Compositions and methods for treating neurological disorders with combination products - Patents.com

Abstract

The present invention relates to a composition comprising a cannabinoid together with additional active ingredients. The present invention also relates to pharmaceutical compositions, dosage forms, and methods of treating neurological disorders by administering the composition to a patient in need thereof. In a first aspect, the present invention broadly resides in a composition comprising the following cannabinoids: about 50 w / w% CBDA, with all other cannabinoids amounting to about 15 w / w%. In a second aspect, the present invention is a pharmaceutical composition comprising the composition of the first aspect of the present invention together with a pharma- ceutically acceptable carrier.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to compositions comprising cannabinoids together with additional active ingredients. The present invention also relates to pharmaceutical compositions, dosage forms, and methods of treating neurological disorders by administering the compositions to a patient in need thereof. [Background technology]

[0002] background The following background discussion is intended only to facilitate an understanding of the present invention. The discussion is not an admission or acknowledgement that any of the material referred to is or was part of the common general knowledge as of the priority date of the application.

[0003] A. Neuroinflammation Neuroinflammation refers to the process by which the brain's innate immune system is triggered following an inflammatory challenge, such as that brought about by injury, infection, exposure to toxins, neurodegenerative disease, or aging. Neuroinflammation has been implicated in contributing to a variety of neurological and physical diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, traumatic brain injury, rheumatoid arthritis, chronic migraine, epilepsy, autism spectrum disorder (ASD), attention-deficit hyperactivity disorder (ADHD), cerebral palsy and related subtypes, neuropathic pain, and depression.

[0004] In the central nervous system (CNS), innate immune responses play a significant role in both physiological and pathological conditions. CNS diseases, including traumatic brain injury, ischemic stroke, brain tumors, and cerebrovascular and neurodegenerative diseases, trigger a cascade of events broadly defined as neuroinflammation, which is characterized by the activation of microglial and astroglial populations. On the other hand, microglial and astroglial activation, T lymphocyte infiltration, and overproduction of inflammatory cytokines have been demonstrated in association with neuronal alterations in both animal and human tissues. Thus, neuroinflammation is an important topic in modern neuroscience.

[0005] Inflammatory or pro-inflammatory cytokines / markers are types of signaling molecules secreted by immune cells such as helper T cells and macrophages, as well as certain other cell types, that promote the process of neuroinflammation and general inflammation. These include interleukin-1 (IL-1), IL-12 and IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). These inflammatory cytokines are primarily produced by and involved in the upregulation of inflammatory responses and play an important role in mediating innate immune responses.

[0006] B. Neuropathy Examples of neurological disorders that are "neuroinflammation-based" include Alzheimer's disease (Alzheimer's disease is the most prevalent chronic, progressive neurodegenerative disease and cause of dementia), Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, traumatic brain injury, rheumatoid arthritis, chronic migraine, epilepsy, autism spectrum disorder, attention-deficit hyperactivity disorder, cerebral palsy and related subtypes, neuropathic pain, and depression.

[0007] C. Microglial Activation / Neurodegeneration Microglial cells are resident macrophages unique to the central nervous system (CNS). They play important roles during CNS development and adult homeostasis. They have a major contribution to adult neurogenesis and neuroinflammation (Zhan Y., Paolicelli RC, Sforazzini F., et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nature Neuroscience. 2014;17(3):400-406;Guruswamy R, ElAli A. Complex Roles of Microglial Cells in Ischemic Stroke Pathobiology: New Insights and Future Directions. Int J Mol Sci. 2017;18:18). Hence, they are involved in the pathogenesis of neurodegenerative diseases and contribute to aging. They play a key role in the maintenance and breakdown of the blood-brain barrier. As innate immune cells, they contribute substantially to the immune response against infectious pathogens that invade the CNS (Xiong XY, Liu L, Yang QW. Functions and mechanisms of microglia / macrophages in neuroinflammation and neurogenesis after stroke. Prog Neurobiol. 2016;142:23-44). They also play a major role in the growth of tumors in the CNS. Microglia are, as a result, the main cell population that connects the nervous and immune systems (Xiong XY, Liu L, Yang QW. Functions and mechanisms of microglia / macrophages in neuroinflammation and neurogenesis after stroke. Prog Neurobiol. 2016;142:23-44).

[0008] Under physiological conditions, ramified, dormant microglia provide a neuroprotective environment (David S, Greenhalgh AD, Kroner A. Macrophage and microglial plasticity in the injured spinal cord. Neuroscience. 2015;307:311-18;Bieber K, Autenrieth SE. Insights how monocytes and dendritic cells contribute and regulate immune defense against microbial pathogens. Immunobiology. 2015;220:215-26). However, most CNS pathologies, and regenerative efforts, involve microglial activation with corresponding inflammatory events (Hoogland IC, Houbolt C, van Westerloo DJ, van Gool WA, van de Beek D. Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation. 2015;12:114;Ascoli BM, Gea LP, Colombo R, Barbe-Tuana FM, Kapczinski F, Rosa AR. The role of macrophage polarization on bipolar disorder: identifying new therapeutic targets. Aust NZJ Psychiatry. 2016;50:618-30;Cherry JD, Olschowka JA, O'Banion MK. Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation. 2014;11:98).Thus, activated inflammatory microglia are neurotoxic and kill neurons by engulfing them or by releasing a variety of neurotoxic molecules and factors, including reactive oxygen species (ROS), glutamate, Fas ligand, tumor necrosis factor alpha (TNFα), and others (Loane DJ, Kumar A. Microglia in the TBI brain: the good, the bad, and the dysregulated. Exp Neurol. 2016;275:316-27; Nakagawa Y, Chiba K. Diversity and plasticity of microglial cells in psychiatric and neurological disorders. Pharmacol Ther. 2015;154:21-35).

[0009] Activated microglia, which drive chronic neuroinflammation, have been implicated in CNS aging (Loane DJ, Kumar A. Microglia in the TBI brain: the good, the bad, and the dysregulated. Exp Neurol. 2016;275:316-27), chronic neuropathic pain (Orihuela R, McPherson CA, Harry GJ. Microglial M1 / M2 polarization and metabolic states. Br J Pharmacol. 2016;173:649-65) and psychiatric disorders (Orihuela R, McPherson CA, Harry GJ. Microglial M1 / M2 polarization and metabolic states. Br J Pharmacol. 2016;173:649-65), as well as Alzheimer's disease (Nakagawa Y, Chiba K. Diversity and plasticity of microglial cells in psychiatric and neurological disorders. Pharmacol Ther. 2015;154:21-35), Parkinson's disease (Orihuela R, McPherson CA, Harry GJ. Microglial M1 / M2 polarization and metabolic states. Br J Pharmacol. 2016;173:649-65), amyotrophic lateral sclerosis (ALS) and multiple sclerosis. Aging is paralleled by systemic chronic activation and polarization of the immune system toward a low-level inflammatory state (Ransohoff RM. A polarizing question: do M1 and M2 microglia exist? Nat Neurosci. 2016;19:987-91;Tang Y, Le W. Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases. Mol Neurobiol. 2016;53:1181-94).

[0010] D. Medicinal Applications of Cannabis Cannabis sativa L. has a tradition of medical use. Medicinal cannabis has attracted great interest due to its anti-inflammatory, antioxidant and anti-necrotic protective effects, as well as displaying a favorable safety and tolerability profile in humans, making it a promising candidate in many therapeutic approaches. However, clinical use has been limited due to its adverse effects on the central nervous system and the potential for abuse and addiction. The plant exudes a resin that contains a mix of cannabinoids, with two main components, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). The structure of CBD was described in the 1960s and has attracted attention due to its lack of psychoactive activity. Due to its good tolerance in humans, lack of psychoactive effects and low abuse potential, it seems ideal for clinical trials.

[0011] E. Cannabinoids In addition to its good safety profile and lack of psychoactive effects, CBD also presents a wide range of therapeutic effects. Several experimental in vitro and in vivo studies have demonstrated anti-inflammatory as well as immune-modulating, antipsychotic, analgesic and antiepileptic actions. For these reasons, CBD is currently one of the most studied cannabinoids. Compared to Δ9-THC, CBD exhibits a lower affinity for cannabinoid receptors type 1 (CB1) and type 2 (CB2). CB1 receptors are found primarily in the terminals of central and peripheral neurons, and CB2 receptors primarily in immune cells. Several in vitro studies have shown that CBD, at low concentrations, has a weak CB1 and CB2 antagonistic effect.

[0012] Studies suggest that CBD behaves as a negative allosteric modulator of CB1, meaning that CBD does not directly activate the receptor but modifies the potency and efficacy of CBD1's orthosteric ligands: Δ9-THC and 2-arachidonoylglycerol (2-AG). These preliminary results require further validation but may explain CBD's ability to antagonize some of the effects of Δ9-THC reported in vitro, in vivo and human clinical studies. It has also been suggested that CBD's role as an allosteric modulator of CB1 could explain its therapeutic role in the treatment of central and peripheral nervous system disorders. CBD has also been shown to inhibit neutrophil chemotaxis and proliferation. CBD may also induce arachidonic acid release and reduce prostaglandin E2 (PGE2) and nitric oxide (NO) production.

[0013] However, not all of the physiological effects of CBD are mediated by cannabinoid receptors. CBD has numerous targets outside the endocannabinoid system, and its actions independent of cannabinoid receptors are the subject of recent pharmacological research. Some effects, such as the anti-inflammatory and immunosuppressive effects, are mediated by more than one target. The anti-inflammatory and immunosuppressive effects are probably due to adenosine receptor A activation. 1A and A 2A The activity of CBD is mediated by the activation of strychnine-sensitive α1 and α1β glycine receptors and the inhibition of passive equilibrative nucleoside transporters. Furthermore, the activity of CBD may derive different physiological effects from the same target. For example, the same glycine receptor is involved in both anti-inflammatory and neuropathic pain suppression. Although the effect on the serotonin 5HT1A receptor may generate anxiolytic, panicolytic and antidepressant effects, research has provided a thorough overview of the molecular pharmacology of CBD. Despite the progress in the molecular pharmacology of CBD, many pharmacological mechanisms of CBD remain uncharacterized.

[0014] Published studies in animals have demonstrated that the oral bioavailability of cannabidiol has been shown to be approximately between 13-19%. Plasma and brain concentrations are dose-dependent in animals, and bioavailability is increased with various oil formulations. Cannabinoids undergo extensive first-pass metabolism, with metabolites mostly excreted via the kidneys.

[0015] Cannabinoids are extensively metabolized by the liver, where they are hydroxylated by P450 enzymes, primarily the CYP3A (2 / 4) and CYP2C (8 / 9 / 19) families of isoenzymes, to 7-OH-CBD, which then undergoes extensive further metabolism in the liver and the resulting metabolites are excreted in the feces and, to a much lesser extent, in the urine.

[0016] Cannabidiol is known to act on cannabinoid (CB) receptors (CB1 and CB2) of the endocannabinoid system, which are found in many areas of the body, including the peripheral and central nervous systems, including the brain. The endocannabinoid system regulates many physiological responses of the body, including pain, memory, appetite and mood. More specifically, CB1 receptors can be found in pain pathways in the brain and spinal cord, where they can affect cannabidiol-induced analgesia and anxiolysis, and CB2 receptors have effects on immune cells, where they can affect cannabidiol-induced anti-inflammatory processes.

[0017] Cannabidiol has been shown to act as a negative allosteric modulator of the cannabinoid CB1 receptor, the most abundant G protein-coupled receptor (GPCR) in the body. Allosteric modulation of a receptor is achieved by modulation of the activity of the receptor at a site that is functionally distinct from the agonist or antagonist binding site. The negative allosteric modulatory effects of cannabidiol are therapeutically important because direct agonists are limited by their psychomimetic effects, whereas direct antagonists are limited by their inhibitory effects. There has been some progress in regulatory approval of CBD. Epidiolex® is a plant-derived, pharmaceutical-grade cannabidiol (CBD) drug that gained FDA approval for use in the United States in 2018. Epidiolex® contains 100 mg of cannabidiol per milliliter (mL) of solution and is taken orally twice daily. The Australian Therapeutic Goods Administration (TGA) approved Epidiolex in September 2020 for the treatment of seizures associated with Lennox-Gastaut syndrome (LGS) or Dravet syndrome in patients aged 2 years or older. [Prior art documents] [Non-patent literature]

[0018] [Non-Patent Document 1] Zhan Y., Paolicelli RC, Sforazzini F., et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nature Neuroscience. 2014;17(3):400-406 [Non-Patent Document 2] Guruswamy R, ElAli A. Complex Roles of Microglial Cells in Ischemic Stroke Pathobiology: New Insights and Future Directions. Int J Mol Sci. 2017;18:18 [Non-Patent Document 3] Xiong XY, Liu L, Yang QW. Functions and mechanisms of microglia / macrophages in neuroinflammation and neurogenesis after stroke. Prog Neurobiol. 2016;142:23-44 [Non-Patent Document 4] Xiong XY, Liu L, Yang QW. Functions and mechanisms of microglia / macrophages in neuroinflammation and neurogenesis after stroke. Prog Neurobiol. 2016;142:23-44 [Non-Patent Document 5] David S, Greenhalgh AD, Kroner A. Macrophage and microglial plasticity in the injured spinal cord. Neuroscience. 2015;307:311-18 [Non-Patent Document 6] Bieber K, Autenrieth SE. Insights how monocytes and dendritic cells contribute and regulate immune defense against microbial pathogens. Immunobiology. 2015;220:215-26 [Non-Patent Document 7] Hoogland IC, Houbolt C, van Westerloo DJ, van Gool WA, van de Beek D. Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation. 2015;12:114 [Non-Patent Document 8] Ascoli BM, Gea LP, Colombo R, Barbe-Tuana FM, Kapczinski F, Rosa AR. The role of macrophage polarization on bipolar disorder: identifying new therapeutic targets. Aust NZJ Psychiatry. 2016;50:618-30 [Non-Patent Document 9] Cherry JD, Olschowka JA, O'Banion MK. Neuroinflammation and M2 microglia: the good, the bad, and the inflamed. J Neuroinflammation. 2014;11:98 [Non-Patent Document 10] Loane DJ, Kumar A. Microglia in the TBI brain: the good, the bad, and the dysregulated. Exp Neurol. 2016;275:316-27 [Non-Patent Document 11] Nakagawa Y, Chiba K. Diversity and plasticity of microglial cells in psychiatric and neurological disorders. Pharmacol Ther. 2015;154:21-35 Summary of the Invention [Means for solving the problem]

[0019] There is a need in the art for improved cannabinoid compositions and effective treatments for neurological disorders. It is an object of the present invention to overcome one or more of the problems foreseen by the prior art.

[0020] Summary of the Invention In a first aspect, the present invention broadly resides in a composition comprising the following cannabinoids: about 50% w / w CBDA, with all other cannabinoids amounting to about 15% w / w.

[0021] In a preferred embodiment, the composition comprises the following cannabinoids: w / w% CBDA 40-60%; CBD 1-5%; CBG 1-10%; CBDP 1-5%; CBDB 1~5%; CBGA 1-10%; CBN 1-3%; THC<1% and additional active ingredients Includes.

[0022] In another preferred embodiment, the composition comprises the following cannabinoids: w / w% CBDA 50%; CBD 2%; CBG 5%; CBDP 2%; CBDB 2%; CBGA 5%; CBN 1-3%; THC<0.3% and additional active ingredients Includes.

[0023] In another preferred embodiment, the composition comprises the following cannabinoids: w / w% CBDA 49%; CBD 2%; CBG 5%; CBDP 2%; CBDB 2%; CBGA 5%; CBN 3%; THC<0.3% and additional active ingredients Includes.

[0024] In another preferred embodiment, the composition comprises the following cannabinoids: w / w% CBDA 45%; CBD 1%; CBG 4%; CBDP 1%; CBDB 2%; CBGA 4%; CBN 2%; THC<0.2% and additional active ingredients Includes.

[0025] In another preferred embodiment, the composition comprises the following cannabinoids: w / w% CBDA 45%; CBD 1%; CBG 4%; CBDP 1%; CBDB 2%; CBGA 4%; CBN 1%; THC<0.2% and additional active ingredients Includes.

[0026] In a preferred embodiment, the composition comprises a cannabinoid in an amount selected from the group consisting of any one of the embodiments described above.

[0027] In a second aspect, the present invention is a pharmaceutical composition comprising the composition of the first aspect of the invention together with a pharma- ceutically acceptable carrier.

[0028] In a third aspect, the present invention is a dosage form comprising the composition of the first aspect of the invention.

[0029] In a fourth aspect, the invention is a method of treating a disorder, comprising administering to a patient in need thereof a therapeutically effective amount of a dosage form of the invention.

[0030] In a fifth aspect, the invention is the use of a composition of the invention in the manufacture of a medicament for the treatment of a disorder.

[0031] In a sixth aspect, the present invention provides a process for extracting a composition of the present invention from cannabis plant material comprising: 1) grinding cannabis plant material to a sufficient grind size; 2) contacting the grounds produced by step a) with oil; 3) mixing the grinds and oil for a sufficient period of time to form a mixture; 4) squeezing the mixture to recover the oil; 5) centrifuging the oil to further refine the oil; 6) collecting the oil extract in a suitable container / steel vessel; It is a process that includes:

[0032] In a seventh aspect, the present invention provides a process for extracting a composition of the present invention from cannabis plant material comprising: 1) grinding cannabis plant material to a sufficient grind size; 2) contacting the ground material produced by step a) with an alcohol; 3) mixing the grind and alcohol for a sufficient period of time to form a mixture; 4) sonicating the mixture; 5) centrifuging the mixture; 6) collecting the alcoholic extract in a suitable container / steel vessel; It is a process that includes:

[0033] In an eighth aspect, the invention is a product produced by the process of the invention.

[0034] In a ninth aspect, the invention is a kit comprising a dosage form of the invention together with instructions for its use.

[0035] In a ninth aspect, the present invention includes compositions, methods and processes as described by the following examples.

[0036] Further features of the present invention are more fully described in the following description of certain non-limiting embodiments thereof, which description is included solely for the purpose of illustrating the present invention and should not be understood as a limitation on the broad summary, disclosure or description of the invention as set forth above.

[0037] Below is a brief description of each of the figures and drawings. [Brief description of the drawings]

[0038] [Figure 1] FIG. 1 is a UPLC mass chromatogram of a cannabinoid standard mixture (10 ppm each) in a) positive and b) negative ionization mode.

[0039] [Diagram 2] FIG. 2 is the in-source fragmentation of CBD and CBG from the reference solution.

[0040] [Diagram 3] FIG. 3 shows the mass spectrometry chromatogram for NTI164.

[0041] [Figure 4] FIG. 4 shows quadrupole mass spectrometry chromatograms of CBD variants of NTI164 to identify CBDB and CBDP.

[0042] [Diagram 5] Figure 5 depicts normalization of inflammation-induced iNOS expression by NTI164. The figure shows that NTI164 normalizes inflammation-induced iNOS expression.

[0043] [Figure 6] Figure 6 presents neuronal viability quantified using MTT [3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl-2H-tetrazolium bromide]. The figure shows that NTI164 increases the number of neurons under basal conditions (short-term exposure).

[0044] [Figure 7] Figure 7 demonstrates that NTI164 stimulates the maturation of immature neurons into healthy cells, even in the absence of any glutamate-induced damage. The figure shows the effect of NTI164 alone (without glutamate) on neurons.

[0045] [Figure 8] Figure 8 demonstrates that CBD is toxic in this paradigm, whereas NTI164 is non-toxic and has a positive effect on cell number and cell viability. The figure shows that NTI164 does not increase cell death in an excitotoxic cytotoxicity paradigm.

[0046] [Figure 9] Figure 9 shows the response of microglia under inflammatory conditions assessing Arginase 1 expression. The figure shows that NTI164 normalizes proinflammatory (injured cell) Arg1 expression.

[0047] [Figure 10] FIG. 10 is a schematic outlining arginine metabolism and the effect it has on the overall balance of anti-inflammatory and pro-inflammatory signals (Reference: Review. Goncalo S. Clemente, Aren van Waarde, Ines F. Antunes, Alexander Domling and Philip H. Elsinga. Arginase as a Potential Biomarker of Disease Progression: A Molecular Imaging Perspective. (2020)).

[0048] [Figure 11] FIG. 11 shows the distribution of patients actively using NTI164 for Example 10.

[0049] [Figure 12] FIG. 12 shows the distribution of disease severity of active patients at baseline according to CGI-S severity of disease for Example 10.

[0050] [Figure 13] FIG. 13 shows the maximum tolerated dose for effective patients for Example 10.

[0051] [Figure 14] FIG. 14 shows CGI-S global improvement upon 28 days of NTI164 treatment.

[0052] [Figure 15] FIG. 15 shows the CGI-S severity of disease after 28 days of treatment.

[0053] [Figure 16] FIG. 16 shows the CGI-S severity of disease after 28 days of treatment.

[0054] [Figure 17] FIG. 17 shows the CGI-S therapeutic effect after 28 days of treatment.

[0055] [Figure 18] FIG. 18 shows the age distribution of patients actively using NTI164 for Example 11.

[0056] [Figure 19] Figure 19 shows the distribution of illness severity of valid patients at baseline according to CGI-S severity of illness for Example 11. CGI-S stands for Clinical Global Impression Scale-Severity of Illness.

[0057] [Figure 20] FIG. 20 shows CGI-S global improvement during 20 weeks of NTI164 treatment.

[0058] [Figure 21] FIG. 21 shows CGI-S global improvement over time of NTI164 treatment up to and including 20 weeks.

[0059] [Figure 22] FIG. 22 shows the CGI-S severity of disease over 20 weeks of treatment.

[0060] [Figure 23] FIG. 23 shows the CGI-S severity of disease over time up to and including 20 weeks of treatment.

[0061] [Figure 24] FIG. 24 shows the CGI-S severity of disease over 20 weeks of treatment.

[0062] [Diagram 25] FIG. 25 shows the CGI-S therapeutic effect over time for up to and including 20 weeks of treatment.

[0063] [Figure 26] FIG. 26 shows the effect of CGI-S treatment over 20 weeks of treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Detailed Description of the Invention For convenience, the following section outlines various meanings of terms generally used herein. In accordance with this discussion, general aspects relating to the compositions, pharmaceutical uses and methods of the invention will be discussed, followed by specific examples that demonstrate the properties of various embodiments of the invention and how they may be used.

[0065] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described.The present invention includes all such variations and modifications.The present invention includes all steps, features, formulations and compounds individually or collectively referred to or indicated in the specification, as well as any and all combinations or any two or more of the steps or features.

[0066] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in its entirety by reference, meaning that it should be read and considered as part of this text by the reader. It is only for the sake of brevity that the documents, references, patent applications or patents cited in this text are not repeated in this text. However, neither the cited materials nor the information contained therein should be understood to be general knowledge.

[0067] Manufacturers' instructions, descriptions, product specifications, and product sheets for any products mentioned herein, or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of the present invention.

[0068] The present invention is not limited in scope by any of the specific embodiments described herein. These embodiments are intended for illustrative purposes only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0069] 1.Definition The meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided in the specification shall control.

[0070] Except in the working examples or where specifically indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood in all instances as being modified by the term "about." When used in connection with percentages, the term "about" can mean ±1%.

[0071] The invention described herein may include one or more ranges of values ​​(e.g., size, concentration, etc.). A range of values ​​will be understood to include all values ​​within the range, including the values ​​defining the range and the values ​​adjacent to the range that lead to the same or substantially the same result as the values ​​immediately adjacent to the values ​​defining the range boundary. For example, one skilled in the art will understand that a 10% variation within the upper or lower limit of a range may be entirely appropriate and is encompassed by the present invention. More specifically, a variation within the upper or lower limit of a range will be 5%, or whichever is greater, as is generally recognized in the art.

[0072] In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" and other forms such as "includes" and "comprises" is not limiting. Also, terms such as "element" or "component" are inclusive of both elements and components that contain one unit and elements and components that contain more than one subunit, unless specifically stated otherwise. Also, the use of the term "part" can include a part of a moiety or the entire moiety.

[0073] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0074] "Therapeutically effective amount", as used herein with respect to methods of treatment and particularly drug dosage, shall mean a dosage that provides a specific pharmacological response in a significant number of subjects in need of such treatment, at which the drug is administered. It is emphasized that a "therapeutically effective amount" administered to a particular subject in a particular case will not always be effective in treating the diseases described herein, even if such a dosage is considered a "therapeutically effective amount" by those skilled in the art. It is further understood that drug dosage is measured in a particular case as an oral dosage or with reference to drug levels as measured in blood. The amount effective for such use will depend on the desired therapeutic effect, the potency of the biologically active material, the desired duration of treatment, the stage and severity of the disease being treated, the weight and general health of the patient, and the judgment of the prescribing physician. Treatment dosages need to be titrated to optimize safety and effectiveness. Thus, one of ordinary skill in the art will know that appropriate dosage levels for treatment will vary, in part, depending on the indication for which the active agent is being used, the route of administration, and the size (weight, body surface or organ size) and condition (age and general health) of the patient. Thus, the clinician may titrate the dosage and modify the route of administration to obtain optimal therapeutic effect. Typical dosages may range from about 0.1 μg / kg up to about 100 mg / kg or more, depending on the factors mentioned above. In other embodiments, dosages may range from 0.1 μg / kg up to about 100 mg / kg, or 1 μg / kg up to 100 mg / kg, or 5 μg / kg up to about 100 mg / kg.

[0075] The frequency of dosing will depend on the pharmacokinetic parameters of the active agent and formulation used. Typically, the clinician will administer the composition until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is within the realm of tasks that are routinely made and routinely performed by those skilled in the art. The appropriate dosage can be ascertained by the use of appropriate dose-response data.

[0076] As used herein, "pharmacologically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0077] As used herein, the term "subject" generally includes mammals, such as humans; livestock animals, such as sheep, goats, pigs, cows, horses, llamas, etc.; companion animals, such as dogs and cats; primates; birds, such as chickens, geese, and ducks; fish; and reptiles. The subject is preferably a human.

[0078] Other definitions for selected terms used herein may be found within the detailed description of the invention and may be applied throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0079] Features of the present invention will now be discussed with reference to the following non-limiting descriptions and examples.

[0080] 2. Embodiment composition The present invention relates to the following cannabinoids: Approximately 50 w / w% CBDA Including, All other cannabinoids amount to approximately 15% w / w. A composition is provided.

[0081] Preferably, the composition includes an additional active ingredient.

[0082] In a preferred embodiment, the present invention provides the following cannabinoids: w / w% Approximately 50% CBDA, Approximately 2% CBD, plus additional active ingredients The present invention provides a composition comprising:

[0083] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% Approximately 50% CBDA, Approximately 5% CBG, plus additional active ingredients The present invention provides a composition comprising:

[0084] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% Approximately 50% CBDA, Approximately 2% CBDP, plus additional active ingredients The present invention provides a composition comprising:

[0085] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% Approximately 50% CBDA, Approximately 2% CBDB, plus additional active ingredients The present invention provides a composition comprising:

[0086] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% Approximately 50% CBDA, Approximately 5% CBGA, plus additional active ingredients The present invention provides a composition comprising:

[0087] In a further preferred embodiment, the present invention provides a composition comprising a cannabinoid, wherein the ratio of CBDA to all other cannabinoids is between 4:1 and 2:1.

[0088] In a further preferred embodiment, the present invention provides a composition comprising a cannabinoid, wherein the ratio of CBDA to all other cannabinoids is about 3:1.

[0089] In a further preferred embodiment, the present invention provides a composition comprising a cannabinoid, wherein the ratio of CBDA to all other cannabinoids is about 3.21:1.

[0090] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 40-60%, CBD 1-5%, CBG 1-10%, CBDP 1-5%, CBDB 1~5%, CBGA 1-10%, CBN 1-3%, THC<1% plus additional active ingredients The present invention provides a composition comprising:

[0091] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 45-55%, CBD 1-3%, CBG 3-7%, CBDP 1-3%, CBDB 1~3%, CBGA 3-7%, CBN 1-3%, THC<0.5% plus additional active ingredients The present invention provides a composition comprising:

[0092] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 50%, CBD 2%, CBG 5%, CBDP 2%, CBDB 2%, CBGA 5%, CBN 3%, THC<0.3% plus additional active ingredients The present invention provides a composition comprising:

[0093] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 49%, CBD 2%, CBG 5%, CBDP 2%, CBDB 2%, CBGA 5%, CBN 2%, THC<0.3% plus additional active ingredients The present invention provides a composition comprising:

[0094] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 48.78%, CBD 1.89%, CBG 4.88%, CBDP 1.68%, CBDB 1.76%, CBGA 4.76%, CBN 1%, THC<0.18% plus additional active ingredients The present invention provides a composition comprising:

[0095] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 45%, CBD 1%, CBG 4%, CBDP 1%, CBDB 2%, CBGA 4%, CBN 2%, THC<0.2% plus additional active ingredients The present invention provides a composition comprising:

[0096] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 45.28%, CBD 1.39%, CBG 3.88%, CBDP 1.18%, CBDB 1.56%, CBGA 3.76%, CBN 1%, THC<0.18% plus additional active ingredients The present invention provides a composition comprising:

[0097] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 62.78%, CBD 5.80%, CBG 0.44%, CBGA 1.26%, CBN 1.98%, THC<0.70% plus additional active ingredients The present invention provides a composition comprising:

[0098] In a further preferred embodiment, the present invention provides the following cannabinoids: w / w% CBDA 60.29%, CBD 5.34%, CBG 0.39%, CBGA 1.14%, CBN 0.85%, THC<0.65% plus additional active ingredients The present invention provides a composition comprising:

[0099] In a further preferred embodiment, the present invention relates to a compound comprising the cannabinoid: w / w% CBDA 50%, CBD 2%, CBG 5%, CBDP 2%, CBDB 2%, CBGA 5%, CBN 3%, THC<0.3%, and Additional Active Ingredients Composition 1 comprising And w / w% CBDA 45%, CBD 1%, CBG 4%, CBDP 1%, CBDB 2%, CBGA 4%, CBN 2%, THC<0.2%, and Additional Active Ingredients Composition 2 comprising is present in an amount selected from the group consisting of:

[0100] In a further preferred embodiment, the present invention relates to a method for determining the amount of cannabinoids in a sample, the amount of cannabinoids being determined by high performance chromatography (HPLC), proton nuclear magnetic resonance spectroscopy (H 1 The composition is provided in such a manner that the molecular weight is determined by a method selected from the group consisting of NMR, and mass spectrometry.

[0101] In a further preferred embodiment, the present invention provides compositions derived from cannabis plant material.

[0102] In a further preferred embodiment, the present invention provides a composition wherein the listed cannabinoids are synthetic.

[0103] In a further preferred embodiment, the present invention provides a composition wherein the listed cannabinoids are a mixture of plant-derived and synthetic cannabinoids.

[0104] In a further preferred embodiment, the present invention provides a composition further comprising an oil selected from the group consisting of synthetic oils, vegetable-based oils, mineral oils, canola oil, and olive oil.

[0105] In a further preferred embodiment, the composition comprises less than 5% w / w terpenes.

[0106] In a further preferred embodiment, the composition comprises less than 2% w / w organic plant material.

[0107] In a further preferred embodiment the composition comprises less than 2% w / w plant phenolics.

[0108] In a further preferred embodiment, the composition comprises a component selected from the group consisting of flavonoids, proteins, sterols and esters.

[0109] In a further preferred embodiment, the composition is substantially pure. Preferably, purity is measured by high performance chromatography (HPLC), proton nuclear magnetic resonance spectroscopy (H 1 Preferably, the purity is determined by a method selected from the group consisting of greater than 75% purity, greater than 80% purity, greater than 85% purity, greater than 90% purity, greater than 95% purity, greater than 96% purity, greater than 97% purity, greater than 98% purity, greater than 99% purity, greater than 99.5% purity, greater than 99.6% purity, greater than 99.7% purity, greater than 99.8% purity, greater than 99.9% purity, greater than 99.95% purity, greater than 99.96% purity, greater than 99.97% purity, greater than 99.98% purity and greater than 99.99% purity.

[0110] In a further preferred embodiment, the composition is characterized by high performance chromatography (HPLC), proton nuclear magnetic resonance spectroscopy (H 1 Contains less than 0.1 wt. % organic impurities as measured by a method selected from the group consisting of NMR, and mass spectrometry.

[0111] In a further preferred embodiment, the composition is substantially free of atmospheric oxygen.

[0112] In a further preferred embodiment, the composition is sterile. In an alternative preferred embodiment, the composition is not sterile.

[0113] In a further preferred embodiment, the present invention provides a composition wherein the cannabinoid component of the composition is in a concentration selected from the group consisting of between 1 and 500 mg / ml, between 10 and 100 mg / ml, and 50 mg / ml.

[0114] In a further preferred embodiment, the present invention provides a composition, wherein the CBDA component of the composition is in a concentration selected from the group consisting of between 1 and 500 mg / ml, between 10 and 100 mg / ml, and 50 mg / ml.

[0115] In a further preferred embodiment, the composition is a liquid.

[0116] In a further preferred embodiment, the composition is an oil.

[0117] In a further preferred embodiment, the composition demonstrates no cannabinoid degradation or decarboxylation when measured at a time point selected from the group consisting of 1 day, 2 days, 7 days, 14 days, 28 days, 5 weeks, 6 weeks and 32 weeks.

[0118] In a further preferred embodiment, the composition demonstrates cannabinoid stability when measured at a time point selected from the group consisting of 1 day, 2 days, 7 days, 14 days, 28 days, 5 weeks, 6 weeks and 32 weeks.

[0119] In a further preferred embodiment, the composition demonstrates no mutagenicity, carcinogenicity or genotoxicity when delivered at a concentration that delivers 120 mg / ml CBDA.

[0120] In a further preferred embodiment, the composition is adapted to inhibit the activity of any one of the following biomarkers: COX-2, iNOS, TNF-alpha, IL-2, IL-12 and GS-MCF.

[0121] Preferably, the composition is adapted to inhibit neuroinflammation, more preferably, the composition is adapted to treat a neurological disorder.

[0122] In a further preferred embodiment, the present invention provides a composition having a UPLC mass chromatogram corresponding to FIG.

[0123] In a further preferred embodiment, the composition comprises an additional active ingredient.

[0124] Preferably, the additional active ingredient is selected from the group consisting of: polypeptides; antibodies; NSAIDs; neuromodulators; and neurotransmitters, steroids - all related classes (corticosteroids), analgesics, antipsychotics, antidepressants, immunotherapies.

[0125] In one preferred embodiment, the NSAID is selected from the group consisting of aspirin, ibuprofen, naproxen, diclofenac, celecoxib, ketorolac, meloxicam, esomeprazole, naproxen, diclofenac, misoprostol, nabumetone, indomethacin, mefenamic acid, etodolac, piroxicam, ketoprofen, diflunisal, oxaprozin, flurbiprofen, sulindac, tolmetin, prednisolone, and fenoprofen.

[0126] In one embodiment, the additional active ingredient is selected from the group consisting of diclofenac, prednisone, celecoxib, and psylocibin.

[0127] In further preferred embodiments, the ratio of the cannabinoid component to the additional active ingredient is selected from the group consisting of 1 unit w / w cannabinoid:1 unit w / w of additional active ingredient, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0128] In further preferred embodiments, the ratio of the additional active ingredient to the cannabinoid is selected from the group consisting of 1 unit w / w of the additional active ingredient and 1 unit w / w of the cannabinoid, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0129] In further preferred embodiments, the ratio of CBDA to the additional active ingredient is selected from the group consisting of 1 unit w / w CBDA:1 unit w / w of the additional active ingredient, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0130] In further preferred embodiments, the ratio of the additional active ingredient to CBDA is selected from the group consisting of 1 unit w / w of the additional active ingredient and 1 unit w / w of CBDA, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0131] In one preferred embodiment, the neuromodulator is a hallucinogenic substance.

[0132] Preferably, the neuromodulator is selected from the group consisting of 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and psilocybin. Preferably, the neuromodulator is selected from the group consisting of steroids (all related classes (corticosteroids)), analgesics, antipsychotics, antidepressants, and immunotherapeutics.

[0133] In further preferred embodiments, the compositions demonstrate synergistic biological activity.

[0134] In a further preferred embodiment, the composition demonstrates a level of biological activity that is greater than the sum of (1) the biological activity of the cannabinoid component when delivered in the absence of the additional active ingredient and (2) the biological activity of the additional active ingredient when delivered in the absence of the cannabinoid component.

[0135] In a further preferred embodiment, the biological activity is selected from the group consisting of inhibiting inflammation, inhibiting neuroinflammation, treating neurological disorders, inhibiting the activity of COX-2, inhibiting the activity of iNOS, inhibiting the activity of TNF-alpha, inhibiting the activity of IL-2, inhibiting the activity of IL-12 and inhibiting the activity of GS-MCF.

[0136] In further preferred embodiments, the composition is selected from the group consisting of a therapeutic composition, a pharmaceutical composition, a cosmetic composition, and a veterinary composition.

[0137] Pharmaceutical Compositions The present invention also provides a pharmaceutical composition comprising a composition of the present invention together with a pharma- ceutically acceptable carrier.

[0138] Therapeutic compositions are within the scope of the present invention. Preferably, the composition is combined with a pharma- ceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solution, for example, phosphate buffered saline. As used herein, "pharma- ceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Additional supplementary active ingredients can also be incorporated into the composition. For example, see Remington's Pharmaceutical Sciences, 19th Ed. (1995, Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference.

[0139] Pharmaceutical compositions can contain compounding materials to modify, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeability of the composition. Suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite, vitamin E, vitamin E phosphate-fat soluble vitamins, nanoemulsions, etc.); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), bulking agents; monosaccharides, disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colorants, flavorings (natural and naturally derived products) and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (including artificial sweeteners such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic®; PEG; sorbitan esters; polysorbates such as polysorbate 20, polysorbate 80; Triton; tromethamine, lecithin; cholesterol; tyloxapol); stability enhancers (sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride), delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0140] The optimal pharmaceutical composition will be determined by those skilled in the art, for example, depending on the intended administration route, delivery format and desired dosage.Such composition can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the composition of the present invention.The preferred form of pharmaceutical composition is determined by the intended administration mode and therapeutic application.

[0141] The primary vehicle or carrier in a pharmaceutical composition is aqueous and non-aqueous in nature. For example, suitable vehicles or carriers may be water for injection, saline solution, possibly supplemented with other materials. Neutral buffered saline, or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5 or acetate buffer at about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In one embodiment of the present invention, the pharmaceutical composition may be prepared for storage by mixing the selected composition having the desired purity with optional compounding agents in the form of an aqueous solution.

[0142] The formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0143] Additional pharmaceutical compositions include the formulation of the present invention in sustained or controlled delivery formulations, and will be apparent to those skilled in the art. The techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depot injections, are also known to those skilled in the art. Additional examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl L-glutamate, ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may include liposomes, which may be prepared by any of several methods known in the art.

[0144] Pharmaceutical compositions used for in vivo administration must typically be sterile. This can be accomplished by filtration through a sterile filtration membrane. In addition, the composition is generally placed in a container that has a sterile access port. Once the pharmaceutical composition is formulated, it can be stored as a solution in a sterile vial.

[0145] In yet a further preferred embodiment, the composition retains its effective biological activity for a period selected from the group consisting of more than 24 hours, more than 36 hours and more than 48 hours.Preferably, the composition is stable for a period selected from the group consisting of 6 months, 1 year and 2 years.In one example, the composition is stable at a temperature selected from the group consisting of -4°C, 4°C, 18°C ​​and 25°C. Dosage form

[0146] Dosage forms are within the scope of the present invention. In a preferred embodiment, the present invention provides a dosage form comprising a composition as described in the first aspect of the invention.

[0147] Preferably, the cannabinoid component of the composition in the dosage form is selected from the group consisting of between 1 mg and 1000 mg, between 1 mg and 500 mg, between 1 and 100 mg, less than 400 mg, less than 300 mg, less than 200 mg, and less than 100 mg. More preferably, the cannabinoid component of the composition is selected from the group consisting of 600 mg, 400 mg, 300 mg, 200 mg, 100 mg, 50 mg, 10 mg, 5 mg, 2 mg, and 1 mg. Preferably, the CBDA component of the composition in the dosage form is selected from the group consisting of between 1 mg and 1000 mg, between 1 mg and 500 mg, between 1 and 100 mg, less than 400 mg, less than 300 mg, less than 200 mg, and less than 100 mg. More preferably, the CBDA component of the composition is selected from the group consisting of 600 mg, 400 mg, 300 mg, 200 mg, 100 mg, 50 mg, 10 mg, 5 mg, 2 mg, and 1 mg.

[0148] In a further embodiment, the dosage form is in a form selected from the group consisting of a liquid, a tablet, a capsule, a cachet, a dry powder sachet and a vial / freeze-dried.

[0149] Preferably, the dosage form is stored in a sealed and sterile container.

[0150] Methods for Treating The present invention also provides a method of treating a disorder, comprising administering to a patient in need thereof a therapeutically effective amount of a dosage form of the present invention.

[0151] In a further preferred embodiment, the dosage form is administered in an amount to at least partially treat the disorder.

[0152] In a further preferred embodiment, the therapeutically effective amount is an amount of cannabinoid selected from the group consisting of between 1-100 mg / kg / day, between 2-50 mg / kg / day, between 5-40 mg / kg / day, between 10-30 mg / kg / day, between 20-25 mg / kg / day, and 20 mg / kg / day. Preferably, the therapeutically effective amount is an amount of cannabinoid vis selected from the group consisting of 10 mg / day, 15 mg / day, 40 mg / day, 400 mg / day, 600 mg / day, 800 mg / day, 1280 mg / day, 1500 mg / day.

[0153] In a further preferred embodiment, the therapeutically effective amount is an amount of CBDA selected from the group consisting of between 1-100 mg / kg / day, between 2-50 mg / kg / day, between 5-40 mg / kg / day, between 10-30 mg / kg / day, between 20-25 mg / kg / day, and 20 mg / kg / day. Preferably, the therapeutically effective amount is an amount of CBDA vis selected from the group consisting of 10 mg / day, 15 mg / day, 40 mg / day, 400 mg / day, 600 mg / day, 800 mg / day, 1280 mg / day, 1500 mg / day.

[0154] In a further preferred embodiment, Tmax occurs between 1 and 4 hours.

[0155] In a further preferred embodiment, T1 / 2 occurs between 1.1 and 2.4 hours.

[0156] In a further preferred embodiment, a therapeutically effective amount is administered to a subject to treat a disorder.

[0157] Preferably, the therapeutically effective amount is administered to the subject utilizing a dosing regimen selected from the group consisting of twice hourly, hourly, once 6 hours, once 8 hours, once 12 hours, once daily, twice weekly, once weekly, once every two weeks, once every six weeks, once monthly, every two months, every three months, once every six months, and once yearly.

[0158] Preferably, the therapeutically effective amount is administered to the subject using a method selected from the group consisting of orally, intravenously, intramuscularly, intrathecally, subcutaneously, sublingually, buccally, rectally, vaginally, topically, parenterally, mucosally, by the ocular route, by the aural route, nasally, by inhalation, to the skin, transdermally, and systemically.

[0159] In a further preferred embodiment, the disorder is caused by inflammation.

[0160] In a further preferred embodiment, the disorder is caused by neuroinflammation.

[0161] Preferably, the disorder is a neurological disorder. More preferably, the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, traumatic brain injury, rheumatoid arthritis, chronic migraine, epilepsy, autism spectrum disorder, attention deficit hyperactivity disorder, cerebral palsy and related subtypes, neuropathic pain, and depression.

[0162] In a further preferred embodiment, the ASD is ASD level II / III, either "mildly affecting", "moderately affecting", "markedly affecting" or "severely affecting" on the CGI severity scale.

[0163] In a further preferred embodiment, the treatment reduces neuroinflammation. Preferably, the treatment suppresses the activity of any one of the following biomarkers: COX-2, iNOS, TNF-alpha, IL-2, IL-12, and GS-MCF.

[0164] The subjects which may be treated with the present invention will include humans as well as other mammals and animals.

[0165] In a further preferred embodiment, the method comprises administering to a patient in need thereof a therapeutically effective amount of a dosage form of the present invention together with an additional active ingredient. In a preferred form, the additional active ingredient is administered using a dosing regimen selected from the group consisting of: simultaneously with administration of a dosage form of the present invention; prior to administration of a dosage form of the present invention; after administration of a dosage form of the present invention; concurrently with administration of a dosage form of the present invention; sequentially before administration of a dosage form of the present invention; and sequentially after administration of a dosage form of the present invention.

[0166] Preferably, the additional active ingredient is selected from the group consisting of: polypeptides; antibodies; NSAIDs; neuromodulators; and neurotransmitters, steroids - all related classes (corticosteroids), analgesics, antipsychotics, antidepressants, immunotherapies.

[0167] In one preferred embodiment, the NSAID is selected from the group consisting of aspirin, ibuprofen, naproxen, diclofenac, celecoxib, ketorolac, meloxicam, esomeprazole, naproxen, diclofenac, misoprostol, nabumetone, indomethacin, mefenamic acid, etodolac, piroxicam, ketoprofen, diflunisal, oxaprozin, flurbiprofen, sulindac, tolmetin, prednisolone, and fenoprofen.

[0168] In one embodiment, the additional active ingredient is selected from the group consisting of diclofenac, prednisone, celecoxib, and psilocybin.

[0169] In further preferred embodiments, the ratio of the cannabinoid component to the additional active ingredient is selected from the group consisting of 1 unit w / w cannabinoid:1 unit w / w / additional active ingredient, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0170] In further preferred embodiments, the ratio of the additional active ingredient to the cannabinoid is selected from the group consisting of 1 unit w / w of the additional active ingredient and 1 unit w / w of the cannabinoid, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0171] In further preferred embodiments, the ratio of CBDA to the additional active ingredient is selected from the group consisting of 1 unit w / w CBDA:1 unit w / w / of the additional active ingredient, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0172] In further preferred embodiments, the ratio of the additional active ingredient to CBDA is selected from the group consisting of 1 unit w / w of the additional active ingredient and 1 unit w / w of CBDA, 2:1, 3:1, 4:1, 5:1, between 10,000:1 and 1:1, between 1,000:1 and 1:1, between 500:1 and 1:1, between 100:1 and 1:1, between 50:1 and 1:1, and between 10:1 and 1:1.

[0173] In one preferred embodiment, the neuromodulator is a hallucinogenic substance.

[0174] Preferably, the neuromodulator is selected from the group consisting of 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and psilocybin.

[0175] The effectiveness of the administered therapeutic compositions can be monitored by standard diagnostic procedures.

[0176] Use of the composition in the manufacture of a medicament The use is within the scope of the present invention.The present invention also provides the use of a composition of the first aspect of the invention in the manufacture of a medicament for the treatment of a disorder.

[0177] In one preferred embodiment, the present invention relates to the use of the following cannabinoids in the manufacture of a medicament for the treatment of a disorder: w / w% CBDA 40-60%; CBD 1-5%; CBG 1-10%; CBDP 1-5%; CBDB 1~5%; CBGA 1-10%; CBN 1-3% and THC<1% The present invention relates to the use of a composition comprising:

[0178] In a further embodiment, the cannabinoid of the composition is used in the manufacture of a medicament for the treatment of a disorder:

[0179] w / w% CBDA 50%; CBD 2%; CBG 5%; CBDP 2%; CBDB 2%; CBGA 5%; CBN 3% and THC<0.3%; Composition 1 comprising And w / w% CBDA 45%; CBD 1%; CBG 4%; CBDP 1%; CBDB 2%; CBGA 4%; CBN 2%, and THC<0.2% Composition 2 comprising is present in an amount selected from the group consisting of:

[0180] In a further embodiment, the composition further comprises an oil selected from the group consisting of synthetic oils, vegetable-based oils, mineral oils, canola oil, and olive oil.

[0181] In a further embodiment, the composition comprises less than 5% w / w terpenes.

[0182] In a further embodiment, the composition comprises less than 2% w / w organic plant material.

[0183] In a further embodiment, the composition comprises less than 2% w / w plant phenolics.

[0184] In a further embodiment, the cannabinoid component of the composition is selected from the group consisting of: between 1 and 500 mg / ml, between 10 and 100 mg / ml, and at a concentration of 50 mg / ml.

[0185] In further embodiments, the CBDA component of the composition is selected from the group consisting of: between 1 and 500 mg / ml, between 10 and 100 mg / ml, and at a concentration of 50 mg / ml.

[0186] In a further embodiment, the composition has a UPLC mass chromatogram corresponding to FIG. 3 utilizing the conditions described in Example 1.

[0187] process The present invention relates to a process for extracting the composition of the first aspect of the invention from cannabis plant material comprising: 1) grinding cannabis plant material to a sufficient grind size; 2) contacting the grounds produced by step a) with oil; 3) mixing the grinds and oil for a sufficient period of time to form a mixture; 4) squeezing the mixture to recover the oil; 5) centrifuging the oil to further refine the oil; 6) collecting the oil extract in a suitable container / steel vessel; The present invention also provides a process that includes:

[0188] In a further preferred embodiment, the cannabis plant material is derived from Cannabis sativa L.

[0189] In further preferred embodiments, the sufficient grinding size is selected from the group consisting of between 0.1 mm and 3 mm, between 1 mm and 2 mm, and between 0.5 mm and 2.5 mm.

[0190] In further preferred embodiments, the sufficient period of time is selected from the group consisting of between 30 minutes and 2 hours, between 45 minutes and 1.5 hours, and 1 hour.

[0191] In a further preferred embodiment, the ratio of ground material to oil in step (2) is selected from the group consisting of 400 mg ground material:1 ml oil, 300 mg ground material:1 ml oil, 200 mg ground material:1 ml oil, 100 mg ground material:1 ml oil, and 333 mg ground material:1 ml oil.

[0192] Preferably, the oil is olive oil.

[0193] The present invention relates to an alternative process for extracting the composition of the first aspect of the invention from cannabis plant material, comprising: 1) grinding cannabis plant material to a sufficient grind size; 2) contacting the ground material produced by step a) with an alcohol; 3) mixing the grind and alcohol for a sufficient period of time to form a mixture; 4) sonicating the mixture; 5) centrifuging the mixture; 6) collecting the alcoholic extract in a suitable container / steel vessel; The present invention also provides a process that includes:

[0194] In a further preferred embodiment, the alcohol is ethanol.

[0195] In a further preferred embodiment, the alcohol is selected from the group consisting of ethanol, isopropyl alcohol, methyl alcohol, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol.

[0196] In further preferred embodiments, the sufficient grinding size is selected from the group consisting of between 0.1 mm and 3 mm, between 1 mm and 2 mm, and between 0.5 mm and 2.5 mm. In further preferred embodiments, the sufficient period of time is selected from the group consisting of between 30 minutes and 2 hours, between 45 minutes and 1.5 hours, and 1 hour.

[0197] In a further preferred embodiment, the ratio of ground material to alcohol in step (2) is selected from the group consisting of 400 mg ground material:1 ml alcohol, 300 mg ground material:1 ml alcohol, 200 mg ground material:1 ml alcohol, 100 mg ground material:1 ml alcohol, 100 mg ground material:4 ml alcohol, 100 mg ground material:3 ml alcohol, 100 mg ground material:2 ml alcohol, and 333 mg ground material:1 ml alcohol.

[0198] The product of the process The invention also provides products produced by the above-described processes.

[0199] kit The invention also provides a kit comprising a dosage form of an aspect of the invention together with instructions for its use.

[0200] device A device is within the scope of the present invention. In a preferred embodiment, the present invention provides a device, comprising: (1) a composition as described in the first aspect of the present invention; and (2) an applicator.

[0201] Methods for stabilizing Methods for stabilizing the compositions are within the scope of the present invention.

[0202] In a further preferred embodiment, the method protects the composition from degradation.

[0203] In yet further preferred embodiments, the composition retains its effective biological activity for a period selected from the group consisting of greater than 24 hours, greater than 36 hours, and greater than 48 hours.

[0204] The addition of approved pharmaceutical excipients to stabilize compositions is preferred from a safety standpoint because simpler methodologies are more likely to produce less variable results and excipient choices may be limited to those with Generally Regarded as Safe (GRAS) status. Excipients for stabilization of protein solutions can be classified into four broad categories based on their chemical properties and mechanism of action: salts, sugars, polymers, or proteins / amino acids. Salts (e.g., chlorides, nitrates) stabilize the tertiary structure of proteins by shielding charges through ionic interactions. Sugars (e.g., glycerol, sorbitol, fructose, trehalose) increase the surface tension and viscosity of the solution to prevent protein aggregation. Similarly, polymers (e.g., polyethylene glycol, cellulose derivatives) stabilize protein tertiary structure by increasing the viscosity of the solution to prevent protein aggregation as well as intra- and intermolecular electrostatic interactions between amino acids in proteins. Proteins (e.g., human serum albumin) can stabilize the structure of other proteins through ionic, electrostatic, and hydrophobic interactions. Similarly, small amino acids that have no net charge, such as alanine and glycine, stabilize proteins through the formation of weak electrostatic interactions.

[0205] As discussed above, the medicament of the present invention may include one or more pharmaceutically acceptable carriers.The use of such media and agents for the manufacture of medicaments is well known in the art.Except where any conventional media or agent is incompatible with pharmaceutically acceptable materials, its use in the manufacture of pharmaceutical compositions according to the present invention is contemplated.The pharmaceutically acceptable carrier according to the present invention may include one or more of the following examples: a. surfactants and polymers, including, but not limited to, polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, crospovidone, polyvinylpyrrolidone-polyvinyl acrylate copolymers, cellulose derivatives, HPMC, hydroxypropyl cellulose, carboxymethyl ethyl cellulose, hydroxypropyl methylcellulose phthalate, polyacrylates and polymethacrylates, urea, sugars, polyols, and their polymers, emulsifiers, sugar gums, starches, organic acids and their salts, vinylpyrrolidone, and vinyl acetate; and / or b. Binders, such as various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, and / or (3) fillers, such as lactose monohydrate, anhydrous lactose, microcrystalline cellulose, and various starches, and / or c. bulking agents such as lactose monohydrate, lactose anhydrous, mannitol, microcrystalline cellulose and various starches, and / or d. lubricants, such as agents that act to increase the ability of the dosage form to be extruded from a packaging cavity, and / or e. sweeteners, such as any natural or artificial sweetener, including sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K; and / or f. Flavoring agents, and / or g. preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic chemicals such as phenol, or quaternary compounds such as benzalkonium chloride, and / or h. buffer solutions, and / or i. a diluent, such as a pharma- ceutically acceptable inert filler, e.g., microcrystalline cellulose, lactose, dibasic calcium phosphate, sugars, and / or mixtures of any of the foregoing, and / or j. an absorption enhancer, such as glyceryl trinitrate, and / or k. Other pharma- ceutically acceptable excipients.

[0206] Medicaments of the invention suitable for use in animals, and particularly in humans, typically must be sterile and stable under the conditions of manufacture and storage.

[0207] The present invention also provides compositions, methods and processes as described by the foregoing examples.

[0208] The invention will now be described with reference to the following non-limiting examples, which descriptions do not in any way limit the preceding paragraphs of this specification, but are provided for illustration of the methods and compositions of the present invention. EXAMPLES

[0209] It will be apparent to those skilled in the art of milling and pharmaceutical technology that numerous improvements and modifications can be made to the above process without departing from the basic inventive concept. For example, in some applications, the biologically active material may be pretreated and fed to the process in a pretreated form. All such modifications and improvements are considered to be within the scope of the present invention, the nature of which will be determined from the foregoing description and the appended claims. Furthermore, the following examples are provided for illustrative purposes only and are not intended to limit the scope of the process or composition of the present invention.

[0210] Example 1 A Example 1 - Extraction and purification of NTI164 A.1 Research Aims To extract and identify the most desirable constituents from the NTI164 plant strain using an inert oil-based extraction process. A.2 Materials and Methods A.2.1 NTI164 Plant Material

[0211] The NTI164 plant is a full-spectrum medicinal cannabis plant (genus species Cannabis sativa) that the inventors subsequently identified as containing cannabidiolic acid (CBDA), cannabidiol (CBD), cannabigerolic acid (CBGA) and cannabidivarin (CBDV), cannabinol (CBN), but with greater than 0.03% tetrahydrocannabinol (THC). The NTI164 plant was grown, dried and packaged under license and permit from the Drug Enforcement Administration (ODC) in accordance with Good Manufacturing Practice (GMP) and TGO 93 and 100 guidelines.

[0212] A.2.2 Extraction method – oil-based Equipment: The following equipment was used: 10 mL glass scintillation bottles with lids; Cobram Estate Olive Oil; vegetable grinder (similar to a coffee or food grade grinder) pore size maximum 50 μM; Whatman paper, grade 1; pipettes; weighing scale (transfer boat and spoon); Eppendorf tubes; 50 mL Falcon tubes; benchtop centrifuge (Eppendorf Centrifuge 5702); Oz Design brand 6 liter fruit, wine and cider press.

[0213] Extraction: Pressing and Centrifugation: All operations are undertaken at standard laboratory temperature (18-22°C). The tough stems were removed from the buds of NTI164 and the stems were discarded. The grinder was cleaned with 70% EtOH and the grinding compartment was filled with dried plant material. The material was ground for 10 seconds at the finest of three settings (1-2mm particle size). The grind was then mixed with 100ml olive oil at a vegetable / oil ratio of 333mg / mL in an autoclaved Schott bottle. It was then placed in a stirrer for 1 hour at room temperature and stirred with a magnetic stirrer (50rpm). The oil plus vegetable mixture is then placed in an Oz Design brand 6 liter fruit, wine and cider press to recover the oil components from the plant (mash). The recovered oil was then placed in a 50mL Falcon tube and spun at 300g for 15 minutes at room temperature (Isolation 1). The oil was then removed into a clean shot bottle and the volume recovered was kept tracked. The oil recovery for isolation 1 is approximately 40%. The mash is discarded after each isolation. To the recovered oil, we added an additional 333 mg / mL of ground plant / oil (another 100 ml) material, repeated mixing for 1 hour, and recovered and reused the oil until a total of 999 μg / mL (3×100 ml) of plant / oil mixture had passed (isolation 2). The oil recovery for isolation 2 is approximately 50%. Finally, we placed in a Falcon tube and spun as discussed above (isolation 3). The oil recovery for isolation 3 is approximately 50%. We then collected only the oil and placed the oil in an Eppendorf tube for processing. This triple extraction method resulted in a final product with a total volume of 50 ml, with a concentration of 48 mg CBDA per ml olive oil, determined using a UPLC potency test using the method described below.

[0214] A.2.3 Extraction method – Ethanol based Extraction: Squeezing and Centrifugation: An alternative method involves extraction based on the use of ethanol. In this method, 500 milligrams of NTI164 ground plant material is mixed with 20 ml of ethanol in a 50 ml centrifuge tube. The tube is shaken vigorously for 60 seconds and then placed in a 30°C sonication bath for 10 minutes. The sample is then placed on a shaker (200 rpm) for 30 minutes. Once complete, it is placed in a centrifuge and centrifuged at 4400 rcf for 5 minutes. The supernatant can then be evaluated in various preclinical models.

[0215] A.2.4 Analytical analysis Ultra-performance liquid chromatography (UPLC) reversed phase and liquid chromatography mass spectrometry (LCMS) were used to identify the constituents in the NTI164 concentrate derived from the methods discussed above. The analysis was performed using an integrated (U)HPLC system and a single quadrupole mass spectrometer detector with an electrospray ionization (ESI) interface.

[0216] The UPLC settings and conditions used were as follows: Cortex UPLC shield RP18 (0A 1.6 uM, 2.1 x 100 mm); Analytical flow rate: 0.7 ml / min; Mobile phase A: Water 0.1% TFA; Mobile phase B: Acetonitrile; Isothermal: 41:59 Mobile phase A / Mobile phase B; Temperature: 35°C; Detector: Acquity UPLC PDA; Injection volume: 0.7 uL for 1.0 mg / ml reference standard preparation, appropriately scaled sample solution; Software: Empower 3CDS. Reference standard solutions were obtained from Novachem, Cerilliant Corporation (TX, USA). These were all pre-dissolved solutions previously shown to be suitable for generating calibration curves.

[0217] Cannabidivarin (CBDV), cannabidiol (CBD), cannabigerol (CBG), tetrahydrocannabivarin (THCV), cannabinol (CBN), Δ 9 -Tetrahydrocannabinol (Δ 9 -THC), Δ 8-Tetrahydrocannabinol (Δ 8 A mixture of 16 cannabinoids in methanol was prepared containing 10 ppm each of 1-THC, cannabichromene (CBC), their respective acidic forms and cannabicyclol (CBL). All solvents used were LCMS grade and standards were prepared by dilution with 90% mobile phase B and 10% deionized water. The detailed analytical conditions for UPLC-LCMS analysis are listed in Table 1.

[0218] Table 1: Parameters and conditions for UPLC and LCMS analysis. [Table 1]

[0219] A.3 Results A.3.1 UPLC and LCMS Analysis Results FIG. 1 shows the separation of cannabinoids in a mixed standard solution (i.e., reference solution). Under experimental conditions, Δ 9 Neutral cannabinoids such as -THC, CBD and CBL ionize in positive mode, whereas their respective acidic forms ionize in negative mode. CBD and CBG co-elute from the column, but their molecular weights are different and can be identified by mass spectrometry. In addition, Figure 2 shows the difference between the SID fragmentation patterns obtained for CBD and CBG (i.e. as additional reference solutions). These highly specific results demonstrate the advantages of LCMS over LC-UV for the analysis and identification of cannabinoids.

[0220] Figure 3 presents the UPLC mass chromatogram for NTI164 extracted using an oil-based method. The results found that the NTI164 extract (oil suspension) contained the following constituents presented in Table 2. Additional constituents would include flavonoids, proteins, phenols, sterols and esters. These are known constituents that make up 30-40% of whole plant cannabis material. Table 4 presents the accompanying elution times and areas under the peaks for the identified CBD peaks for the UPLC mass chromatogram of Figure 3.

[0221] Table 2: Components of extracted NTI164 oil (to two decimal places, rounded up if over 0.5, rounded down if less than 0.5) [Table 2-1] [Table 2-2]

[0222] Table 3 presents the NTI164 compositions extracted using ethanol extraction and the components quantified using the methods described herein.

[0223] Table 3: Components in extracted NTI164 ethanol (to two decimal places, rounded up if over 0.5, rounded down if less than 0.5) [Table 3]

[0224] Table 4 presents the associated elution times and areas under the peaks for the identified CBD peaks for the UPLC mass chromatogram of FIG. 3 (NTI164, extracted oil).

[0225] Table 4: Elution times and areas under the peaks [Table 4]

[0226] Note that the rarer cannabinoids such as CBDB and CBDP are only detected using quadrupole MS (unlike the routine HPLC used for other cannabinoids). These results are presented in Figure 4.

[0227] Example 2 B. Example 2 - Characterization of the stability properties of NTI164 B.1 Research Aims To evaluate the stability of NTI164 samples suspended in oil formulations at room temperature.

[0228] B.2 Materials and Methods B.2.1 Sample preparation Triplicate samples of NTI164 were prepared using the methods described above.

[0229] For control samples, three representative pre-prepared concentrated samples NTI164 (oil and dried flowers) were obtained as follows: Oil S = Samples were prepared as outlined above: For flowers, a portion of the homogenized plant material was added to acetonitrile or ethanol and sonicated for 20 minutes. The ensuing extract was filtered through a 0.22 μm syringe tip filter directly into a 2 mL sample vial for analysis. Concentrates were prepared similarly using isopropanol as the extraction solvent.

[0230] B.2.2 Sampling NTI164 samples were assayed on a weekly basis with CBDA used as the primary marker / stability indicator. An ACQUITY UPLC H-Class System coupled with Cortex UPLC Shield RP18 particle chemistry was used to provide a UPLC isocratic separation of the primary cannabinoids with a cycle time of 10.5 minutes. The analytical method using UPLC was used as described above.

[0231] Reference standard solutions were obtained from Cerilliant Corporation (Round Rock, Tex.) These pre-dissolved solutions have previously been shown to be suitable for generating calibration curves.

[0232] The preparation of the standard curve was performed as follows: Primary cannabinoid (-) Δ 9 - Linearity of THC and CBD was verified at 10 concentrations between 0.004mg / mL and 1.000mg / mL prepared via serial dilution in methanol using appropriate standards as a representative demonstration of linearity. Table 5 outlines the cannabinoids used in the isolation.

[0233] Table 5: Cannabinoids used in isolation [Table 5]

[0234] B.3 Results B.3.1 UPLC / mass spectrometry analysis results An ACQUITY UPLC H-class system coupled with Cortex UPLC Shield RP18 particle chemistry was used to provide a UPLC homogeneous separation of the major cannabinoids with a cycle time of 10.5 minutes. Samples of NTI164 were assayed on a weekly basis, with CBDA as the primary marker and stability indicator. The results presented in Table 6 demonstrate that NTI164 is stable in an inert oil medium at room temperature for 6 weeks. No decarboxylation or product degradation is observed over this time frame.

[0235] Table 6: Stability of NTI164 at room temperature [Table 6-1] [Table 6-2]

[0236] Example 3 C. Example 3 - Characterization of the biological properties of NTI164 C.1 Research Aims To evaluate the anti-inflammatory and neuroprotective effects of NTI164 in neuronal and microglial cell lines.

[0237] Neuroinflammation is one of the main triggers of neurodegeneration. Investigation of the factors and pathways that can induce the first steps of the inflammatory response will lead to the identification of potential therapeutic targets to halt the progression of many disorders.

[0238] C.2 Materials and Methods C.2.1 Sample preparation and dilution 500mg of dried plant material of NTI164 is suspended in 20ml absolute ethanol (using a 50ml blue top Falcon tube suitable for centrifugation) and vigorously stirred / shaken for 60 seconds. The tube is then placed in a sonication bath at 35-40°C for 10 minutes. Once sonication is complete, the sample is then placed on a tray shaker (200 rpm) for 30 minutes at room temperature. Once complete, the sample is then centrifuged at 4400 rpm for 5 minutes. The supernatant is collected for testing and development.

[0239] Units used to describe the treatment and concentration of NTI164 for the test product a. 1 / 1000 dilution of extract - 10UL (stock material is NTI164-10UL, which is equivalent to 2μg / ml CBDA) b. 1 / 3000 dilution of extract-3UL (stock material is NTI164-3UL, which is equivalent to 6 μg / ml CBDA) c. 1 / 10000 dilution of extract-1UL (stock material is NTI164-1UL, which is equivalent to 0.1 μg / ml of CBDA).

[0240] For the CBD samples, pure standards (in powder form) were used. CBD 98% isolate was purchased from LGC Standards (London UK) as a reference standard (CAS number 13956-29-1). The CBD standard reference was prepared at a concentration of 1 mg / ml (in acetonitrile). CBD dilutions were made in acetonitrile as follows: 2 μg / ml, 6 μg / ml and 0.1 μg / ml.

[0241] The final concentrations of NTI164 (CBDA equivalent) and CBD used in these studies were 2 μg / ml.

[0242] C.2.2 Microglial BV2 culture The immortalized microglial cell line, BV2, was purchased from the American Tissue Culture Collection. BV2 was cultured in RPMI medium containing gentamicin and supplemented with 10% FBS for expansion and 5% fetal bovine serum (FBS) when plated for experiments. All cells were derived from passages between 39 and 45. Cells were cultured at 45,000 cells / mm 2 and 24 hours after plating, were treated with phosphate-buffered saline (PBS, as a control) or interleukin-1B plus interferon-y (IL-1B+IFNy, to induce inflammation). To test the effect of NTI164 in modifying the inflammatory response, NTI164 was applied 1 hour before (pre-treatment) or 1 hour after (post-treatment) inflammation. NTI164 was applied at 10uL, 3uL or 1uL from isolates obtained using the original extraction protocol: range = 1.0-0.1ug CBDA as determined from mass spectrometry data.

[0243] C.2.3 Multiplex cytokine / chemokine assays Microglial cultures harvested after treatment initiation were briefly centrifuged to remove particulate matter (300 g for 10 min). Cytokine and chemokine levels in microglial cultures were measured using a Bio-Plex 200 in a 96-well magnetic plate assay according to the manufacturer's instructions (Bio-Rad). Cytokines and chemokines measured included IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 (p70), IL-13, IL-17, G-CSF, GM-CSF, IFNγ, TNFα, CXCL1 (KC), CCL2 (MCP-1), and CCL5 (RANTES). All samples were run in duplicate and data were analyzed using Bio-Plex manager software.

[0244] C.2.4 Immunohistochemistry (protein level) assays Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. After 3×5 min washing with PBS, cells were incubated with primary antibodies (anti-COX2, anti-ARG1) 1:1000 overnight at 4° C., and after 3×5 min washing in PBS, cells were then incubated in the appropriate fluorescent secondary antibodies 1:250 (Invitrogen) for 2 h at room temperature. After the final wash, cell nuclei were stained with DAPI in mounting medium as before. Photomicrographs of cells were taken from duplicate wells in 3 fields per well and analyzed for area coverage of each marker using Fiji.

[0245] C.2.5 Cell viability (mitochondrial activity) assay Microglial viability was quantified using MTT [3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl-2H-tetrazolium bromide; Sigma]. In this assay, the tetrazolium dye MTT is bioreduced by mitochondria to a formazan product that is insoluble in tissue culture medium. Briefly, MTT was added to cells at various times after treatment with PBS, LPS or IL-4 with or without test products to a final concentration of 250 μg / ml. After 30 min, the formazan was dissolved in DMSO and absorbance was measured at 490 nm using a spectrophotometer (Glomax Multi+; Promega, UK).

[0246] C.2.6 Statistics Data for replicates within an experiment were averaged, and data from at least three independent experiments were then analyzed using Graph Pad Prism or Student's t-test.

[0247] C.3 Results C.3.1 iNOS expression NTI164 normalized inflammation-induced iNOS expression. iNOS expression is increased by inflammation and in inflammation-activated microglial cells, and NTI164 normalized expression toward control levels, thus reducing the inflammatory process triggered by iNOS. Inducible nitric oxide synthase (iNOS) is one of the three main enzymes that generate nitric oxide (NO) from the amino acid L-arginine. Inducible nitric oxide synthase (iNOS) plays a critical role in regulating multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Previous studies have shown that iNOS plays pathogenic and regulatory roles in MS and EAE as well as many other neuroinflammatory disorders. Figure 5 demonstrates that NTI164 normalized inflammation-induced iNOS expression.

[0248] C.3.2 Neuronal viability NTI164 increased the number of surviving neurons under basal conditions (short-term exposure). Neuronal viability was quantified using MTT [3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl-2H-tetrazolium bromide; Sigma]. NTI164-treated cells were able to increase the number of "healthy" cells under basal conditions after short-term glutamate exposure. Cell excitotoxicity was achieved via glutamate activation (3 mM). NTI164 was able to stimulate cell growth after short-term glutamate-induced "injury".

[0249] The cell viability (mitochondrial activity) assay (or MTT assay) was used to determine the viability or metabolic activity of cells in intracellular microcapsules. It is based on the ability of metabolically active cells to convert a water-soluble dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to an insoluble formazan. Cell viability is a measure of the proportion of live, healthy cells in a population, and cell viability assays are used to determine the overall health of cells. Figure 6 presents the viability of neurons quantified using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide].

[0250] C.3.3 Maturation of immature neurons NTI164 stimulated the maturation of immature neurons into healthy cells, even in the absence of any glutamate-induced damage. This study demonstrates that NTI164 can stimulate the "healthy maturation" of immature neurons, a process that may be essential after trauma or damage. NTI164 can provide healthy neuronal cytogenesis, an essential process in recovery from neuroinflammation, neuronal damage. Figure 7 demonstrates that NTI164 stimulates the maturation of immature neurons into healthy cells, even in the absence of any glutamate-induced damage.

[0251] C.3.4 Cell death NTI164 does not increase cell death in an excitotoxic cytotoxicity paradigm.

[0252] FIG. 7 demonstrates that CBD is toxic in this paradigm, whereas NTI164 is non-toxic and has a positive effect on cell number and cell viability.

[0253] C.3.5 ARG1 expression NTI164 (NTI) normalizes proinflammatory (injured cell) Arg1 expression. Macrophage-specific upregulation of arginase-1 is generally thought to promote inflammation. Arginase 1 expression is still increased by inflammation in inflammatory activated cells, and NTI164 normalizes expression toward control levels. Figure 9 shows the microglial response under inflammatory conditions evaluating arginase 1 expression.

[0254] FIG. 10 reviews arginine metabolism and the effect it has on the overall balance of anti-inflammatory and pro-inflammatory signals.

[0255] Example 4 D Example 4 - Preclinical studies related to biomarkers involved in neuroinflammation D.1 Research Aims To determine the effects of NTI164 on the levels of COX-2, IL2 and TNF-alpha in human-derived microglial cells.

[0256] Many neurological disorders arise due to inflammation induced by dysregulated immune responses.

[0257] For example, multiple sclerosis (MS) is a progressive inflammatory disease characterized by the loss of myelin sheaths in the central nervous system. Typical symptoms include fatigue, difficulty walking, and speech and vision abnormalities. Cyclooxygenase-2 (COX-2) is considered the main enzyme responsible for causing inflammation, a common mechanism of disease involved in MS. COX-2 is a powerful clinical biomarker in the assessment of disease progression and overall therapeutic management.

[0258] IL2 plays a key role in immune regulation and in MS progression. IL-12 is a cytokine that plays a major role in the pathogenesis of multiple sclerosis. Blocking this cytokine via neutralizing antibodies causes dramatic improvements in animal models of the disease and in multiple human trials.

[0259] TNF-alpha plays a key role in the dysregulation of acute inflammation involved in MS pathogenesis.

[0260] D.2 Materials and Methods D.2.1 COX-2

[0261] Immunohistochemistry (protein level) assay: Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. After 3×5 min washing with PBS, cells were incubated with primary antibody (anti-COX2) 1:1000 overnight at 4° C., and after 3×5 min washing in PBS, cells were then incubated in appropriate fluorescent secondary antibody 1:250 (Invitrogen) for 2 h at room temperature. After the final wash, cell nuclei were stained with DAPI in mounting medium as before. Photomicrographs of cells were taken in 3 fields per well from duplicate wells and analyzed for area coverage of each marker using Fiji.

[0262] D.2.2 IL-2 and TNF-alpha Multiplex cytokine / chemokine assay: Microglial cultures harvested after treatment initiation were briefly centrifuged (300 g for 10 min) to remove particulate matter. Cytokine and chemokine levels in microglial cultures were measured using a Bio-Plex 200 in a 96-well magnetic plate assay according to the manufacturer's instructions (Bio-Rad). Cytokines and chemokines measured included IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 (p70), IL-13, G-CSF, GM-CSF, IFNγ, TNFα, CXCL1 (KC), CCL2 (MCP-1), and CCL5 (RANTES). All samples were run in duplicate and data were analyzed using Bio-Plex manager software.

[0263] D.2.3 Sample preparation and dilution 500mg of dried plant material of NTI164 is suspended in 20ml absolute ethanol (using a 50ml blue top Falcon tube suitable for centrifugation) and vigorously stirred / shaken for 60 seconds. The tube is then placed in a sonication bath at 35-40°C for 10 minutes. Once sonication is complete, the sample is then placed on a tray shaker (200 rpm) for 30 minutes at room temperature. Once complete, the sample is then centrifuged at 4400 rpm for 5 minutes. The supernatant is collected for testing and development.

[0264] Units used to describe the treatment and concentration of NTI164 for the test product a. 1 / 1000 dilution of extract - 10UL (stock material is NTI164-10UL, which is equivalent to 2μg / ml CBDA) b. 1 / 3000 dilution of extract-3UL (stock material is NTI164-3UL, which is equivalent to 6 μg / ml CBDA) c. 1 / 10000 dilution of extract-1UL (stock material is NTI164-1UL, which is equivalent to 0.1 μg / ml of CBDA).

[0265] For the CBD samples, pure standards (in powder form) were used. CBD 98% isolate was purchased from LGC Standards (London UK) as a reference standard (CAS number 13956-29-1). The CBD standard reference was prepared at a concentration of 1 mg / ml (in acetonitrile). CBD dilutions were made in acetonitrile as follows: 2 μg / ml, 6 μg / ml and 0.1 μg / ml.

[0266] The final concentrations of NTI164 (CBDA equivalent) and CBD used in these studies were 2 μg / ml.

[0267] D.3 Results D.3.1 COX-2 Preclinical studies performed on cells using immunohistochemistry demonstrated that NTI164 can suppress and inhibit the expression of COX-2 in human-derived microglial cells.Compared to CBD alone, NTI164 was up to 3 times more potent in suppressing COX-2 both before and after inflammatory injury.See Table 7 below.

[0268] Table 7: Summary of COX-2 inhibition in cells when treated with NTI164 versus CBD alone. [Table 7]

[0269] D.3.2 IL2 and TNF-alpha NTI164 is statistically more potent in suppressing key biomarkers: IL-12 and TNF-alpha when compared to CBD alone and a CBD / THC (1:1) mixture.

[0270] These results demonstrate that NTI164 IL-12 P=0.0011 vs. CBD alone was highly significant; NTI164 TNF-alpha P=0.0575 vs. CBD alone trended positive; NTI164 IL-12 P=0.0069 vs. CBD / THC combination was highly significant; NTI164 TNF-alpha P=0.0446 vs. CBD / THC combination was significant.

[0271] Table 8 summarizes the significance between NTI164 versus CBD alone and CBD / THC combination (1:1 concentration ratio) in suppressing TNF-alpha and IL-12. [Table 8]

[0272] D.4 Discussion D.4.1 COX-2, IL2 and TNF-alpha These results showing inhibition of COX-2, IL2 and TNF-alpha reaffirm the potent properties of NTI164 in modulating inflammatory processes in neurological disorders where inflammation induced by immune responses is dysregulated.

[0273] Example 5 E. Example 5 - Further characterization of NTI164 E.1 Research Aims Compositional analysis of NTI164 extracted via oil using UPLC / MS methods (as outlined in the examples above).

[0274] E.2 Materials and Methods An ACQUITY UPLC H-class system coupled with Cortex UPLC Shield RP18 particle chemistry was used to provide a UPLC homogeneous separation of the major cannabinoids with a cycle time of 10.5 minutes. Samples of NTI164 were assayed on a weekly basis, with CBDA as the primary marker and stability indicator. The results presented in Table 6 demonstrate that NTI164 is stable in an inert oil medium at room temperature for 6 weeks. No decarboxylation or product degradation is observed over this time frame.

[0275] Equipment: The following equipment was used: 10 mL glass scintillation bottles with lids; Cobram Estate Olive Oil; vegetable grinder (similar to a coffee or food grade grinder) pore size maximum 50 μM - 80 μM; Whatman paper, grade 1; pipettes; weigh scale (transfer boat and spoon); Eppendorf tubes; 50 mL Falcon tubes; benchtop centrifuge (Eppendorf Centrifuge 5702); Oz Design brand 6 liter fruit, wine and cider press.

[0276] Extraction: Pressing and Centrifugation: All operations are undertaken at standard laboratory temperature (18-22°C). The tough stems were removed from the buds of NTI164 and the stems were discarded. The grinder was cleaned with 70% EtOH and the grinding compartment was filled with dried plant material. The material was ground for 10 seconds at the finest of three settings (1-2mm particle size). The grind was then mixed with 100ml olive oil at a vegetable / oil ratio of 333mg / mL in an autoclaved Schott bottle. It was then placed in a stirrer for 1 hour at room temperature and stirred with a magnetic stirrer (50rpm). The oil plus vegetable mixture is then placed in an Oz Design brand 6 liter fruit, wine and cider press to recover the oil components from the plant (mash). The recovered oil was then placed in a 50mL Falcon tube and spun at 300g for 15 minutes at room temperature (Isolation 1). The oil was then removed into a clean shot bottle and the volume recovered was kept tracked. The oil recovery for isolation 1 is approximately 40%. The mash is discarded after each isolation. To the recovered oil, we added an additional 333 mg / mL of ground plant / oil (another 100 ml) material, repeated mixing for 1 hour, and recovered and reused the oil until a total of 999 μg / mL (3×100 ml) of plant / oil mixture had passed (isolation 2). The oil recovery for isolation 2 is approximately 50%. Finally, we placed in a Falcon tube and spun as discussed above (isolation 3). The oil recovery for isolation 3 is approximately 50%. We then collected only the oil and placed the oil in an Eppendorf tube for processing. This triple extraction method resulted in a final product with a total volume of 50 ml, with a concentration of 48 mg CBDA per ml olive oil, determined using a UPLC potency test using the method described below.

[0277] E.3 Results The results of the UPLC analysis are presented below in Table 9.

[0278] Table 9 presents NTI164 compositions extracted using ethanol extraction and the components quantified using the UPLC method described herein. [Table 9]

[0279] By increasing the pore size in the grinder system from 50 μM to 80 μM, we were able to extract 1-3% cannabinol (CBN) in the final oil extract product. Example 6 F. Example 6 - Evaluating the anti-inflammatory properties of NTI164 in combination with diclofenac F.1 Research Aims

[0280] To evaluate the anti-inflammatory properties of NTI164 in combination with diclofenac compared to CBD in combination with diclofenac.

[0281] Diclofenac is a nonsteroidal anti-inflammatory drug (NSAID). It is used to treat mild to moderate pain or the signs and symptoms of osteoarthritis, rheumatoid arthritis and neuropathic pain. F.2 Materials and Methods F.2.1 Immunohistochemistry (protein level) assays

[0282] Human derived microglial cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. After 3 × 5 min washes with PBS, cells were incubated with primary antibody (anti-COX2) 1:1000 overnight at 4°C, and after 3 × 5 min washes in PBS, cells were then incubated in the appropriate fluorescent secondary antibody 1:250 (Invitrogen) for 2 h at room temperature. After the final wash, cell nuclei were stained with DAPI in mounting medium as before. Photomicrographs of cells were taken from duplicate wells in 3 fields per well and analyzed for area coverage of each marker using Fiji.

[0283] COX-2 is measured using immunohistochemistry assay as described above.As part of positive control, cells are treated with interleukin and interferon, which makes cells enter "active inflammatory state".In treatment group, these cells are also treated with various treatments, and then COX-2 expression is evaluated after these treatments (1 hour after treatment). F.2.2 Sample preparation and dilution

[0284] The following samples were prepared: CBD alone, NTI164 alone, diclofenac alone, diclofenac + NTI164.

[0285] In this example, ethanol extraction was used. 500 mg of dried plant material of NTI164 is suspended in 20 ml of absolute ethanol (using a 50 ml blue top Falcon tube suitable for centrifugation) and vigorously stirred / shaken for 60 seconds. The tube is then placed in a sonication bath at 35-40°C for 10 minutes. Once sonication is complete, the sample is then placed on a tray shaker (200 rpm) for 30 minutes at room temperature. Once complete, the sample is then centrifuged at 4400 rpm for 5 minutes. The supernatant is collected for testing and development.

[0286] Units used to describe the treatment of test products and concentrates of NTI164. a. 1 / 1000 dilution of extract - 10UL (stock material is NTI164-10UL, which is equivalent to 2μg / ml CBDA) b. 1 / 3000 dilution of extract-3UL (stock material is NTI164-3UL, which is equivalent to 6 μg / ml CBDA) c. 1 / 10000 dilution of extract-1UL (stock material is NTI164-1UL, which is equivalent to 0.1 μg / ml of CBDA).

[0287] For the CBD samples, pure standards (in powder form) were used. CBD 98% isolate was purchased from LGC Standards (London UK) as a reference standard (CAS number 13956-29-1). The CBD standard reference was prepared at a concentration of 1 mg / ml (in acetonitrile). CBD dilutions were made in acetonitrile as follows: 2 μg / ml, 6 μg / ml and 0.1 μg / ml.

[0288] Diclofenac (CAS number 15307-86-5) was purchased from Merck Chemicals (purity, 98% HPLC). A reference stock standard was made at 1 mg / ml using acetonitrile and the following dilutions were made: 2 μg / ml, 6 μg / ml, 0.1 μg / ml.

[0289] The final concentrations of NTI164 (CBDA equivalent), CBD and diclofenac used in this study were 2 μg / ml. F.3 Results F.3.1 Synergies

[0290] Synergy was observed with the NTI164 + diclofenac combination: COX-2 inflammation was reduced by 8% with the NTI164 + diclofenac combination compared to 24% with diclofenac alone and 55% with NTI164 alone.

[0291] Treatment with NTI164 alone is highly effective in reducing cellular inflammatory processes, but when combined with diclofenac, these anti-inflammatory effects are even stronger.

[0292] Table 10 presents the inflammation results for the combination study (N=9), NTI164 plus diclofenac. [Table 10] Example 7 G. Example 7 - Evaluating the anti-inflammatory properties of NTI164 in combination with celecoxib G.1 Research Aims

[0293] To evaluate the anti-inflammatory properties of NTI164 in combination with diclofenac compared to CBD in combination with celecoxib.

[0294] Celecoxib is a nonsteroidal anti-inflammatory drug (NSAID). Diclofenac is used to treat mild to moderate pain or the signs and symptoms of conditions such as osteoarthritis or rheumatoid arthritis. G.2 Materials and Methods G.2.1 Microglial BV2 culture

[0295] The immortalized microglial cell line, BV2, was purchased from the American Tissue Culture Collection. BV2 was cultured in RPMI medium containing gentamicin and supplemented with 10% fetal bovine serum (FBS) for expansion and 5% FBS at the time of plating for experiments. All cells were derived from passages between 39 and 45. Cells were cultured at 45,000 cells / mm 2 and 24 hours after plating, were treated with phosphate buffered saline (PBS, as control) or interleukin-1B + interferon-y (IL-1B + IFNy, to induce inflammation). To test the effect of NTI164 in modifying the inflammatory response, NTI164 and synergistic treatments (see below) were applied 1 hour after inflammation. Extracted NTI164 was applied at 10uL, 3uL or 1uL from isolates obtained using the original extraction protocol: range = 1.0-0.1ug CBDA as determined from mass spectrometry data. Treatments were celecoxib 5, 25 and 125μM. G.2.2 Multiplex cytokine / chemokine assays

[0296] Microglial cultures harvested after treatment initiation were briefly centrifuged to remove particulate matter (300 g for 10 min). Cytokine and chemokine levels in microglial cultures were measured in a 96-well magnetic plate assay using a Bio-Plex 200 according to the manufacturer's instructions (Bio-Rad). Cytokines and chemokines measured included IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 (p70), IL-13, G-CSF, GM-CSF, IFNγ, TNFα, CXCL1 (KC), CCL2 (MCP-1), and CCL5 (RANTES). All samples were run in duplicates averaged for analysis, with at least three samples run per group. G.2.3 Immunohistochemistry (protein level) assays

[0297] Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. After 3×5 min washing with PBS, cells were incubated with primary antibody (anti-COX2) 1:1000 overnight at 4° C., and after 3×5 min washing in PBS, cells were then incubated in the appropriate fluorescent secondary antibody 1:250 (Invitrogen) for 2 h at room temperature. After the final wash, cell nuclei were stained with DAPI in mounting medium as before. Photomicrographs of cells were taken from duplicate wells in 3 fields per well and analyzed for area coverage of each marker using Fiji. G.2.4 Cell viability (mitochondrial activity) assay

[0298] Microglial viability was quantified using MTT [3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl-2H-tetrazolium bromide; Sigma]. In this assay, the tetrazolium dye MTT is bioreduced by mitochondria to a formazan product that is insoluble in tissue culture medium. Briefly, MTT was added to cells at various time points after treatment with PBS, LPS or IL-4 with or without test products to a final concentration of 250 μg / ml. After 30 min, the formazan was dissolved in DMSO and absorbance was measured at 490 nm using a spectrophotometer (Glomax Multi+; Promega, UK). G.3 Results G.3.1 Synergy

[0299] Synergistic effects were observed with the NTI164 + celecoxib combination. Inflammation, as observed by MTT assay, cytokine release (TNF-alpha, G-CSF, IL1a, IL6) and COX2 protein, was reduced with the NTI164 + celecoxib combination versus celecoxib alone and NTI164 alone.

[0300] Treatment with NTI164 alone is highly effective in reducing cellular inflammatory processes, but when combined with celecoxib, these anti-inflammatory effects are even stronger.

[0301] Table 11 presents the inflammation results for the combination study to demonstrate the synergistic effect of NTI164 and Celebrex. [Table 11-1] [Table 11-2] Example 8 H. Example 8 - Evaluating the anti-inflammatory properties of NTI164 in combination with prednisone H.1 Research Aims

[0302] To evaluate the synergistic suppression of inflammatory responses using combined prednisone and NTI164 treatment in a preclinical in-vitro study. H.2 Materials and Methods

[0303] Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min. After 3×5 min washing with PBS, cells were incubated with primary antibody (anti-COX2) 1:1000 overnight at 4° C., and after 3×5 min washing in PBS, cells were then incubated in the appropriate fluorescent secondary antibody 1:250 (Invitrogen) for 2 h at room temperature. After the final wash, cell nuclei were stained with DAPI in mounting medium as before. Photomicrographs of cells were taken from duplicate wells in 3 fields per well and analyzed for area coverage of each marker using Fiji.

[0304] The treatment groups were:

[0305] (1) Control: PBS buffer

[0306] (2) Positive control: Inflammatory stimulation with interferon gamma and interleukin-1B-activated prednisone (PDN) concentrate 5uM

[0307] (3) Combination therapy: Prednisone (PDN) 5uM + NTI164 concentrate 7.5ug / ml Includes. H.3 Results

[0308] Results analysis: PDN vs. combination therapy (Prednisone 5uM + NTI164 concentrate 7.5ug / ml) calculated as % reduction in inflammation. Student's t-test was used for statistical analysis.

[0309] Treatment with NTI164 alone is highly effective in reducing cellular inflammatory processes, but when combined with prednisone, these anti-inflammatory effects are even stronger. Table 12 presents inflammation results for combination studies to demonstrate synergy of NTI164 and prednisone. [Table 12] Example 9 I Example 9 - Evaluating the anti-inflammatory properties of NTI164 in combination with psilocybin I.1 Research Aims

[0310] To evaluate the synergistic suppression of inflammatory responses using combined psilocybin and NTI164 treatments in preclinical in-vitro studies. I.2 Materials and Methods

[0311] Microglial media harvested after treatment initiation were briefly centrifuged to remove particulate matter (300 g for 10 min). Levels of TNFα in microglial culture media were measured using a Bio-Plex 200 in a 96-well magnetic plate assay according to the manufacturer's instructions (Bio-Rad). Samples were run in duplicates that were averaged for analysis. At least three samples were run per group. I.3 Results

[0312] Result analysis: Table 13 presents the TNF-alpha ratings for the psilocybin combination studies. Table 13: TNF-Alpha Ratings [Table 13] Treatment with NTI164 alone is highly effective in reducing cellular inflammatory processes, but when combined with psilocybin, these anti-inflammatory effects are even stronger. Example 10 J Example 10 - NTI164 for treatment of ASD level II / III - 4 weeks J.1 ASD Research Design and Methods

[0313] Example 6 F. Example 6 - NTI164 for treatment of ASD level II / III - 4 weeks F.1 ASD Research Design and Methods Of the 18 patients enrolled in the study, those who received NTI164 constituted 94% (n=17). Effective patients constituted 78% (n=14), patients who discontinued after receiving the first dose of NTI164 constituted 16% (n=3), and patients who discontinued before receiving NTI164 but after enrollment constituted 6% (n=1). The mean age of effective patients was 13.4 years, with the youngest patient being 10 years and the oldest being 17 years (Figure 11).

[0314] All eligible patients were diagnosed with ASD level II / III and rated as either "mildly morbid," "moderately morbid," "markedly morbid," or "severely morbid" at baseline on the CGI severity scale (Figure 12).

[0315] Patients were initiated on NTI164 treatment at 5 mg / kg / day and increased by 5 mg / kg / day weekly for a period of 4 weeks until 20 mg / kg / day or the maximum tolerated dose was achieved. The daily dose was calculated by multiplying the dosage by the patient's weight and then dividing by the concentration of CBDA in the oil (53 mg / mL). This resulted in a total daily volume in mL (Table 14), which was divided into twice-daily (BD) AM and PM doses.

[0316] Table 14 - Calculation of daily dose for each patient. [Table 14]

[0317] The mean maximum daily dose for effective patients was 16.7 mg / kg / day, with 64% of patients tolerating a maximum dose of 20 mg / kg / day and 36% of patients tolerating maximum daily doses ranging between 6 mg / kg / day and 19 mg / kg / day (Figure 13). J.2 Research results

[0318] The Clinical Global Impression-Severity (CGI-S) scale was used to assess:

[0319] Global Improvement: The clinician's judgement is to rate the overall improvement as being entirely attributable to drug treatment or not;

[0320] Severity of disease: Comparison of baseline and post-baseline (28 days of NTI164 treatment); and

[0321] Efficacy index: scored based on drug efficacy only. This is a score calculated based on the degree of therapeutic effect and side effects.

[0322] General Improvements

[0323] 93% of effective patients showed improvement after 28 days of daily treatment with NTI164. 64% of these patients had a global improvement of "much improved", 29% had a global improvement of "minimally improved", and only one patient (7%) had "no change" (Figure 14). Statistical significance was assessed using Wilcoxon signed rank test and paired t-test:

[0324] Paired t-test: The mean difference in CGI-S between 28-day treatment and baseline was -0.714, 95% confidence interval = -1.332, -0.097, p-value = 0.027. The Wilcoxon signed rank test statistic was -15 and the corresponding p-value was 0.047.

[0325] Severity of illness

[0326] The mean score for disease severity at baseline was 4.4 (Figure 12), which was reduced to a mean score of 3.6 after 28 days of NTI164 treatment (Figures 15, 16).

[0327] Treatment effect

[0328] After 28 days of daily treatment with NTI164, 14% of patients responded, demonstrating the second highest possible efficacy index of 2: a significant therapeutic effect accompanied by side effects that did not significantly interfere with the patient's functioning.

[0329] 72% of effective patients had an efficacy index of either 5 or 6: half of these patients had no side effects and the other half had a moderate therapeutic effect with side effects that did not significantly interfere with the patient's functioning, 7% had an efficacy index of 9: a minimal therapeutic effect with no side effects, and only one patient 7% had an efficacy index of 13: unchanged or worsening, based on no change in condition and no side effects (Figure 17).

[0330] Example 11 K Example 11 - NTI164 for treatment of ASD Level II / III - 20 weeks K.1 ASD Research Design and Methods Example 10 above presents treatment results at 4 weeks (28 days) (n=14 effective). Example 11 here presents treatment results at 20 weeks (n=12 effective). As discussed in Example 10 above, patients began treatment with NTI164 at 5 mg / kg / day, which was increased by 5 mg / kg / day per week for a period of 4 weeks until 20 mg / kg / day or the maximum tolerated dose was reached, and (in this study) the maximum tolerated dose was continued for 16 weeks (providing a total daily dosing period of 20 weeks).

[0331] The overall objective of this study was to evaluate the ongoing safety and efficacy of NTI164 administered daily over a 20-week period. A secondary objective was to evaluate the efficacy of NTI164 in treating symptoms associated with autism spectrum disorder. Efficacy was measured using a variety of standard physician- and patient-directed questionnaires used in the art.

[0332] Patients (n=12) The mean age of valid patients at week 20 was 13.3 years, with the youngest patient being 10 years old and the oldest 17 years old (Error! Reference not found). All valid patients were diagnosed with ASD level II / III and rated as either "mildly affected", "moderately affected", "markedly affected" or "severely affected" at baseline on the CGI severity scale (Figure 19).

[0333] dose Based on pediatric trials undertaken worldwide, the maximum dose selected for this study was 20 mg / kg / day.

[0334] To reduce the risk of side effects, the study drug was titrated over the course of 4 weeks, starting at 5 mg / kg / day and increasing by 5 mg per week until the maximum tolerated dose or 20 mg / kg / day was achieved. The maximum tolerated dose was then administered over the course of 20 weeks. The daily volume was administered in two doses, AM and PM.

[0335] The formula used to calculate each patient's dose was body weight x dose / NTI164 concentration = daily dose / 2 = twice daily dose. During the first week of treatment, each patient received 5 mg / kg / day of NTI164. During the second week of treatment, each patient received 10 mg / kg / day of NTI164. During the third week of treatment, each patient received 15 mg / kg / day of NTI164. During the fourth week of treatment, each patient received 20 mg / kg / day of NTI164. During weeks 5-20 of treatment, each patient received their maximum tolerated dose or 20 mg / kg / day of NTI164.

[0336] NTI164 was formulated in oil for oral administration. The total oil concentration was 53 mg / ml.

[0337] At the end of week 20, participants had the option to either terminate participation and taper off study drug at 5 mg / kg / week until discontinuation, or continue on their maximum tolerated dose until week 52.

[0338] Primary endpoint Safety was monitored and measured using standard procedures in the art. Parent / caregiver and physician questionnaires completed at baseline and every 4 weeks through week 20, as well as complete blood tests, liver and kidney function tests, and vital signs were used to monitor and measure safety.

[0339] Secondary endpoints Efficacy was monitored and measured using standard procedures in the art.

[0340] Clinical Global Impression Scale-Severity of Illness (CGI-S), reflects the clinician's impression of the severity of illness on a 7-point scale ranging from 1 = not at all to 7 = most extremely ill [Timeframes: baseline, weeks 4, 8, 12, and 20].

[0341] Vineland Adaptive Behavior Scales, Third Edition (Vineland-3). Used to measure adaptive functioning across three core domains (communication, daily living skills, and socialization) and two need-specific domains (motor skills and maladaptive behaviors), with items rated on a 3-point scale (0=none; 1=occasionally; 2=usually or frequently). Core domains sum to a total Adaptive Behavior Composite [Time Frames: Baseline, Week 20].

[0342] Social Responsiveness Scale, 2nd Edition-School Age (SRS-2). Assess five domains including social awareness, social cognition, social communication, social motivation, as well as restricted interests and repetitive behaviors. Items are scored on a 4-point scale (ranging from 1=not true to 4=almost always true). [Timeframe: Baseline, Week 20].

[0343] Clinical Global Impression Scale-Improvement-Caregiver (CGI-I-Ca). This is a 7-point scale measuring symptom change from baseline. Provided as baseline and post-baseline caregiver and clinician questionnaires [Timeframe: baseline, weeks 4, 8, 12, and 20].

[0344] Clinical Global Impression Scale-Improvement-Clinician (CGI-I-Cl). This is a 7-point scale that measures symptom change from baseline. It is administered as baseline and post-baseline caregiver and clinician questionnaires [Timeframe: baseline, weeks 4, 8, 12, and 20].

[0345] Clinical Global Impression Scale-Change in Target Behavior (CGI-C). Reflects clinician's impression of behavior change on a 7-point scale ranging from 1=not at all to 7=very severe problems. Provided as baseline and post-baseline questionnaires [Timeframe: baseline, weeks 4, 8, 12, and 20].

[0346] Clinical Global Impression Scale-Attention Change (CGI-CA). Reflects clinician's impression of attention change on a 7-point scale ranging from 1=not at all to 7=very severe problem. Provided as baseline and post-baseline questionnaires [Timeframe: baseline, weeks 4, 8, 12, and 20].

[0347] Anxiety Scale for Children-Autism Spectrum Disorder-Parent Version (ASC-ASD-P). A parent / caregiver version developed to detect symptoms of anxiety in youth with ASD. It consists of four subscales (Performance Anxiety, Uncertainty, Anxious Arousal, and Separation Anxiety) and items are rated on a 4-point scale (0=never and 3=always). The sum of the subscales equals a total score [Timeframe: Baseline, Week 4, Week 8, Week 12, Week 20].

[0348] Anxiety Scale for Children-Autism Spectrum Disorder-Child Version (ASC-ASD-C). A pediatric version developed to detect symptoms of anxiety in young people with ASD. It consists of four subscales (performance anxiety, uncertainty, anxious arousal, and separation anxiety) and items are rated on a 4-point scale (0=never and 3=always). The sum of the subscales equals the total score [Timeframe: baseline, week 4, week 8, week 12, week 20].

[0349] Sleep Disorders Scale for Children (SDSC). Six subscales include Disorders of Sleep Onset and Maintenance, Sleep-Disordered Breathing, Arousal Disorder, Sleep-Wake Transition Disorder, Disorder of Excessive Somnolence, and Hyperhydrosis. Items are rated on a 5-point scale, where 1=never and 5=always (every day). The sum of the subscale scores equals the total score [Timeframe: Baseline, Week 4, Week 8, Week 12, Week 20].

[0350] K.2 Research results safety results The safety data conclude that NTI164 at doses of 5, 10, 15 and 20 mg / kg administered twice daily was safe and well tolerated in this study population. This conclusion is further supported by laboratory values. No changes were observed in the patients' complete blood work or in liver or kidney function tests. No changes were observed in the patients' vital signs.

[0351] Efficacy Results Wilcoxon signed-rank test and paired t-test were used to assess statistical significance of the analyzed data sets.

[0352] Paired t-test: The mean difference in CGI-S between 20 weeks of treatment and baseline was -1.08, 95% confidence interval = -1.772, -0.3948, p-value = 0.005303.

[0353] The Wilcoxon signed rank test statistic was -15 and the corresponding p-value was 0.009654.

[0354] One hundred percent of patients (n=12) showed "much improved" improvement in symptoms related to disease severity after 20 weeks of daily treatment with NTI164.

[0355] Table 15 below summarizes the results from the Wilcoxon signed rank test and paired t-test for the analyzed data set at week 20. Table 15 - Summary of Wilcoxon Signed Rank Test and Paired T-Test for Analyzed Data Set at Week 20 [Table 15]

[0356] Global Improvement. 100% of effective patients (n=12) showed improvement after 20 weeks of daily treatment with NTI164. All patients had a global improvement of "2. Much improved." Three of these patients had previously been scored as "3. Minimally improved" after 4 weeks of treatment. See Figures 20 and 21.

[0357] Severity of disease. The mean score for severity of disease at baseline was 4.3. This was reduced to a mean score of 3.3 after 20 weeks of daily NTI164 treatment. See Figures 22-24.

[0358] Treatment Efficacy. After 20 weeks of daily NTI164 treatment, 67% of effective patients demonstrated the highest possible efficacy index of 1 and 2: marked treatment effect - great improvement. Complete or near-complete remission of all symptoms. 33% of patients had an efficacy index of either 5, 6 or 7: moderate treatment effect - definitive improvement. Partial remission of symptoms. See Figures 25 and 26.

[0359] Conclusion.

[0360] NTI164 was shown to be safe and well tolerated at doses up to 20 mg / kg / day. NTI164 demonstrated statistically significant efficacy in improving symptoms associated with autism spectrum disorder after 20 weeks of daily therapy.

Claims

1. The following cannabinoids: w / w% CBDA 40-60%, CBD 1-5%, CBG 1-10%, CBDP 1-5%, CBDB 1-5%, CBGA 1-10%, CBN 1-3%, THC < 1%, and Additional Active Ingredients A composition comprising:

2. The cannabinoid is w / w% CBDA 50%, CBD 2%, CBG 5%, CBDP 2%, CBDB 2%, CBGA 5%, CBN 3%, THC < 0.3%, and Additional Active Ingredients Composition 1 comprising and w / w% CBDA 45%, CBD 1%, CBG 4%, CBDP 1%, CBDB 2%, CBGA 4%, CBN 2%, THC < 0.2%, and Additional Active Ingredients Composition 2 comprising 10. The composition of claim 1, wherein the amount of hydroxybenzoates is selected from the group consisting of:

3. 10. The composition of claim 1, further comprising an oil selected from the group consisting of synthetic oils, vegetable-based oils, mineral oils, canola oil, and olive oil.

4. 10. The composition of claim 1, comprising less than 5% w / w terpenes.

5. 10. The composition of claim 1, comprising less than 2% w / w organic plant material.

6. 10. The composition of claim 1, comprising less than 2% w / w of plant phenolics.

7. 10. The composition of claim 1, wherein the cannabinoid component of the composition is at a concentration selected from the group consisting of between 1 and 500 mg / ml, between 10 and 100 mg / ml, and 50 mg / ml.

8. 10. The composition of claim 1, wherein the CBDA component of the composition is at a concentration selected from the group consisting of between 1 and 500 mg / ml, between 10 and 100 mg / ml, and 50 mg / ml.

9. 10. The composition of claim 1, having a UPLC mass chromatogram corresponding to FIG. 3 utilizing the conditions described in Example 1.

10. 10. The composition of claim 1, wherein the additional active ingredient is selected from the group consisting of diclofenac, prednisolone, and Celebrex.

11. A pharmaceutical composition comprising the composition of any one of claims 1 to 10 together with a pharmaceutically acceptable carrier.

12. A dosage form comprising the composition of any one of claims 1 to 10.

13. 13. The dosage form of claim 12, wherein the CBDA component of the composition is selected from the group consisting of between 1 mg and 1000 mg, between 1 mg and 500 mg, between 1 and 100 mg, less than 400 mg, less than 300 mg, less than 200 mg, and less than 100 mg.

14. 13. The dosage form of claim 12, wherein the CBDA component of the composition is selected from the group consisting of 600 mg, 400 mg, 300 mg, 200 mg, 100 mg, 50 mg, 10 mg, 5 mg, 2 mg, and 1 mg.

15. The dosage form of claim 12 for treating a disorder in a patient in need thereof.

16. 16. The dosage form of claim 15, wherein the disorder is a neurological disorder.

17. 16. The dosage form of claim 15, wherein the neurological disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, traumatic brain injury, rheumatoid arthritis, chronic migraine, epilepsy, autism spectrum disorder, attention deficit hyperactivity disorder, cerebral palsy and related subtypes, neuropathic pain, and depression.

18. 11. Use of the composition of claims 1 and 10 in the manufacture of a medicament for the treatment of a disorder.

19. 11. A process for extracting a composition according to claims 1 to 10 from cannabis plant material, comprising: (1) grinding the cannabis plant material to a sufficient grind size; (2) contacting the grounds produced by step a) with oil; (3) mixing the grounds and oil for a sufficient period of time to form a mixture; (4) pressing the mixture to recover the oil; (5) centrifuging the oil to further refine the oil; (6) collecting the oil extract in a suitable container; The process includes:

20. 11. A process for extracting a composition according to claims 1 to 10 from cannabis plant material, comprising: (1) grinding the cannabis plant material to a sufficient grind size; (2) contacting the ground material produced by step a) with alcohol; (3) mixing the grind and alcohol for a sufficient period of time to form a mixture; (4) sonicating the mixture; (5) centrifuging the mixture; (6) collecting the alcoholic extract in a suitable container; The process includes:

21. 20. A product produced by the process of claim 19.

22. 13. A kit comprising the dosage form of claim 12 together with instructions for use.

23. 10. The composition, and process as hereinbefore described by the preceding examples.

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