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WO2020232350A1 - Dosages pour cannabinoïdes synthétiques - Google Patents

Dosages pour cannabinoïdes synthétiques Download PDF

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Publication number
WO2020232350A1
WO2020232350A1 PCT/US2020/033121 US2020033121W WO2020232350A1 WO 2020232350 A1 WO2020232350 A1 WO 2020232350A1 US 2020033121 W US2020033121 W US 2020033121W WO 2020232350 A1 WO2020232350 A1 WO 2020232350A1
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human
cell line
cannabinoid receptor
cells
cannabinoid
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Andrew A. MONTE
Robert SCHEINMAN
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University of Colorado System
University of Colorado Colorado Springs
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University of Colorado Colorado Springs
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • This disclosure relates to the field of drug screening assays.
  • Marijuana has long been known to possess psychoactive properties, with the first known reference describing a cannabis product as a psychoactive agent dating to before the year 2000 BC.
  • THC tetrahydrocannabinol
  • CB1 and CB2 cannabinoid receptors in the 1980s, there was a pharmaceutical effort to synthesize cannabinoid receptor agonists for potential therapeutic uses, such as combating nausea and pain.
  • the vast majority of these efforts never reached commercial fruition.
  • independent chemists now use this publicly available research to produce synthetic cannabinoids.
  • Spice is the street name for one such group of designer drugs and produces effects similar to cannabis. These drugs consist of psychoactively inert dry plant material sprayed with synthetic cannabinoid receptor agonists. Spice is often consumed like cannabis, i.e. applied to plant material, rolled into cigarettes, and smoked. It is sold in small, colorful packets with evocative names like“Arctic Spice,”“Dream,” and“K-2.”
  • Synthetic cannabinoid receptor agonists consist of at least four different groups of chemical structures, each discussed in greater detail below. These chemicals bind to the same CB1 and CB2 cannabinoid receptors as THC, although they often do so much more efficiently. In total, thousands of synthetic cannabinoid receptor agonists have been characterized. Furthermore, a skilled chemist could use publicly available information to modify these known compounds to produce previously uncharacterized molecules that would have a high likelihood of binding to the cannabinoid receptors.
  • the potentially lethal dilemma presented above is overcome by the assay of the present invention that combines the accuracy of the current drug screening methods with the ability to simultaneously screen for any cannabinoid receptor agonist that may be present in a sample.
  • an assay for the detection of cannabinoid related compounds comprising a cell line, wherein the cell line comprises a cannabinoid receptor, wherein activation of the cannabinoid receptor results in a reporter reaction within the cell line.
  • reporter reaction comprises a reduction in intracellular cyclic adenosine monophosphate (cAMP), wherein the intracellular cAMP comprises a fluorescent tag.
  • cAMP intracellular cyclic adenosine monophosphate
  • the reporter reaction comprises a calcium flux detection assay.
  • the cell line comprises at least one of Chinese hamster ovary (CHO)cells, Human embryonic kidney 293 (HEK-293) cells, human brain astrocytoma (1321 N1), human T-cell leukemia (1301), human glioblastoma astrocytoma (U373 MG (Uppsala)), human monocytic leukemia (THP 1), metastatic rhabdomyosarcoma (RH-30), telomerase- immortalized human microvascular endothelial cells (TIME), sinoatrial node (Shox2), and NHCF-A human cardiac fibroblasts-atrial cells.
  • acquiring a sample from a human b. applying the sample to a cell line; i. wherein the cell line comprises a cannabinoid receptor; and ii. wherein binding of the cannabinoid related compounds to the cannabinoid receptor results in a reporter reaction within the cell line.
  • the sample comprises at least one of blood, urine, stool, hair, subcutaneous fat, and saliva.
  • the reporter reaction comprises a reduction in intracellular cyclic adenosine monophosphate (cAMP), wherein the intracellular cAMP comprises a fluorescent tag.
  • cAMP intracellular cyclic adenosine monophosphate
  • reporter reaction comprises a calcium flux detection assay.
  • the cell line comprises at least one of human brain astrocytoma (1321 N1), human T-cell leukemia (1301), human glioblastoma astrocytoma (U373 MG (Uppsala)), human monocytic leukemia (THP 1), metastatic rhabdomyosarcoma (RH- 30), telomerase-immortalized human microvascular endothelial cells (TIME), sinoatrial node (Shox2), and NHCF-A human cardiac fibroblasts-atrial cells.
  • human brain astrocytoma 1321 N1
  • human T-cell leukemia (1301)
  • human glioblastoma astrocytoma U373 MG (Uppsala)
  • human monocytic leukemia THP 1
  • RH- 30 metastatic rhabdomyosarcoma
  • TIME telomerase-immortalized human microvascular endothelial cells
  • CBD1 human cannabinoid receptor 1
  • CBD2 human cannabinoid receptor 2
  • FIG. 1 is an image showing the traditional method of drug screening and one embodiment of the screen of the present invention.
  • FIG. 2 shows results from a BRET probe wherein CAMYEL and CB1 expression plasmids were transfected into HEK cells. The addition of 5F-ADB-PINACA (black dots) against methanol control (white dots) is shown.
  • FIG. 3 shows results from a BRET probe where CAMYEL was transfected into HEK cells, but without the transfection of CB1.
  • the addition of 5F-ADB-PINACA (black dots) against methanol control (white dots) is shown.
  • FIG. 4 shows results from a BRET probe where CAMYEL and CB1 transfected into HEK cells.
  • Cells treated with 5F-ADB-PINACA (ADB) are shown as black dots.
  • Cells treated with both 5F-ADB-PINACA (ADB) and AM251 are shown as white dots.
  • FIG. 5 shows the aggregate of different concentrations of 5F-ADB-PINCA. The change in inverse BRET ratio at plateau was plotted against concentration of
  • FIG. 6 shows an example of 2-photon microscopy, where photos are detected during 800 mHz laser pulses emitted by CFP.
  • FIG. 7 shows a representative Phasor plot.
  • FIG. 8 shows the experimental design of adding the fluorescent protein CFP to the C-terminus domain of CB1 , with interacts with FIAsh bound to the third intracellular loop of CB1.
  • FIG. 9 shows the changes in fluorescence lifetime of the CFP protein (half-life measured in nanoseconds) with the binding of FIAsH at two different constructs - either 940 (left bars in black) or 958 (right bars in white).
  • the first set of bars shows the measure of CFP without any additions of FIAsH.
  • the second set of bars shows the addition of the FIAsH constructs after 20 minutes.
  • the third set of bars show the further addition of 100 nM THC.
  • the fourth set of bars shows the addition of 1 mM THC.
  • An“assay” is an analysis performed to determine the presence, absence, quantity, or activity level of one or more components within a sample.
  • potency refers to the concentration of a particular agonist necessary to elicit a particular biological response. This measure may be independent of the binding affinity of said agonist for the receptor in question.
  • sample is any part or composition removed from a larger whole and potentially representative of that whole.
  • suitable biological samples from a test subject may include, but are not limited to, blood, saliva, urine, and other bodily fluids; skin; hair, with or without a skin tag; subcutaneous fat; or any other biological sample which may contain the targeted composition. It will be apparent to those having skill in the art that certain samples may not suitable for direct use in an assay. Therefore, the sample may require suitable processing prior to use, such as, for example, pH balancing, cell lysis, or filtration.
  • the first, human cannabinoid receptor type 1 (CB1/Cnr1) was initially cloned in 1990. (“Structure of a cannabinoid receptor and functional expression of the cloned cDNA”. Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner Tl Nature. 1990 Aug 9; 346(6284):561-4.)
  • the full-length protein (SEQ ID No. 1) and two shorter isoforms (SEQ ID Nos. 2 and 3) are G-protein coupled receptors.
  • the second receptor, human cannabinoid receptor type 2 (CB2/Cnr2) (SEQ ID No. 4) was initially cloned 3 years later (“Molecular
  • peripheral receptor for cannabinoids.
  • Munro S Thomas KL, Abu- Shaar M Nature. 1993 Sep 2; 365(6441 ):61 -5.
  • these receptors may be used to detect the presence of cannabinoid receptor agonists within a sample.
  • the assay will make use of the human CB1 receptor.
  • THC is the predominant psychoactive compound found in cannabis. However, it is only a partial CB1 agonist. As a result, many of the compounds disclosed below have significantly higher binding affinities for the CB1 or CB2 receptors, and are also significantly more efficient at activating the receptor. THC has the following chemical structure:
  • the CP class of cannabinoid receptor agonists are structurally distinct from THC.
  • this class of cannabinoid receptor agonists is defined as“2-(3-hydroxycyclohexyl)phenol with substitution at the 5-position of the phenolic ring by alkyl or alkenyl, whether or not substituted on the cyclohexyl ring to any extent.”
  • the CP class of cannabinoid receptor agonists is structurally distinct from THC, and comprises the following compounds as examples:
  • CP-55,244 ((2S,4S,4aS,6R,8aR)-6-(hydroxymethyl)-4-[2-hydroxy-4-(2- methyloctan-2-yl)- phenyl]-1 ,2,3,4,4a,5,6,7,8,8a-decahydronaphthalen-2-ol) has the following chemical formula:
  • Otenabant (CP-945,598) (1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H- purin-6-yl]- 4-(ethylamino)piperidine-4- carboxamide) has the following chemical formula:
  • the JWH class of cannabinoid receptor agonists include members of the naphthoylindole, naphthylmethane, naphthoylpyrrole, and naphthylmethylene families and under the proposed Synthetic Drug Prevention Act of 2012 include“3-(1- naphthoyl)indole or 3-(1-naphthylmethane)indole by substitution at the nitrogen atom of the indole ring, whether or not further substituted on the indole ring to any extent, whether or not substituted on the naphthoyl or naphthyl ring to any extent; 3-(1- naphthoyl)pyrrole by substitution at the nitrogen atom of the pyrrole ring, whether or not further substituted in the pyrrole ring to any extent, whether or not substituted on the naphthoyl ring to any extent; 1-(1-naphthylmethylene)indene by substitution of the 3- position of
  • JWH-018 (1-pentyl-3-(1-naphthoyl)indole) has the following chemical formula:
  • JWH-073 (1-butyl-3-(1-naphthoyl)indole) has the following chemical formula:
  • JWH-019 (1-hexyl-3-(1-naphthoyl)indole) has the following chemical formula:
  • JWH-081 (4-methoxynaphthalen- 1-yl- (1 -pentylindol- 3-yl)methanone) has the following chemical structure:
  • JWH-200 (1-[2-(4-morpholinyl)ethyl]-3-(1-naphthoyl)indole) has the following chemical structure:
  • JWH-250 (1-pentyl-3-(2-methoxyphenylacetyl)indole) has the following chemical structure:
  • JWH-122 (1-pentyl-3-(4-methyl-1-naphthoyl)indole) has the following chemical structure:
  • JWH-398 (1-pentyl-3-(4-methyl-1-naphthoyl)indole) has the following chemical structure:
  • JWH-203 (1-pentyl-3-(2-chlorophenylacetyl)indole) has the following chemical structure:
  • the HU class of cannabinoid receptor agonists are generally structurally similar to THC. This class comprises the following molecules as examples:
  • HU-210 ((6aR,10aR)- 9-(Hydroxymethyl)- 6,6-dimethyl- 3-(2-methyloctan- 2-yl)- 6a,7,10,10a-tetrahydrobenzo [c]chromen- 1 -ol) has between 100 to 800 times the affinity for the CB1 receptor than THC, and has the following chemical structure:
  • HU-211 ((6aS, 10aS)-9-(Hydroxymethyl)- 6,6-dimethyl- 3-(2-methyloctan- 2-yl)- 6a,7, 10, 10a-tetrahydrobenzo [c]chromen-1-ol) has the following structure:
  • HU-243 ((6aR,8S,9S, 10aR)-9-(hydroxymethyl)-6,6-dimethyl-3-(2- methyloctan-2- yl)-8,9-ditritio-7,8,10, 10a-tetrahydro-6aH-benzo[c]chromen-1-ol) has the following structure:
  • HU- 308 ([(1 R,2R,5R)-2-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-7,7- dimethyl-4-bicyclo[3.1.1]hept-3-enyl] methanol) has the following structure:
  • cannabinoid receptor agonists are commonly referred to as the benzoylindoles.
  • This class includes 3-phenylacetylindole or 3-benzoylindole by substitution at the nitrogen atom of the indole ring, whether or not further substituted in the indole ring to any extent, whether or not substituted on the phenyl ring to any extent.
  • This class comprises the following molecules as examples: AM-2201 (1-(5-fluoropentyl)-3-(1-naphthoyl)indole) has the following chemical structure:
  • AM-694 (1-(5-fluoropentyl)-3-(2-iodobenzoyl)indole) has the following chemical structure:
  • RCS-4 (1-pentyl-3-(4-methoxybenzoyl)indole) has the following chemical structure:
  • RCS-8 or 1-(2-cyclohexylethyl)-3-(2-methoxyphenylacetyl)indole has the following chemical structure:
  • Additional classes include the AB, WIN, ADB, PB and UR classes. Representative molecules from these classes are shown below:
  • AB-001 has the following chemical structure:
  • WIN 55,212-2 has the following chemical structure:
  • ABD-PINACA has the following chemical structure:
  • PB-22 has the following chemical structure:
  • UR-144 has the following chemical structure:
  • panel 101 illustrates the traditional method of detecting cannabinoid receptor agonists in a patient sample.
  • Triangles 103 represent known cannabinoid receptor agonists present in the patent sample, while arrows 104 represent newly developed cannabinoid receptor agonists, for which effective detection tests have not yet been designed.
  • a panel of antibodies (105) specific to the known cannabinoid receptor agonists are added to the sample. If the target compounds are present in the sample, the antibodies will bind to the target and undergo a reporter reaction (106), thus allowing the person conducting the assay to determine if the target compound is present.
  • this method is only capable of detecting cannabinoid receptor agonists for which specific antibodies have been developed. Therefore, it will not detect novel or otherwise uncharacterized cannabinoid receptor agonists (104), regardless of the ability of those compounds to activate the cannabinoid receptors (107) of the patient’s cells (108).
  • Panel 102 illustrates one embodiment of the present invention. In this
  • the binding of any cannabinoid receptor agonist, whether known or unknown, to the cannabinoid receptor (107) of the target cell (109) results in a reporter reaction.
  • the reporter reaction may trigger a protein (111) to undergo a colorimetric change (110).
  • a transgenic cell line may be generated by inserting the required gene into the genome of the target cell line to produce a cell line useful for this assay.
  • the process of producing such a transgenic cell line will vary based on the type of cells being modified, but the standard procedures for doing so are well known in the art.
  • the assay of the present invention will make use of one or more of the following cell types: Chinese hamster ovary (CHO) cells, Human embryonic kidney 293 (HEK-293) cells, human brain astrocytoma ( 1321 N 1 ) , Human T-cell leukemia (1301), Human glioblastoma astrocytoma (U373 MG (Uppsala)), human monocytic leukemia (THP 1), Metastatic rhabdomyosarcoma cell line (RH-30), Telomerase-immortalized human microvascular endothelial cells (TIME), sinoatrial node (Shox2), and NHCF-A Human Cardiac Fibroblasts-Atrial. If the chosen cell line naturally expresses a
  • genome editing techniques such as CRISPR, may be used to remove the gene in order to create a control cell line.
  • the receptors of the present invention may be present in synthetic lipid bilayer, rather than the cellular membrane of a living cell.
  • Methods of producing such a system are known in the art, and one example is disclosed in US. Pat. No. 6,790,632 to Zweig, which is incorporated herein by reference. Use of such a synthetic system would remove the necessity of culturing live cells while also allow for more rapid screening techniques, such as affixing the assay to a dipstick.
  • the activation of cannabinoid receptors may be detected by assaying, for example, phosphatidylinositol turnover, cyclic adenosine monophosphate (cAMP) levels, or changes in intracellular calcium.
  • activation of CB1 could be assayed by detecting the amount of fluorescently tagged cAMP that is present in the target cells.
  • the CB1 receptor exhibits its effects mainly through activation of Gi, thus activation of the CB1 receptor results in a reduction of intracellular cAMP.
  • activation of the receptor by a cannabinoid receptor agonist would result in a reduction in the fluorescent signal in the target cells, relative to untreated control cells.
  • changes in intracellular calcium levels may be used as a reporter of CB1 activation.
  • embodiments of the present invention may be used to detect more than the mere presence or absence of a cannabinoid receptor agonist. They may also be used to detect the relative potency of said cannabinoids in the sample. By comparing the relative change in cannabinoid receptor signaling activity of the test sample that of a known control, such a THC, the relative potency of the cannabinoid receptor agonist in the sample can be determined. As such, this novel system has numerous benefits over the existing method of detecting cannabinoid related compounds.
  • Detection method Bioluminescence resonance energy transfer (BRET) was utilized for detection of cannabinoids.
  • HEK-293 cells were transfected with CAMYEL and the human CB1 Receptor.
  • hCB1 receptor binds cannabinoids, decreasing cAMP.
  • CAMYEL binds cAMP allowing for BRET.
  • Coelenterazine is used as the substrate to provide excitation for BRET probe.
  • Cells are treated with forskolin prior to addition of cannabinoid in order to increase cAMP levels.
  • cAMP levels are determined using the mean bioluminescence ratio. Fluorescence sampled every 54 seconds.
  • CAMYEL and CB1 expression plasmids were co-transfected into HEK cells.
  • the CAMYEL protein contains both luciferase and YFP connected by a high affinity cAMP binding site.
  • the presence of cAMP is detected by measuring the emission at 460 nm (Rluc) and 535 nm (YFP).
  • the inverse BRET ratio is calculated by dividing the donor signal (460nm) by the acceptor signal (535nm).
  • CAMYEL was transfected into HEK cells. Unlike Example 1 , CB1 was not transfected. As forskolin caused an increase in cAMP signal, it demonstrates (1) that CAMYEL was successfully transfected and (2) that adenylate cyclase is active and can make cAMP (FIG. 3). However, as the HEK cells do not express CB1 , this demonstrates that the presence of CB1 is required to see increased cAMP upon 5F-ADB-PINACA addition.
  • AM251 blocks the cAMP signal
  • Example 1 through Example 4 strongly demonstrate that this method is measuring activation of CB1. Others have seen cAMP levels decrease. Our hypothesis is that the cell line chosen contained a Gs protein capable of interacting with CB1 and activating adenylate cyclase. While non-physiological, this observation demonstrates that the present method is capable of detecting a synthetic cannabinoid via changes in cAMP in real time.
  • FRET analysis of movement of the 3 rd Intracellular Loop in Response to THC 2 photon microscopy is a means of obtaining imaging data from a very thin slice of tissue - similar to confocal microscopy.
  • the detector only accepts data if it comes in bursts of 2 photons from the same pixel that arrive at virtually the same time. This generally only happens if the two photons originated at the site of the exact focal plane. Single photon hits on the detector are excluded/ignored. Thirty pictures need to be collected from the same spot on the plate to collect enough photons to give an image (FIG. 6).
  • FRET Forster Resonance Energy Transfer
  • FRET FRET occurs, the detectable acceptor emission increases (excitation) while at the same time, the detectable donor emission decreases (quenching).
  • CFP the quenching of the donor
  • the detector in the 2-photon microscope is filtered to look at emissions within the CFP energy window. If decrease in emission is detected, then FRET is occurring.
  • FRET measurement Fluorescence Lifetime Imaging
  • the fluorescent protein, CFP is expressed as a domain at the C-terminus of CB1.
  • CFP will function as the donor.
  • a fluorescent dye called FIAsH-Edt2 functions as the acceptor.
  • FIAsH binds to a 6 amino acid sequence -CCGPCC.
  • An engineered -CCGPCC- sequence is expressed either at position 940 or at position 958 in CB1. This places FIAsH on the 3rd intracellular loop of CB1. In this experiment, it is hypothesized that different parts of the 3rd intracellular loop will move in response to cannabinoid binding.
  • cells are transfected with a plasmid encoding the CB1 FRET construct (either 940 or 958).
  • Cells are imaged using 2-photon microscopy and data collected on CFP emission in the absence of FIAsH.
  • FIAsH (1 mM) is added to the culture well and allowed to diffuse into the cell for 20 min (approx.).
  • CFP emission is measured again. If CFP emission falls, FRET is occurring.
  • a cannabinoid is then added to the culture well and the measure is repeated. If the FRET signal changes, then the 3rd intracellular loop is moving in response to the cannabinoid binding to CB1.
  • CFP has a lifetime emission of approximately 2 ns. This is true for both the 940 construct and the 958 construct.

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Abstract

La présente invention concerne un nouveau dosage pour la détection d'agonistes du récepteur cannabinoïde dans un échantillon, sans qu'il ne soit nécessaire d'avoir préalablement caractérisé la molécule. De plus, la présente invention concerne un nouveau dosage capable de cribler tous les agonistes du récepteur cannabinoïde qui peuvent être présents dans un échantillon simultanément. En outre, la présente invention concerne un procédé de criblage d'un échantillon de patient pour la présence de cannabinoïdes naturels ou synthétiques à l'aide d'un nouveau dosage.
PCT/US2020/033121 2019-05-15 2020-05-15 Dosages pour cannabinoïdes synthétiques Ceased WO2020232350A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002640A1 (fr) * 1990-08-08 1992-02-20 The United States Of America, Represented By The Secretary, United States Department Of Commerce Recepteur de cannabinoides
WO2005021547A2 (fr) * 2003-08-28 2005-03-10 Pharmaxis Pty Ltd. Nouveaux agonistes des recepteurs cannabinoides cb2 et utilisations desdits agonistes
US20050059135A1 (en) * 1993-03-31 2005-03-17 Cadus Pharmaceutical Corporation Methods and compositions for identifying receptor effectors
US20080234293A1 (en) * 2007-03-02 2008-09-25 The University Of Tennessee Research Foundation Tri-aryl/heteroaromatic cannabinoids and use thereof
US20130158025A1 (en) * 2010-08-31 2013-06-20 Amorepacific Corporation Novel compound acting as a cannabinoid receptor-1 inhibitor
WO2019144126A1 (fr) * 2018-01-22 2019-07-25 Pascal Biosciences Inc. Cannabinoïdes et leurs dérivés pour favoriser l'immunogénicité des cellules tumorales et infectées

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992002640A1 (fr) * 1990-08-08 1992-02-20 The United States Of America, Represented By The Secretary, United States Department Of Commerce Recepteur de cannabinoides
US20050059135A1 (en) * 1993-03-31 2005-03-17 Cadus Pharmaceutical Corporation Methods and compositions for identifying receptor effectors
WO2005021547A2 (fr) * 2003-08-28 2005-03-10 Pharmaxis Pty Ltd. Nouveaux agonistes des recepteurs cannabinoides cb2 et utilisations desdits agonistes
US20080234293A1 (en) * 2007-03-02 2008-09-25 The University Of Tennessee Research Foundation Tri-aryl/heteroaromatic cannabinoids and use thereof
US20130158025A1 (en) * 2010-08-31 2013-06-20 Amorepacific Corporation Novel compound acting as a cannabinoid receptor-1 inhibitor
WO2019144126A1 (fr) * 2018-01-22 2019-07-25 Pascal Biosciences Inc. Cannabinoïdes et leurs dérivés pour favoriser l'immunogénicité des cellules tumorales et infectées

Non-Patent Citations (2)

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Title
KUMAR PRITESH; SONG ZHAO-HUI: "Identification of Raloxifene as a Novel CB2 Inverse Agonist", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 435, no. 1, 24 May 2013 (2013-05-24), pages 76 - 81, XP028552953, DOI: 10.1016/j.bbrc.2013.04.040 *
MICHELINI ELISA; CEVENINI LUCA; CALABRETTA MARIA MADDALENA; CALABRIA DONATO; RODA ALDO: "Exploiting In Vitro and In Vivo Bioluminescence for the Implementation of the Three Rs Principle (Replacement, Reduction, and Refinement) in Drug Discovery", ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 406, no. 23, 1 September 2014 (2014-09-01), pages 5531 - 5539, XP035377903, DOI: 10.1007/s00216-014-7925-2 *

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