 () between days 12 and 19 Animals treated with cannabinoids lived significantly longer than controls (P <0 01 by the log-rank test) THC was also administered and WIN-55,212-2 to 5 rats each in which no tumor was induced (- -)

(b) Magnetic resonance imaging in axial (upper) and coronal (lower) projections of a rat's brain before (left) and after

(right) of THC treatment A 100 mm 3 glioblastoma (arrow) was eradicated with 500 μg of THC The image was taken 7 days after the end of THC treatment

Figure 4

Anti-tumor action of cannabinoids in immunodeficient mice (a) Glioblastomas were induced in 18 mice When the tumors reached the desired size (day 0), 6 animals were treated with vehicle (o), while another 6 were treated with THC (•) and another 6 with WIN- 55,212-2 (z) for 7 days The size of tumors in animals treated with cannabinoids was significantly smaller than in controls a all times (P <0 01 by Student's f test) (b) Examples of glioblastomas in mice (above) and dissected

(bottom, 1 cm bar) after treatment for 7 days with vehicle, THC or WIN-55,212-2 (WIN)

Figure 5

Involvement of cannabinoid receptors in cell death

(a) C6 glioblastoma cells were cultured for 5 days in the absence or presence of 1 μM THC, 1 μM SR141716 (SR1) and / or 1 μM SR144528 (SR2) (n = 6) * Sιgnιfιcatιvably different from incubations without additions ( P <0 01 by Student's r test)

(b) Presence of CB T and CB 2 cannabinoid receptors in C6 glioblastoma cells. The detection of the receptors was performed by Western or / or with specific antibodies for each of the two receptors.

Embodiment of the invention

The present invention is further illustrated by the examples set forth below.

E | emplo 1

Glioblastoma cure in rats

Male Wistar rats (250-300 g body weight) were anesthetized with

3% isofluorane in a mixture of oxygen (0 8 l / mm) and protoxide (0 4 l / mm)

5x10 6 cells of C6 glioblastoma were prepared in 100 µl phosphate buffered saline (PBS) supplemented with 0 1% glucose and injected stereotaxically into the frontal-paputal lobe of the hemisphere right brain (4 mm to the right of bregma, 4.5 mm deep from the skull) (Left, M et al, Gene Ther 2, 66-69, 1995) Rats received dexamethasone (2 mg / l) and tetracycline ( 75 mg / kg of body weight) in the water for 3 days before and 7 days after the inoculation of the cells A thorough follow-up of the tumors was performed by magnetic resonance by methods described by other authors (Izquierdo et al, op αt, Cortés, ML, by Felipe, P, Martín, V, Hughes, MA & Izquierdo, M, Gene Ther 5, 1499-1507, 1998)

Cannabinoid administration to rats began 12 days after cell inoculation. At this time, the average tumor size was 70 mm 3 (range 25-100 mm 3 ) estimated by magnetic resonance imaging (Izquierdo et al, op cit, Cortés, by Felipe, Martin Hughes & Izquierdo, op αt) The cannabinoids were administered by a cannula located at the site of inoculation of the tumor and fixed to the skull with dental cement, a small stainless steel thyme secured the cannula and cement Dental The cannula was connected subcutaneously through a catheter to an osmotic mini-pump (Alzet 2001) that operated at a flow of 1 μl / h for 7 days The osmotic pump was filled with 500-2500 μg THC or 50-250 μg WIN-55,212-2 in 200 μl of PBS supplemented with 5 mg / ml of ordered and dialyzed bovine serum albumin (BSA) The doses of cannabinoids used depended on the characteristics of the tumor to be treated, the higher doses were used aron for large, dense and invasive tumors

As seen in Fig 3a, all animals that were left untreated died uniformly 12-18 days after cell inoculation. Animals treated with cannabinoids had a significantly longer life than control animals. Moreover, cannabinoids eradicated completely the tumor in a significant percentage of animals In Fig 3b, an MRI image of one of the animals cured with THC is shown, after the administration of the cannabinoid the tumor mass disappears completely, and a residual hypointense zone is observed that is interpreted as a fibrous scar at the site of inoculation No recurrence was observed in animals cured with cannabinoids

Example 2

Healing of qlioblastomas in immunodeficient mice

Tumors were induced in RAG-2 '" mice by subcutaneous inoculation of 5x10 6 cells of C6 ghoblastoma in 100 µl of PBS supplemented with 0 1% glucose About 10 days later, when the average volume of the tumors was 250 mm 3 (range 200-300 mm 3 ), the animals were randomly divided into 3 groups and injected for 7 days vehicle,

500 mg of THC or 50 μ g of WIN 55,212-2-day in 100 .mu.l of PBS supplemented with 5 mg / ml of delipidized and dialyzed BSA Dimensions of the tumors were measured with a caliper and its volume was calculated as (4π / 3) x (width / 2) 2 x (length / 2)

As shown in Fig 4a, the tumor size was extraordinarily smaller in animals treated with THC or WIN-

55,212-2 than in control animals Examples of mice bearing tumors and dissected tumors after treatment with or without cannabinoids for 7 days are shown in Fig 4b

Example 3

Implication of cannabinoid receptors in cell death qlioblastoma were cultured at 37 ° C and 5% CO 2 C6 glioblastoma cells in medium

F12 supplemented with 10% fetal calf serum, 24 h before the start of the experiment, the cells were transferred to serum free F12 medium and supplemented with insulin (5 μg / ml), transfernna (5 μg / ml), sodium selenite (5 μg / ml) and elliptical and dialyzed BSA (10 mg / ml) The medium was renewed every 48 h and cell viability was determined by the MTT method (Sánchez, C, Galve-Ropem, I, Canova, C, Brachet, P & Guzman M, op at) As shown in Fig 5a, THC was capable of inducing the death of C6 glioblastoma cells also when added simultaneously SR141716 medium (selective antagonist of CBi) and SR144528 ( a selective antagonist of CB 2) prevented THC-induced cell death

To verify that both receptors were present in C6 cells, the cells were washed with PBS, the plates were scratched in lysis medium and the particulate fraction was obtained by centrifugation at 40 OOOg for 60 min (Sánchez, C, Galve-Roperh, I, Canova, C, Brachet, P & Guzmán, M, op at) The samples were electrophoresed in polyacrylamide gels with sodium dodecyl sulfate and the proteins were transferred from the gels to nitrocellulose membranes The membranes were blocked with dehpidized BSA and 1% dialyzed, and incubated with an antibody against residues 1-14 of the rat CBi receptor (diluted 1 5000) or with an antibody against residues 350-361 of the human CB 2 receptor (diluted 1 2000) The samples were finally developed with an electrochemiluminescence "kit" (Amersham, Bucks, United Kingdom) As shown in Fig 5b, C6 glioblastoma cells expressed both the CB Í receptor and CB 2 receptor

Example 4

Secondary treatment with live m cannabinoids Cannabmoids (2500 μg THC or 250 μg WIN-55,212-2) were administered to rats without tumors for 7 days as described above. Rats were then sacrificed and their brains fixed. with 4% paraformaldehyde in PBS Death by apoptosis was determined in 40 µm thick sections of the brain using a "TUNNEL staining kit" according to the supplier's instructions (Boehπnger, Mannheim, Germany) DNA stranding with Deoxyundin triphosphate labeled with fluorescein was visualized by a confocal microscope (excitation wavelength 488 nm, length of 525 nm emission wave) The laser intensity and photodetector sensitivity were kept constant to allow comparison between treatments At least 5 optical fields were analyzed per animal

TUNNEL stains were performed in the subventπcular area of the rat brain, which continues to proliferate in the adult animal. The administration of cannabinoids not only did not produce any significant apoptotic effect on the living brain, but also the slight dizziness observed in the caudate putamen of control animals was not observed in animals treated with cannabinoids

Claims

Claims
1 - Use of natural and synthetic cannabinoids in the manufacture of a medicament for therapeutic treatment, in mammals including humans, of brain tumors selected from the group comprising glioblastomas, medulloepitehomas, medulloblastomas, neuroblastomas, germinomas, embryonic carcinomas, astrocytomas, astroblastomas, ependymomas, ohgodendrogliomas, plexal carcinomas, neuroepitheliomas, pineoblastomas, epandimoblastomas, neuroectodermal tumors, malignant meningiomas, chondrosarcomas, meningeal sarcomatosomes, malignant melanomas and malignant schwannomas
2 - Use according to claim 1, wherein the brain tumors are ghoblastomas
3 - Use according to claims 1 and 2, wherein the natural cannabinoids are selected from the group consisting of Δ 9 -tetrahιdrocannabιnol (THC), Δ 8 -tetrahιdrocannabιnol, cannabinol and cannabidiol
4 - Use according to any one of the preceding claims, wherein the natural cannabinoid is Δ 9 -tetrahιdrocannabιnol (THC)
5 - Use according to claims 1 and 2, wherein the synthetic cannabinoids are selected from the group consisting of WIN-55,212-2, HU-210, CP-
55,940 and CP-50,556 (levonantradol)
6 - Use according to claims 1 and 5, wherein the synthetic cannabinoid is WIN-55,212-2 7 - Medication for the treatment, in mammals including humans, of brain tumors selected from the group comprising glioblastomas, meduloepite omas, medulloblastomas, neuroblastomas germinomas, embryonic carcinomas, astrocytomas, ependymoma astroblastomas, oligodendroghomas, plexal carcinomas, neuroepitehomas, pineoblastbiaomas, pineoblastbiaomas tumors neuroectodermal malignant meningiomas, chondrosarcomas, meningeal sarcomatosomes, malignant melanomas and malignant schwannomas, characterized in that it comprises, as an active ingredient, a natural or synthetic cannabinoid, and a pharmaceutically acceptable excipient
8 - A medicament according to claim 7, wherein the natural cannabinoid is selected from the group consisting of Δ 9 -tetrahιdrocannabιnol (THC) Δ 8 -tetrahιdrocannabιnol, cannabinol and cannabidiol
9 - A medicament according to claim 7, wherein the synthetic cannabinotde is selected from the group consisting of WIN-55,212-2, HU-210, CP-55,940 and CP-50,556 (levonantradol)
10 - A medicament according to any one of claims 7 to 9 wherein the excipient is one suitable for intratumoral (intracranial) or systemic administration, such as oral, intravenous or intrapentoneal
11 - A medicament according to claim 10, wherein the excipient for intratumoral administration is a phosphate buffered saline (PBS) supplemented with bovine serum albumin (BSA) slipped and dialyzed
12 - A medicament according to any one of claims 10 to 11, wherein the concentration of the cannabinoid in the liquid of intratumoral administration is 10 to 10,000 μg / ml for the natural cannabinoid and 1 to 1000 μg / ml for the synthetic cannabinoid
13 - Procedure for therapeutic treatment, in mammals including humans, of brain tumors selected from the group comprising glioblastomas, meduloepitehomas, medulloblastomas, neuroblastomas, germinomas, embryonic carcinomas, astrocytomas, astroblastomas, ependymomas, oligodendrogliomas, plexal carcinomas, pineappleoomas epandimoblastomas neuroectodermal tumors, malignant meningiomas, chondrosarcomas, meningeal sarcomatosomes, malignant melanomas and malignant schwannomas, characterized in that it comprises administering, to the animal affected by one of said tumors, a therapeutically effective amount of a medicament as defined in any one of claims 7 to 12
14 - Method according to claim 13, characterized in that the administration is carried out intratumorally
15 - Method according to claim 14, characterized in that the administered amount of cannabinoid (active ingredient) varies from 100 to 50,000 μg for natural cannabinoids and from 10 to 5000 μg for synthetic cannabinoids
PCT/ES2000/000450 2000-02-11 2000-11-22 Therapy with cannabinoids in the treatment of cerebral tumor WO2001058445A1 (en)

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DE60020111T DE60020111T2 (en) 2000-02-11 2000-11-22 Therapeutic application of cannabinoides for brain tumor treatment
AU13979/01A AU1397901A (en) 2000-02-11 2000-11-22 Therapy with cannabinoids in the treatment of cerebral tumor
EP00976087A EP1177790B1 (en) 2000-02-11 2000-11-22 Therapy with cannabinoids in the treatment of cerebral tumor
ES00976087T ES2241670T3 (en) 2000-02-11 2000-11-22 Cannabinoid therapy for the treatment of cerebral tumors.
AT00976087T AT295162T (en) 2000-02-11 2000-11-22 Therapeutic application of cannabinoides for brain tumor treatment

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US10/647,739 Division US20040039048A1 (en) 2000-02-11 2003-08-25 Therapy with cannabinoid compounds for the treatment of brain tumors

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Cited By (12)

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EP1307188A1 (en) * 2000-05-17 2003-05-07 Atlantic Technology Ventures, Inc. Cannabinoid drugs
EP1307186A1 (en) * 2000-05-17 2003-05-07 Atlantic Technology Ventures, Inc. Methods for decreasing cell proliferation based on (3r, 4r)-delta8-tetrahydrocannabinol-11-oic acids
EP1307188A4 (en) * 2000-05-17 2005-07-06 Atlantic Technology Ventures I Cannabinoid drugs
EP1307186A4 (en) * 2000-05-17 2005-10-19 Atlantic Technology Ventures I Methods for decreasing cell proliferation based on (3r, 4r)-delta8-tetrahydrocannabinol-11-oic acids
US9084771B2 (en) 2007-05-17 2015-07-21 Sutter West Bay Hospitals Methods and compositions for treating cancer
GB2478072A (en) * 2008-06-04 2011-08-24 Gw Pharma Ltd THC and CBD for use in the treatment of brain tumours
GB2478072B (en) * 2008-06-04 2012-12-26 Gw Pharma Ltd Anti-tumoural effects of cannabinoid combinations
GB2478074B (en) * 2008-06-04 2012-12-26 Gw Pharma Ltd Anti-tumoural effects of cannabinoid combinations
US8632825B2 (en) 2008-06-04 2014-01-21 Gw Pharma Limited Anti-tumoural effects of cannabinoid combinations
GB2478074A (en) * 2008-06-04 2011-08-24 Gw Pharma Ltd THC and CBD for use in the treatment of tumours
US8790719B2 (en) 2010-03-12 2014-07-29 Gw Pharma Limited Phytocannabinoids in the treatment of cancer
US9675654B2 (en) 2010-03-12 2017-06-13 Gw Pharma Limited Phytocannabinoids in the treatment of cancer

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