CN118103052A

CN118103052A - Compositions and methods for treating neurological disorders with combination products

Info

Publication number: CN118103052A
Application number: CN202280068671.4A
Authority: CN
Inventors: A·W·克里普斯; E·伊斯格尔; A·E·M·安德鲁斯; T·G·杜西
Original assignee: Neurotechnology International Ltd
Current assignee: Neurotechnology International Ltd
Priority date: 2021-10-11
Filing date: 2022-10-11
Publication date: 2024-05-28

Abstract

The present invention relates to compositions comprising cannabinoids and additional active ingredients. The invention also relates to pharmaceutical compositions, dosage forms and methods of treating neurological disorders by administering the compositions to a patient in need thereof.

Description

Compositions and methods for treating neurological disorders with combination products

Technical Field

Background

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

A. neuroinflammation

Neuroinflammation refers to the process of triggering the innate immune system of the brain following an inflammatory attack, for example, an inflammatory attack caused by injury, infection, exposure to toxins, neurodegenerative diseases or aging. Neuroinflammation has been implicated in the development of a variety of neurological and somatic 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 Disorders (ASD), attention Deficit Hyperactivity Disorder (ADHD), cerebral palsy and related subtypes, neuropathic pain and depression.

In the Central Nervous System (CNS), the innate immune response plays an important role in both physiological and pathological conditions. CNS disorders, including traumatic brain injury, ischemic stroke, brain tumors, and cerebrovascular and neurodegenerative diseases, trigger a cascade of events broadly defined as neuroinflammation, characterized by activation of microglial and astrocyte populations. In another aspect, microglial and astrocyte activation, T lymphocyte infiltration, and inflammatory cytokine overproduction have been shown to be associated with neuronal changes in animal and human tissues. Thus, neuroinflammation is an important topic in contemporary neuroscience.

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

B. Neurological disorders

Examples of "neuroinflammatory" based neurological conditions include: alzheimer's disease (Alzheimer's disease is the most common chronic progressive neurodegenerative disease and is the 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.

C. Microglial activation/neurodegeneration

Microglia are unique resident macrophages of the Central Nervous System (CNS). They play an important role during CNS development and adult homeostasis. They have a major contribution to adult neurogenesis and neuroinflammation (Zhan y., paolicelli r.c., 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)., therefore, they are involved in the pathogenesis of neurodegenerative diseases and promote aging they play an important role in maintaining and breaking the blood brain barrier as innate immune cells they substantially contribute to the immune response (Xiong XY,Liu L,Yang QW.Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke.Prog Neurobiol.2016,142:23–44). against infectious agents affecting the CNS they also play a major role in the growth of CNS tumors (Xiong XY,Liu L,Yang QW.Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke.Prog Neurobiol.2016,142:23–44).

Under physiological conditions, the branched resting 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 activation of microglia and 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,Géa LP,Colombo R,Barbé-Tuana FM,Kapczinski F,Rosa AR.The role of macrophage polarization on bipolar disorder:identifying new therapeutic targets.Aust N Z JPsychiatry.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). whereby the activated inflammatory microglia are neurotoxic, killing neurons by phagocytizing them or releasing various neurotoxic molecules and factors including Reactive Oxygen Species (ROS), glutamate, fas-ligand, tumor necrosis factor alpha (tnfa) and the like (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).

Activated microglial cells driving chronic neuroinflammation have also been shown to have a substantial contribution to 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/M2polarization and metabolic states.Br J Pharmacol.2016,173:649–65) and psychiatric diseases (Orihuela R,McPherson CA,Harry GJ.Microglial M1/M2 polarization and metabolic states.Br JPharmacol.2016,173:649–65), neurodegenerative diseases including alzheimer's (Nakagawa Y,Chiba K.Diversity and plasticity of microglial cells in psychiatric and neurological disorders.Pharmacol Ther.2015,154:21–35)、 parkinson's (Orihuela R,McPherson CA,Harry GJ.Microglial M1/M2polarization and metabolic states.Br J Pharmacol.2016,173:649–65)、 Amyotrophic Lateral Sclerosis (ALS) and multiple sclerosis. Aging occurs in parallel with systemic chronic activation of the immune system and polarization towards low-level inflammatory states (Ransohoff RM.A polarizing question:do M1 and M2 microglia existNat 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).

D. medical application of cannabis

Cannabis (Cannabis sativa l.) has traditional medical uses. Medical cannabis has attracted significant interest due to its anti-inflammatory, antioxidant and anti-necrotic protective effects and the favorable safety and tolerability profile exhibited in humans, making it a promising candidate in a variety of therapeutic approaches. However, clinical use is limited due to adverse reactions to the central nervous system and the potential for abuse and addiction. The plant exudes a resin containing a mixture of cannabinoids having two major components Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD). The structure of CBD was described in the 60 s of the 20 th century, and has received attention because of its lack of mental activity. It appears to be the ideal choice for clinical trials due to its excellent tolerability in humans, lack of psychoactive effects and low potential for abuse.

E. Cannabinoids

In addition to its good safety and lack of psychoactive effects, CBD also shows a broad range of therapeutic effects. Some in vitro and in vivo experimental studies have demonstrated anti-inflammatory and immunomodulatory, antipsychotic, analgesic and antiepileptic effects. For these reasons, CBD is one of the most studied cannabinoids at present. CBD shows low affinity for cannabinoid receptors type 1 (CB ₁) and type 2 (CB ₂) compared to Δ9-THC. The CB ₁ receptor is mainly present at the ends of central and peripheral neurons and the CB ₂ receptor is mainly present in immune cells. Several in vitro studies have shown that low concentrations of CBD have weak CB ₁ and CB ₂ antagonistic effects.

Studies have shown that CBD behaves as a negative allosteric modulator of CB ₁, meaning that CBD does not directly activate the receptor, but rather alters the potency and efficacy of the positive ligand Δ9-THC and 2-arachidonylglycerol (2-AG) of CBD 1. These preliminary results require further validation, but can explain the ability of CBD to antagonize some of the effects of Δ9-THC reported in vitro, in vivo and in human clinical studies. It has also been proposed that the role of CBD as an allosteric modulator of CB ₁ may 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. It can also induce arachidonic acid release and reduce prostaglandin E2 (PGE 2) and Nitric Oxide (NO) production.

However, not all physiological effects of CBD are mediated by cannabinoid receptors. CBD has many targets outside the endogenous cannabinoid system, whose role independent of the cannabinoid receptor is the subject of recent pharmacological studies. Some effects, such as anti-inflammatory and immunosuppressive effects, are mediated by more than one target. The anti-inflammatory, immunosuppressive effects may be mediated by activating the adenosine receptors a _1A and a _2A and the strychnine-sensitive α1 and α1β glycine receptors and inhibiting the balanced nucleoside transporter. Furthermore, CBD activity may elicit different physiological effects from the same target. For example, the same glycine receptor is involved in both anti-inflammatory and neuropathic pain inhibition. The effects on serotonin 5HT1A receptors may produce anxiolytic, anti-panic and antidepressant effects, and thus studies have shown an extensive overview of the molecular pharmacology of CBD. Despite advances in molecular pharmacology of CBD, many of the pharmacological mechanisms of CBD remain uncharacterized.

Studies published in animals have demonstrated that oral bioavailability of cannabidiol has been shown to be about 13-19%. Plasma and brain concentrations are dose-dependent in animals, with bioavailability increasing with various oil formulations. Cannabinoids undergo extensive first pass metabolism, with metabolites thereof excreted primarily through the kidneys.

Cannabinoids are extensively metabolized by the liver, where they are hydroxylated to 7-OH-CBD by the P450 enzymes, mainly the CYP3A (2/4) and CYP2C (8/9/19) isozymes families. The metabolite then undergoes significant further metabolism in the liver, with the resulting metabolite excreted in the feces and to a much lesser extent in the urine.

Cannabidiol is known to act on the Cannabinoid (CB) receptors (CB 1 and CB 2) of the endogenous cannabinoid system, which receptors are present in various areas of the body, including the peripheral and central nervous systems, including the brain. The endogenous cannabinoid system regulates a variety of physiological responses of the body, including pain, memory, appetite, and emotion. More particularly, CB1 receptors can be found within the painful pathways of the brain and spinal cord, where they can affect cannabidiol-induced analgesia and anxiolytic effects, and CB2 receptors have an effect on immune cells, where they can affect cannabidiol-induced anti-inflammatory processes.

Cannabidiol has been shown to act as a negative allosteric modulator of the cannabinoid CB1 receptor, which is the most abundant G-protein coupled receptor (GPCR) in vivo. Allosteric modulation of the receptor is achieved by modulating the activity of the receptor at functionally different sites from the binding site of an agonist or antagonist. Negative allosteric modulation of cannabidiol is therapeutically important because direct agonists are limited by their psychotropic effects, whereas direct antagonists are limited by their inhibitory effects.

There have been some developments in the regulatory approval of CBDs.Is a plant-derived pharmaceutical grade Cannabidiol (CBD) drug, and was approved for use in the united states by the FDA in 2018. /(I)Contains 100mg cannabidiol/milliliter (mL) solution, and is orally administered twice a day. The Australian therapeutic administration (TGA) approved Epidiolex in 9 months 2020 for the treatment of Lennox-Gastaut syndrome (LGS) or Dravet syndrome-related episodes in patients two years or older.

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

Brief description of the invention

In a first aspect, the invention broadly consists in a composition comprising: about 50w/w% CBDA; and wherein all other cannabinoids add up to about 15w/w%.

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 an additional active ingredient.

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 an additional active ingredient.

w/w％

CBDA 49％；

CBD 2％；

CBG 5％；

CBDP 2％；

CBDB 2％；

CBGA 5％；

CBN 3％；

THC <0.3%; and an additional active ingredient.

w/w％

CBDA 45％；

CBD 1％；

CBG 4％；

CBDP 1％；

CBDB 2％；

CBGA 4％；

CBN 2％；

THC <0.2%; and an additional active ingredient.

w/w％

CBDA 45％；

CBD 1％；

CBG 4％；

CBDP 1％；

CBDB 2％；

CBGA 4％；

CBN 1％；

THC <0.2%; and an additional active ingredient.

In a preferred embodiment, the composition comprises an amount of cannabinoid selected from any of the embodiments described above.

In a second aspect, the invention is a pharmaceutical composition comprising the composition of the first aspect of the invention and a pharmaceutically acceptable carrier.

In a third aspect, the invention is a dosage form comprising the composition of the first aspect of the invention.

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

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 condition.

In a sixth aspect, the invention is a method of extracting the composition of the invention from cannabis plant material, the method comprising the steps of:

1) Grinding cannabis plant material to a sufficient grind size;

2) Contacting the grind resulting from step a) with oil;

3) Mixing the grind and the oil for a time sufficient to form a mixture;

4) Extruding the mixture to recover oil;

5) Centrifuging the oil to further refine the oil; and

6) The oil extract was collected in a suitable container/steel vessel.

In a seventh aspect, the present invention is a method of extracting the composition of the present invention from cannabis plant material, the method comprising the steps of:

1) Grinding cannabis plant material to a sufficient grind size;

2) Contacting the grind resulting from step a) with an alcohol;

3) Mixing the grind and alcohol for a time sufficient to form a mixture;

4) Sonicating the mixture;

5) Centrifuging the mixture; and

6) The alcohol extract was collected in a suitable container/steel vessel.

In an eighth aspect, the invention is a product produced by the method of the invention.

In a ninth aspect, the invention is a kit comprising a dosage form of the invention and instructions for its use.

In a ninth aspect, the invention includes compositions, methods and processes as described in the following examples.

Other features of the invention are more fully described in the following description of various non-limiting embodiments. The description is included for the purpose of illustrating the invention. It is not to be interpreted as limiting the broad summary, disclosure, or description of the invention as set forth above.

Brief Description of Drawings

The following is a brief description of each figure and accompanying drawing.

FIG. 1 is a UPLC mass chromatogram of a cannabinoid standard mixture (10 ppm each); a) Positive ionization mode; and b) a negative ionization mode.

FIG. 2 is in-source fragmentation of CBD and CBG from a reference solution.

Figure 3 shows a mass spectrometry chromatogram of NTI 164.

Figure 4 shows a quadruple mass spectrometry chromatogram of CBD variants of NTI164 to identify CBDB and CBDP.

FIG. 5 shows the normalization of the inflammation-induced iNOS expression of NTI 164. The figure shows that NTI164 normalizes inflammation-induced iNOS expression.

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

Fig. 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 on neurons (no glutamate).

Fig. 8 demonstrates that CBD is toxic in this example, while 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 the excitotoxic cell injury paradigm.

Figure 9 shows microglial response under inflammatory conditions assessing arginase 1 expression. The figure shows that NTI164 normalizes inflammation-induced (injured cell) Arg1 expression.

Fig. 10 is a graph summarizing arginine metabolism and its effect on the overall balance of anti-inflammatory and pro-inflammatory signals. (ref: review).S.Clemente,Aren van Waarde,Inês F.Antunes,Alexander />And Philip H.Elsinga.Arginase as a Potential Biomarker of Disease Progression:A Molecular Imaging Perspective.(2020)).

Fig. 11 shows the distribution of patients actively using NTI164 for example 10.

Figure 12 shows the disease severity profile of the active patient according to CGI-S disease severity at baseline for example 10.

Figure 13 shows the maximum tolerated dose for the active patient of example 10.

Figure 14 shows the overall improvement in CGI-S at 28 days of NTI164 treatment.

Figure 15 shows the CGI-S severity of the disease after 28 days of treatment.

Figure 16 shows the CGI-S severity of the disease after 28 days of treatment.

Figure 17 shows the effect of CGI-S treatment after 28 days of treatment.

Fig. 18 shows an age distribution of a patient who actively uses NTI164 in example 11.

Figure 19 shows the disease severity profile of positive patients according to CGI-S disease severity at baseline for example 11. CGI-S refers to the clinical global impression scale-disease severity.

Figure 20 shows the overall improvement in CGI-S at 20 weeks of NTI164 treatment.

Figure 21 shows the overall improvement in CGI-S over time up to and including NTI164 treatment for 20 weeks.

Figure 22 shows the CGI-S severity of the disease at 20 weeks of treatment.

Figure 23 shows disease CGI-S severity over time up to and including 20 weeks of treatment.

Figure 24 shows the CGI-S severity of the disease at 20 weeks of treatment.

Figure 25 shows the effect of CGI-S treatment over time up to and including 20 weeks of treatment.

Figure 26 shows the effect of CGI-S treatment at 20 weeks of treatment.

Detailed Description

For convenience, the following section generally lists various meanings of the terms used herein. Following this discussion, general aspects concerning the compositions, pharmaceutical uses and methods of the invention are discussed, followed by specific examples demonstrating the nature of the various embodiments of the invention and how they can be carried out.

It will be appreciated by those skilled in the art that the invention described herein may be subject to variations and modifications other than those specifically described. The present invention includes all such variations and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of said steps or features or any two or more of said steps or features.

Each document, reference, patent application or patent cited herein is expressly incorporated by reference in its entirety, meaning that the reader should read and consider it as part of this document. The documents, references, patent applications or patents cited herein are not repeated herein for the sake of brevity only. However, neither the materials referred to nor the information contained in the materials should be construed as being common general knowledge.

Manufacturer specifications, descriptions, product descriptions, and product pages for any product mentioned herein or in any document incorporated by reference herein are incorporated by reference and may be used in the practice of the invention.

The scope of the invention is not limited to any particular embodiments described herein. These embodiments are for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the invention as described herein.

1. Definition of the definition

The following provides a means for some of the terms and phrases used in the specification, examples, and appended claims. If there is a clear difference in the usage of terms in the art from its definition provided herein, the definition provided within the specification controls.

Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about". The term "about" when used in conjunction with a percentage may refer to ± 1%.

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 values defining the range, as well as values adjacent to the range that produce the same or substantially the same result as values immediately adjacent to the value defining the boundary of the range. For example, those skilled in the art will appreciate that a 10% variation of the upper or lower limit of the range may be entirely suitable and is encompassed by the invention. More particularly, the upper or lower limit of the range will vary by 5% or as generally recognized in the art, whichever is greater.

In the present application, the use of the singular also includes the plural unless specifically stated otherwise. In the present application, the use of "or" means "and/or" unless otherwise indicated. Furthermore, the use of the term "include" and other forms such as "include" and "included" are not limiting. Furthermore, unless specifically stated otherwise, terms such as "element" or "component" encompass both elements and components comprising one unit as well as elements and components comprising more than one subunit. Furthermore, the use of the term "portion" may include a portion of a portion or the entire portion.

Throughout the 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.

As used herein, a "therapeutically effective amount" with respect to a method of treatment and in particular a dosage of a drug shall mean a dosage that provides a particular pharmacological response when the drug is administered in a significant amount of a subject in need of such treatment. It is emphasized that a "therapeutically effective amount" administered to a particular subject in a particular situation is not always effective in treating the diseases described herein, even though such a dose is considered by those skilled in the art to be a "therapeutically effective amount". It will also be appreciated that in certain circumstances, the drug dosage is measured as an oral dosage, or with reference to a measured drug level in the blood. The amount effective for this use will depend on: a desired therapeutic effect; efficacy of the bioactive material; a desired duration of treatment; the stage and severity of the disease being treated; the weight and general health status of the patient; judgment of prescribing physician. Titration of therapeutic doses is required to optimize safety and efficacy. Those skilled in the art will appreciate that the appropriate dosage level for treatment will therefore vary, depending in part on the indication in which the active agent is used, the route of administration, and the size (body weight, body surface or organ size) and condition (age and general health) of the patient. Thus, the clinician can titrate the dose and modify the route of administration to obtain the best therapeutic effect. Typical dosages may range from about 0.1 μg/kg up to about 100mg/kg or more, depending on the factors described above. In other embodiments, the dosage may be in the range of 0.1 μg/kg to about 100mg/kg; or 1 μg/kg up to about 100mg/kg; or 5 μg/kg up to about 100mg/kg.

The frequency of administration will depend on the pharmacokinetic parameters of the active agent and the formulation used. Typically, the clinician will administer the composition until a dose is reached that achieves the desired effect. Thus, the composition may be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implant device or catheter. Further refinement of the appropriate dosage is routinely performed by those of ordinary skill in the art and is within the scope of the tasks they routinely perform. The appropriate dose may be determined by using the appropriate dose-response data.

As used herein, "pharmaceutically 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.

The term "subject" as used herein generally includes mammals, such as: a person; farm animals such as sheep, goats, pigs, cattle, horses, llamas; companion animals such as dogs and cats; a primate; birds such as chickens, geese and ducks; fish; and reptiles. The object is preferably a person.

Other definitions of selected terms used herein may be found in the detailed description of the invention and are applicable throughout. Unless defined otherwise, 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.

Features of the present invention will now be discussed with reference to the following non-limiting description and examples of implementation.

2. Description of the embodiments

Composition and method for producing the same

The present invention provides a composition comprising the following cannabinoids:

About 50w/w% CBDA; and

Wherein all other cannabinoids total about 15w/w%.

Preferably, the composition comprises an additional active ingredient.

In a preferred embodiment, the present invention provides a composition comprising the following cannabinoids and additional active ingredients: w/w%

About 50% CBDA;

About 2% CBD.

In a further preferred embodiment, the present invention provides a composition comprising the following cannabinoids and additional active ingredients: w/w%

About 50% CBDA;

About 5% CBG.

About 50% CBDA;

About 2% CBDP.

About 50% CBDA;

About 2% CBDB.

About 50% CBDA;

About 5% CBGA.

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.

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.

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.

In a further preferred embodiment, the present invention provides a composition comprising the following cannabinoids and additional active ingredients:

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％。

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％。

w/w％

CBDA 50％；

CBD 2％；

CBG 5％；

CBDP 2％；

CBDB 2％；

CBGA 5％；

CBN 3％；

THC<0.3％。

w/w％

CBDA 49％；

CBD 2％；

CBG 5％；

CBDP 2％；

CBDB 2％；

CBGA 5％；

CBN 2％；

THC<0.3％。

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％。

w/w％

CBDA 45％；

CBD 1％；

CBG 4％；

CBDP 1％；

CBDB 2％；

CBGA 4％；

CBN 2％；

THC<0.2％。

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％。

w/w％

CBDA 62.78％；

CBD 5.80％；

CBG 0.44％；

CBGA 1.26％；

CBN 1.98％；

THC<0.70％。

w/w％

CBDA 60.29％；

CBD 5.34％；

CBG 0.39％；

CBGA 1.14％；

CBN 0.85％；

THC<0.65％。

In a further preferred embodiment, the present invention provides a composition wherein the cannabinoid is present in an amount selected from the group consisting of:

Composition 1 comprising

w/w％

CBDA 50％；

CBD 2％；

CBG 5％；

CBDP 2％；

CBDB 2％；

CBGA 5％；

CBN 3％；

THC <0.3%; and

The presence of an additional active ingredient(s),

And

Composition 2 comprising

w/w％

CBDA 45％；

CBD 1％；

CBG 4％；

CBDP 1％；

CBDB 2％；

CBGA 4％；

CBN 2％；

THC <0.2%; and

Additional active ingredients.

In a further preferred embodiment, the present invention provides a composition wherein the amount of cannabinoid is determined by a method selected from the group consisting of: high performance chromatography (HPLC), proton nuclear magnetic resonance spectroscopy (H ¹ NMR) and mass spectrometry.

In a further preferred embodiment, the present invention provides a composition derived from cannabis plant material.

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

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

In a further preferred embodiment, the present invention provides a composition further comprising an oil selected from the group consisting of: synthetic oil; a plant-based oil; mineral oil; canola oil; and olive oil.

In a further preferred embodiment, the composition comprises less than 5% w/w terpene.

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

In a further preferred embodiment, the composition comprises less than 2% w/w plant phenol.

In a further preferred embodiment, the composition comprises a component selected from flavonoids, proteins, sterols and esters.

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

In a further preferred embodiment, the composition comprises less than 0.1wt% of organic impurities, as measured by a method selected from the group consisting of: high performance chromatography (HPLC), proton nuclear magnetic resonance spectroscopy (H ¹ HMR); and mass spectrometry.

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

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

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

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

In a further preferred embodiment, the composition is a liquid.

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

In a further preferred embodiment, the composition shows no degradation or decarboxylation of cannabinoids when measured at a time point selected from the group consisting of: at 1 day; at 2 days; at 7 days; at 14 days; at 28 days; at 5 weeks; at 6 weeks; and at 32 weeks.

In a further preferred embodiment, the composition exhibits cannabinoid stabilization when measured at a time point selected from the group consisting of: at 1 day; at 2 days; at 7 days; at 14 days; at 28 days; at 5 weeks; at 6 weeks; and at 32 weeks.

In a further preferred embodiment, the composition does not exhibit mutagenicity, carcinogenicity, or genotoxicity when delivered at a concentration that delivers 120mg/ml CBDA.

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.

Preferably, the composition is suitable for inhibiting neuroinflammation. More preferably, the composition is suitable for treating neurological disorders.

In a further preferred embodiment, the present invention provides a composition having a UPLC mass spectrum corresponding to that of fig. 3, determined using the conditions described in example 1.

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

Preferably, the additional active ingredient is selected from: a polypeptide; an antibody; NSAIDs; neuromodulatory agents; and neurotransmitters, steroids-all related classes (corticosteroids), analgesics, antipsychotics, antidepressants, immunotherapy.

In a 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 (oxaprozin), flurbiprofen, sulindac, tolmetin, prednisolone, and fenoprofen.

In one embodiment, the additional active ingredient is selected from: diclofenac, prednisone, celecoxib, and psylocibin.

In a further preferred embodiment, the ratio of the cannabinoid component and the additional active ingredient is selected from the group consisting of: 1 unit w/w cannabinoid: 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.

In a further preferred embodiment, the ratio of the additional active ingredient and 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.

In a further preferred embodiment, the ratio of CBDA and the further active ingredient is selected from: 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.

In a further preferred embodiment, the ratio of the additional active ingredient to CBDA is selected from: 1 unit w/w of the additional active ingredient and 1 unit w/w/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.

In a preferred embodiment, the neuromodulatory agent is a fanciful agent.

Preferably, the neuromodulatory agent is selected from: 3, 4-methylenedioxymethamphetamine; lysergic acid diethylamine; and stropharia rugoso-annulata. Preferably, the neuromodulatory agent is selected from: steroids (all relevant classes (corticosteroids)), analgesics, antipsychotics, antidepressants and immunotherapeutic drugs.

In a further preferred embodiment, the composition exhibits synergistic biological activity.

In a further preferred embodiment, the composition exhibits a level of biological activity greater than the sum of: (1) Biological activity of the cannabinoid component when delivered in the absence of additional active ingredient; and (2) the biological activity of the additional active ingredient when delivered in the absence of the cannabinoid component.

In a further preferred embodiment, the biological activity is selected from: inhibiting inflammation; inhibiting neuroinflammation; treating a neurological disorder; inhibiting COX-2 activity; inhibit iNOS activity; inhibiting TNF-alpha activity; inhibiting IL-2 activity; inhibit IL-12 activity and inhibit GS-MCF activity.

In a further preferred embodiment, the composition is selected from the group consisting of: a therapeutic composition; a pharmaceutical composition; a cosmetic composition; and veterinary compositions.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising the composition of the invention and a pharmaceutically acceptable carrier.

Therapeutic compositions are within the scope of the present invention. Preferably, the composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate buffered saline. As used herein, "pharmaceutically 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 pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients may also be incorporated into the compositions. See, e.g., remington' sPharmaceutical Sciences, 19 th edition (1995,Mack Publishing Co., easton, pa.), incorporated herein by reference.

The pharmaceutical composition may contain formulation materials for altering, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition. Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); an antimicrobial agent; antioxidants (e.g., ascorbic acid, sodium sulfite or sodium bisulfite, vitamin E phosphate-fat-soluble vitamins, nanoemulsions); buffers (e.g., borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediamine tetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), bulking agents; monosaccharides, disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrins); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavors (natural and naturally derived products) and diluents; an emulsifying agent; hydrophilic polymers (e.g., polyvinylpyrrolidone); a low molecular weight polypeptide; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (including artificial sweeteners such as mannitol or sorbitol); a suspending agent; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (sucrose or sorbitol); tonicity enhancing agents (e.g., alkali metal halides, preferably sodium chloride or potassium chloride), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants.

The optimal pharmaceutical composition will be determined by one skilled in the art based on, for example, the intended route of administration, the form of delivery, and the desired dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the compositions of the present invention. The preferred form of the pharmaceutical composition depends on the intended mode of administration and therapeutic application.

The primary vehicle or carrier in the pharmaceutical composition is aqueous and non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, possibly supplemented with other substances. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Other exemplary pharmaceutical compositions comprise tris buffer at about pH 7.0-8.5 or acetate buffer at about pH4.0-5.5, which may also comprise sorbitol or a suitable substitute thereof. In one embodiment of the invention, the pharmaceutical composition may be prepared for storage by mixing the selected composition of the desired purity with an optional formulation in the form of an aqueous solution.

The formulation components are present at an acceptable concentration at the site of application. For example, buffers are used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

Additional pharmaceutical compositions, including formulations of the invention in the form of sustained or controlled release formulations, will be apparent to those skilled in the art. Techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Other examples of sustained-release formulations include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. The sustained release matrix 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 also include liposomes, which may be prepared by any of a variety of methods known in the art.

Pharmaceutical compositions for in vivo administration must generally be sterile. This can be achieved by filtration through sterile filtration membranes. In addition, the composition is typically placed in a container having a sterile inlet. Once the pharmaceutical composition is formulated, it may be stored as a solution in a sterile vial.

In another preferred embodiment, the composition retains its effective biological activity for a period of time selected from the group consisting of: for greater than 24 hours; for more than 36 hours; and greater than 48 hours. Preferably, the composition stabilizes for a period of time 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 ℃, 18 ℃ and 25 ℃.

Dosage forms

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

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

In a further embodiment, the dosage form is selected from the following forms: solutions, tablets, capsules, wafers, dry powder sachets and vials/freeze-dried.

Preferably, the dosage forms are stored in sealed and sterile containers.

Therapeutic method

The invention also provides a method of treating a disease comprising administering to a patient in need thereof a therapeutically effective amount of a dosage form of the invention.

In a further preferred embodiment, the dosage form is administered in an amount that at least partially treats the disease.

In a further preferred embodiment, the therapeutically effective amount is an amount of a cannabinoid selected from the group consisting of: between 1 and 100 mg/kg/day; between 2 and 50 mg/kg/day; between 5 and 40 mg/kg/day; between 10 and 30 mg/kg/day; between 20 and 25 mg/kg/day; and 20 mg/kg/day. Preferably, the therapeutically effective amount is an amount of a cannabinoid 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.

In a further preferred embodiment, the therapeutically effective amount is an amount of CBDA selected from the group consisting of: between 1 and 100 mg/kg/day; between 2 and 50 mg/kg/day; between 5 and 40 mg/kg/day; between 10 and 30 mg/kg/day; between 20 and 25 mg/kg/day; and 20 mg/kg/day. Preferably, the therapeutically effective amount is an amount of CBDA 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.

In a further preferred embodiment, tmax occurs between 1 and 4 hours.

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

In a further preferred embodiment, a therapeutically effective amount is administered to a subject to treat a disease.

Preferably, a therapeutically effective amount is administered to a subject using a dosing regimen selected from the group consisting of: twice per hour; every hour; once every six hours; once every 8 hours; once every 12 hours; once a day; twice a week; once a week; once every 2 weeks; once every 6 weeks; once a month; every 2 months; every 3 months; once every 6 months; and once a year.

Preferably, a therapeutically effective amount is administered to a subject using a method selected from the group consisting of: oral, intravenous, intramuscular, intrathecal, subcutaneous, sublingual, buccal, rectal, vaginal, topical, parenteral, mucosal, by ocular route, by otic route, nasal, by inhalation, transdermal and systemic.

In a further preferred embodiment, the disease is caused by inflammation.

In a further preferred embodiment, the disease is caused by neuroinflammation.

Preferably, the disorder is a neurological disorder. More preferably, the neurological disorder is selected from: 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.

In a further preferred embodiment, the ASD is ASD grade II/III and is "mildly ill", "moderately ill", "significantly ill" or "severely ill" on the CGI severity scale.

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

Subjects that can be treated with the present invention will include humans as well as other mammals and animals.

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 invention together with additional active ingredients. In a preferred form, the additional active ingredient is administered using a dosing regimen selected from the group consisting of: simultaneously with administration of the dosage form of the invention; prior to administration of the dosage form of the invention; after administration of the dosage form of the invention; concurrent with administration of the dosage forms of the present invention; sequentially prior to administration of the dosage forms of the invention; and sequentially following administration of the dosage forms of the invention.

In a 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.

In a further preferred embodiment, the ratio of the cannabinoid component and the additional active ingredient is selected from the group consisting of: 1 unit w/w of 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.

In a further preferred embodiment, the ratio of the additional active ingredient to the cannabinoid is selected from the group consisting of: 1 unit w/w of additional active ingredient and 1 unit w/w of 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.

In a further preferred embodiment, the ratio of CBDA and the further active ingredient is selected from: 1 unit w/w CBDA 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.

In a further preferred embodiment, the ratio of the additional active ingredient to CBDA is selected from: 1 unit w/w of additional active ingredient and 1 unit w/w/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.

In a preferred embodiment, the neuromodulatory agent is a fanciful agent.

Preferably, the neuromodulatory agent is selected from: 3, 4-methylenedioxymethamphetamine; lysergic acid diethylamine; and stropharia rugoso-annulata.

The effect of the administered therapeutic composition can be monitored by standard diagnostic methods.

Use of a composition in the preparation of a medicament

The use is within the scope of the invention. The invention also provides the use of a composition according to the first aspect of the invention in the manufacture of a medicament for the treatment of a condition.

In a preferred embodiment, the invention is the use of a composition comprising the following cannabinoids in the manufacture of a medicament for use in treating 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％。

In a further preferred embodiment, in the preparation of a medicament for treating a condition, the cannabinoid of the composition is present in an amount selected from the group consisting of:

Composition 1 comprising

w/w％

CBDA 50％；

CBD 2％；

CBG 5％；

CBDP 2％；

CBDB 2％；

CBGA 5％；

CBN 3%; and

THC<0.3％；

And

Composition 2 comprising

w/w％

CBDA 45％；

CBD 1％；

CBG 4％；

CBDP 1％；

CBDB 2％；

CBGA 4％；

CBN 2%

THC<0.2％。

In a further embodiment, the composition further comprises an oil selected from the group consisting of: synthetic oil; a plant-based oil; mineral oil; canola oil; and olive oil.

In a further embodiment, the composition comprises less than 5% w/w terpene.

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

In a further embodiment, the composition comprises less than 2% w/w plant phenol.

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

In a further embodiment, the CBDA component of the composition is selected from the group consisting of: a concentration of between 1 and 500 mg/ml; between 10 and 100 mg/ml; the concentration was 50mg/ml.

In a further embodiment, the composition has a UPLC mass spectrum corresponding to that of fig. 3, as determined using the conditions described in example 1.

Method of

The present invention also provides a process for extracting a composition of the first aspect of the invention from cannabis plant material, the process comprising the steps of:

1) Grinding cannabis plant material to a sufficient grind size;

2) Contacting the grind resulting from step a) with oil;

3) Mixing the grind and the oil for a time sufficient to form a mixture;

4) Extruding the mixture to recover oil;

5) Centrifuging the oil to further refine the oil; and

6) The oil extract was collected in a suitable container/steel vessel.

In a further preferred embodiment, the Cannabis plant material is derived from Cannabis (Cannabis sativa l.).

In a further preferred embodiment, sufficient grinding size is selected from: between 0.1mm and 3 mm; between 1mm and 2 mm; and between 0.5mm and 2.5 mm.

In a further preferred embodiment, the sufficient time is selected from: between 30 minutes and 2 hours; between 45 minutes and 1.5 hours; and 1 hour.

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

Preferably, the oil is olive oil.

The present invention also provides an alternative method of extracting the composition of the first aspect of the invention from cannabis plant material, the method comprising the steps of:

1) Grinding cannabis plant material to a sufficient grind size;

2) Contacting the grind resulting from step a) with an alcohol;

3) Mixing the grind and alcohol for a time sufficient to form a mixture;

4) Sonicating the mixture;

5) Centrifuging the mixture; and

6) The alcohol extract was collected in a suitable container/steel vessel.

In a further preferred embodiment, the alcohol is ethanol.

In a further preferred embodiment, the alcohol is selected from: ethanol, isopropanol, methanol, benzyl alcohol, 1, 4-butanediol, 1,2, 4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butanol.

In a further preferred embodiment, sufficient grinding size is selected from: between 0.1mm and 3 mm; between 1mm and 2 mm; and between 0.5mm and 2.5 mm. In a further preferred embodiment, the sufficient time is selected from: between 30 minutes and 2 hours; between 45 minutes and 1.5 hours; and 1 hour.

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

Product of the process

The invention also provides a product produced by the method.

Medicine box

The invention also provides a kit comprising a dosage form according to one aspect of the invention and instructions for its use.

Device and method for controlling the same

The device is within the scope of the invention. In a preferred embodiment, the present invention provides an apparatus, wherein the apparatus comprises: (1) a composition as described in the first aspect of the invention; and (2) an applicator.

Method for stabilization

Methods for stabilizing the composition are within the scope of the invention.

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

In another further preferred embodiment, the composition retains its effective biological activity for a period of time selected from the group consisting of: for greater than 24 hours; for more than 36 hours; for greater than 48 hours.

From a safety standpoint, the addition of approved pharmaceutical excipients to stabilize the composition is preferred, as simpler methods may produce less varying results, and the choice of excipients may be limited to those with a generally recognized safety (GRAS) state. Excipients used to stabilize protein solutions can be divided into four general classes based on their chemical nature 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 solution viscosity to prevent protein aggregation and intra-and intermolecular electrostatic interactions between amino acids in proteins. Proteins (e.g., human serum albumin) are capable of stabilizing the structure of other proteins through ionic, electrostatic, and hydrophobic interactions. Similarly, small amino acids that do not have a net charge, such as alanine and glycine, stabilize proteins by forming weak electrostatic interactions.

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 preparation of medicaments is well known in the art. The use of any conventional medium or agent in the preparation of the pharmaceutical compositions of the present invention is contemplated unless it is incompatible with the pharmaceutically acceptable material. Pharmaceutically acceptable carriers of the 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 copolymer, cellulose derivatives, HPMC, hydroxypropyl cellulose, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose phthalate, polyacrylates and polymethacrylates, urea, sugar, polyols and polymers thereof, emulsifiers, gums, starches, organic acids and salts thereof, vinylpyrrolidone and vinyl acetate; and/or

B. binders such as various celluloses and crosslinked polyvinylpyrrolidone, microcrystalline cellulose; and/or (3) fillers such as lactose monohydrate, anhydrous lactose, microcrystalline cellulose, and various starches; and/or

C. Fillers such as lactose monohydrate, lactose anhydrous, mannitol, microcrystalline cellulose, and various starches; and/or

D. Lubricants, e.g. agents acting on the increase of the ability of the dosage form to be expelled from the packaging chamber, and/or

E. sweeteners, such as any natural or artificial sweetener, including sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K; and/or

F. A flavoring agent; and/or

G. Preservatives, for example potassium sorbate, methyl paraben, propyl paraben, benzoic acid and salts thereof, other esters of parahydroxybenzoic acid such as butyl paraben, alcohols such as ethanol or benzyl alcohol, phenolic chemicals such as phenol, or quaternary compounds such as benzalkonium chloride; and/or

H. a buffer; and/or

I. diluents, such as pharmaceutically acceptable inert fillers, e.g., microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing; and/or

J. absorption promoters, such as glyceryl trinitrate; and/or

K. Other pharmaceutically acceptable excipients.

The medicaments of the present invention suitable for use in animals and particularly humans must generally be sterile and stable under the conditions of manufacture and storage.

The invention also provides compositions, methods and processes as described in the preceding examples.

The invention will now be described with reference to the following non-limiting examples. The description of the embodiments in no way limits the preceding paragraphs of this specification, but rather is provided to illustrate the methods and compositions of the invention.

Examples

It will be apparent to those skilled in the art of milling and pharmaceutical technology that numerous enhancements and modifications can be made to the above-described process without departing from the basic inventive concepts. For example, in some applications, the bioactive material may be pre-treated and provided to the method in a pre-treated form. All such modifications and enhancements are considered to be within the scope of the present invention, the nature of which is to be determined from the foregoing description and the appended claims. Moreover, the following examples are provided for illustrative purposes only and are not intended to limit the scope of the methods or compositions of the present invention.

EXAMPLE 1 extraction and purification of NTI164

A.1 study objectives

The most desirable components were extracted and identified from NTI164 plant strain using an inert oil based extraction method.

A2 materials and methods

A2.1NTI164 plant Material

NTI164 plants are full spectrum medical Cannabis plants (Cannabis sativa species), which the inventors subsequently identified contained Cannabidiol (CBDA), cannabidiol (CBD), cannabigerol (CBGA) and Cannabidiol (CBDV), cannabidiol (CBN), but which had > 0.03% Tetrahydrocannabinol (THC). NTI164 plants were grown, dried and packaged according to Good Manufacturing Process (GMP) and TGO 93 and 100 guidelines according to the license and license of the pharmaceutical control Office (ODC).

A.2.2 extraction method-oil based

The device comprises: the following devices were used: 10mL glass scintillation bottle with cover; cobram's Estate olive oil; plant grinders (similar to coffee or food grade grinders) with a pore size of up to 50 μm; a wattle paper (WHATMAN PAPER), grade 1; a pipette; weight scales (transfer boats and spoons); eppendorf tubes; 50mL Falcon tube; a tabletop centrifuge (Eppendorf Centrifuge 5702); oz Design Brand 6Litre Fruit,Wine and Cider Press.

Extracting: extrusion and centrifugation: all work was done at standard laboratory temperature (18-22 ℃). The shoots of NTI164 were peeled from the hard stems and the stems were discarded. The mill was cleaned with 70% EtOH and the milling chamber was filled with dry plant material. The material was milled for 10 seconds (1-2 mm particle size) at the finest of the three settings. The grind was then mixed with 100mL olive oil in an autoclaved Schott bottle at a plant/oil ratio of 333 mg/mL. Next, it was placed on a stirrer for 1 hour at room temperature, and stirred with a magnetic stirrer (50 rpm). The oil plus plant mixture was then placed into Oz Design bran 6Litre Fruit,Wine and Cider Press to recover the oil component (mash) from the plants. Next, the recovered oil was placed in a 50mL Falcon tube and spun at 300g for 15 minutes at room temperature (separation 1). The oil was then transferred to a clean Schott bottle and the volume recovered was recorded. The recovery of oil for separation 1 was about 40%. The mash is discarded after each separation. To the recovered oil was added an additional 333mg/mL of ground plant/oil (additional 100 mL) material, mixing was repeated for 1-hour, and the oil was recovered and reused until a total of 999. Mu.g/mL (3X 100 mL) of plant/oil mixture passed (separation 2). The recovery of oil for separation 2 was about 50%. For the last time we placed in a Falcon tube and rotated as described above (separation 3). Recovery of the oil from separation 3 was about 50%. Then we collect only the oil and place it in Eppendorf tubes for treatment. This triple extraction procedure produced a total volume of 50ml of final product at a concentration of 48mg CBDA in 1ml olive oil, as determined by the UPLC potency test, using the method described below.

A.2.3 extraction method-ethanol-based

Extracting: extrusion and centrifugation: an alternative method includes extraction based on the use of ethanol. In this method, 500 milligrams of ground NTI164 plant material are mixed with 20mL of ethanol in a 50mL centrifuge tube. The tube was vigorously shaken for 60 seconds and then placed in an ultrasonic bath at 30℃for 10 minutes. Next, the sample was placed on a shaker (200 rpm) for 30 minutes. Once complete, place in a centrifuge and centrifuge for 5 minutes at 4400 rcf. The supernatant may then be evaluated in various preclinical models.

A.2.4 analytical analysis

The components in the NTI164 concentrate obtained by the above method were identified using ultra high performance liquid chromatography (UPLC) reversed phase and Liquid Chromatography Mass Spectrometry (LCMS). Analysis was performed using an integrated (U) HPLC system and a single quadrupole mass spectrometer detector with an electrospray ionization (ESI) interface.

The UPLC settings and conditions used were: cortecs UPLC Shield RP 18, (0A 1.6. Mu.M, 2.1X100 mm); analytical flow rate: 0.7mL/min; mobile phase a: water 0.1% tfa; mobile phase B: acetonitrile; equal concentration: 41:59 mobile phase A/mobile phase B; temperature: 35C; a detector: an Acquity UPLC PDA; sample injection volume: for a reference standard of 1.0mg/ml, 0.7uL, sample solution scale was scaled appropriately; software: empower 3CDS. Reference standard solutions were obtained from Novachem, CERILLIANT CORPORATION (TX, USA). These are pre-dissolved solutions, all of which were previously shown to be suitable for generating calibration curves.

A mixture of 16 cannabinoids in methanol was prepared containing 10ppm each of Cannabidiol (CBDV), cannabidiol (CBD), cannabigerol (CBG), tetrahydrocannabinol (THCV), cannabinol (CBN), Δ ⁹ -tetrahydrocannabinol (Δ ⁹-THC)、Δ⁸ -tetrahydrocannabinol (Δ ⁸ -THC), cannabinol (CBC), their respective acid forms and cannabinol (cannabicyclol, CBL). All solvents used were LCMS grade, standard prepared by dilution with 90% mobile phase B and 10% deionized water.

Table 1: parameters and conditions for UPLC and LCMS analysis

A3 results

A.3.1UPLC and LCMS analysis results

Figure 1 presents the isolation of cannabinoids in a mixed standard solution (i.e., a reference solution). Under experimental conditions, neutral cannabinoids such as Δ9-THC, CBD and CBL ionize in positive mode, while their respective acidic forms ionize in negative mode. Although CBD and CBG co-elute from the column, they differ in molecular weight and they can be identified by mass spectrometry. In addition, fig. 2 shows the difference between SID fragmentation patterns obtained for CBD and CBG (i.e., as further reference solutions). These highly specific results show the advantage of LCMS over LC-UV in analyzing and identifying cannabinoids.

Fig. 3 shows a UPLC mass spectrum of NTI164 extracted using an oil-based method. These results found that the NTI164 extract (oil suspension) contained the following components shown in table 2. Additional components will include flavonoids, proteins, phenols, sterols and esters. These are known components, constituting 30-40% of the whole plant cannabis material. Table 4 presents the concomitant elution times and the subpeak areas of the identified CBD peaks of the UPLC mass spectrum of fig. 3.

Table 2: the extracted NTI164 oil contains components (two bits after decimal point, more than 0.5 round up, less than 0.5 round down)

Component (A)	w/w％	w/w％
			Cannabidiol (CBD-A)	48.78％	49％
Cannabidiol (CBD)	1.89％；	2％；
			Cannabigerol (CBG)	4.88％；	5％；
Cannabidimenol (Cannabidiphorol) (CBDP)	1.68％；	2％；
			Canabidibutol(CBDB)	1.76％；	2％；
Cannabigerol acid (CBGA)	4.76％；	5％；
			Tetrahydrocannabinol (THC)	<0.18％	<0.3％
Terpenes	<5％	<5％
			Organic plant material-including phenols	2％	2％

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

Table 3: the extracted NTI164 ethanol component (two bits after decimal point, more than 0.5 round up, less than 0.5 round down)

Component (A)	w/w％	w/w％
			Cannabidiol (CBD-A)	45.28％	45％
Cannabidiol (CBD)	1.39％；	1％；
			Cannabigerol (CBG)	3.88％；	4％；
Cannabidimenol (Cannabidiphorol) (CBDP)	1.18％；	1％；
			Canabidibutol(CBDB)	1.56％；	2％；
Cannabigerol acid (CBGA)	3.76％；	4％；
			Tetrahydrocannabinol (THC)	<0.18％	<0.2％
Terpenes	<5％	<5％
			Organic plant material-including phenols	2％	2％

Table 4 presents the concomitant elution times and the subpeak areas of the identified CBD peaks of the UPLC mass spectrum of FIG. 3 (NTI 164, oil extraction).

Table 4: elution time and area under peak

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Note that the less rare cannabinoids such as CBDB and CBDP were detected using just Quad MS (which is different from conventional HPLC for other cannabinoids). These results are presented in fig. 4.

Characterization of stability Properties of example 2-NTI164

B.1 study objectives

To assess the stability of NTI164 samples suspended in oil formulations at room temperature.

B.2 materials and methods

B.2.1 sample preparation

Triplicate NTI164 samples were prepared using the method described above.

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

B.2.2 sampling

NTI164 samples were assayed weekly using CBDA as the primary marker/stability indicator. Upc equiconcentration isolation of the main cannabinoid was provided at a cycle time of 10.5 minutes using an ACQUITY upc H-Class system in combination with CORTECS UPLC Shield RP particle chemistry. Analytical methods employing UPLC were used as described above.

Reference standard solutions were obtained from CERILLIANT CORPORATION (Round Rock, TX). These pre-dissolved solutions have previously proven suitable for generating calibration curves.

The preparation of the standard curve is performed as follows. The linearity of the major cannabinoids (-) Δ ⁹ -THC and CBD at 10 concentrations (prepared using serial dilutions of appropriate standards in methanol) between 0.004mg/mL and 1.000mg/mL was determined as a representative demonstration of the linearity of the process. Table 5 summarizes the cannabinoids used in the isolation.

Table 5: cannabinoids for use in separations

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B.3 results

B.3.1 UPLC/Mass Spectrometry analysis results

Upc equiconcentration isolation of the main cannabinoid was provided at a cycle time of 10.5 minutes using an ACQUITY upc H-Class system in combination with CORTECS UPLC Shield RP particle chemistry. NTI164 samples were assayed weekly and CBDA was used as the primary marker as a stability indicator. The results presented in table 6 demonstrate that NTI164 is stable in inert oil medium for more than 6 weeks at room temperature. No decarboxylation or product degradation was observed at this time frame.

Table 6: stability of NTI164 at Room temperature

Example 3 characterization of biological Properties of NTI164

C.1 research objectives

To evaluate the anti-inflammatory and neuroprotective effects of NTI164 in neuronal and microglial cell lines.

Neuroinflammation is one of the main triggers of neurodegeneration. The first step in the study of factors and pathways capable of inducing inflammatory responses will lead to the identification of potential therapeutic targets through which to stop the progression of many diseases.

C.2 materials and methods

C.2.1. Sample preparation and dilution

500Mg of dry NTI164 plant material was suspended in 20ml absolute ethanol (using a 50ml blue top Falcon tube suitable for centrifugation), vigorously stirred/shaken for 60 seconds. The tube was then placed in an ultrasonic bath at 35-40C for 10 minutes. After the sonication was completed, the samples were then placed in a disk stirrer (200 rpm) for 30 minutes at room temperature. Once completed, the samples were centrifuged at 4400rpm for 5 minutes. The supernatant was collected for testing and development.

The units used to describe the disposition of the test product and the concentration of NTI 164.

A. 1/1000 dilution of extract-10 UL (stock substance is NTI164-10uL, which is equal to 2. Mu.g/ml CBDA)

B. 1/3000 dilution of extract-3 UL (stock substance is NTI164-3UL, which is equal to 6. Mu.g/ml CBDA)

C. 1/10000 dilution of extract-1 UL (stock material is NTI164-1UL, which is equal to 0.1 μg/ml CBDA).

For CBD samples, pure standards (in powder form) were used. CBD 98% isolate was purchased from LGC STANDARDS (london, uk) (CAS No. 13956-29-1) as a reference standard. A CBD standard reference was prepared at a concentration of 1mg/ml in acetonitrile. CBD dilutions in acetonitrile were prepared as follows: 2. Mu.g/ml; 6. Mu.g/ml; and 0.1. Mu.g/ml.

The final concentration of NTI164 (CBDA equivalent) and CBD used in these studies was 2. Mu.g/ml.

C.2.2. Microglial BV2 cultures

Immortalized microglial cell line BV2 was purchased from american type culture collection (American Tissue Culture collection). BV2 was cultured in RPMI medium containing gentamicin and supplemented with 10% FBS (for amplification) and 5% Fetal Bovine Serum (FBS) (when plated for experiments). All cells were from between passage numbers 39 and 45. Cells were plated at 45,000 cells/mm ², and after plating treated with phosphate buffered saline (PBS as control) or interleukin-1b+interferon-gamma (IL-1b+ifnγ to induce inflammation) for 24 hours. To test the effect of NTI164 on altering inflammatory responses, NTI164 was applied 1 hour before inflammation (pretreatment) or 1 hour after inflammation (post-treatment). NTI164 was applied at 10uL, 3uL or 1uL from the isolate obtained using the original extraction protocol: range = 1.0-0.1ug CBDA determined from mass spectral data.

C.2.3. Multiplex cytokine/chemokine assays

Microglial media were harvested after the start of the treatment, centrifuged briefly to remove particles (300 g,10 min). Cytokine and chemokine levels in microglial media were measured using a Bio-Plex 200 with 96-well magnetic plate assay according to the manufacturer's instructions (Bio-Rad). Measured cytokines and chemokines include IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 (p 70), IL-13, IL-17, G-CSF, GM-CSF, IFNγ, TNFa, CXCL1 (KC), CCL2 (MCP-1), and CCL5 (RANTES). All samples were run in duplicate and data was analyzed using Bio-Plex Manager software.

C.2.4. Immunohistochemical (protein level) assay

Cells were fixed with 4% Paraformaldehyde (PFA) in PBS for 10 min. After washing 3X 5min with PBS, cells were incubated with primary antibody (anti-COX 2, anti-ARG 1) 1:1000 at 4℃overnight, and after washing 3X 5min in PBS, cells were then incubated at room temperature for 2 hours in the appropriate fluorescent secondary antibody 1:250 (Invitrogen). After the final wash, nuclei were stained with DAPI in a mounting medium as described previously. A micrograph of the cells was taken in three fields of view per well from duplicate wells and the regional coverage of each marker was analyzed using Fiji.

C.2.5. Determination of cell Activity (mitochondrial Activity)

Use of MTT [3- (4, 5-dimethylthiazol-2-yl-) -2, 5-diphenyl-2H-tetrazolium bromide; sigma ] to quantify microglial viability. In this assay, MTT (a tetrazolium dye) is biologically reduced by mitochondria to formazan products that are insoluble in tissue culture medium. Briefly, MTT was added to cells to a final concentration of 250 μg/ml at various time points after treatment with PBS, LPS or IL-4 (with or without test product). After 30 minutes, formazan was dissolved in DMSO and absorbance was measured at 490nm using a spectrophotometer (Glomax multi+, promega, UK).

C.2.6. Statistics of

The data repeated within the experiment was averaged and then analyzed for data from at least three independent experiments using GRAPH PAD PRISM or students-t-test.

C.3 results

C.3.1INOS expression

NTI164 normalizes inflammation-induced iNOS expression. iNOS expression increased due to inflammation, and in inflammation-activated microglia, NTI164 normalized expression toward control levels, thus reducing the inflammatory processes triggered by iNOS. Inducible Nitric Oxide Synthase (iNOS) is one of three key enzymes for the production of Nitric Oxide (NO) from the amino acid L-arginine. Inducible Nitric Oxide Synthase (iNOS) plays an important role in the regulation of Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE). Previous studies have demonstrated that iNOS plays a pathogenic and regulatory role in MS and EAE as well as in a variety of other neuroinflammatory diseases. Fig. 5 demonstrates that NTI164 normalizes inflammation-induced iNOS expression.

C.3.2 neuron Activity

The NTI164 increases the number of viable neurons under basal conditions (short term exposure). Use of MTT [3- (4, 5-dimethylthiazol-2-yl-) -2, 5-diphenyl-2H-tetrazolium bromide; sigma ] to quantify neuronal viability. NTI164 treated cells were able to increase the number of "healthy" cells under basal conditions after short-term glutamate exposure. Cell excitotoxicity was achieved by glutamate activation (3 mM). NTI164 is able to stimulate cell growth following short-term glutamate-induced "damage".

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

C.3.3 maturation of immature neurons

NTI164 stimulates the maturation of immature neurons into healthy cells even in the absence of any glutamate-induced damage. This study demonstrates that NTI164 can stimulate "healthy maturation" of immature neurons. This process may be critical after a wound or injury. NTI164 is capable of providing healthy neuronal cell development, an important process for recovery from neuroinflammation, neuronal damage. Fig. 7 shows that NTI164 stimulates the maturation of immature neurons into healthy cells even in the absence of any glutamate-induced damage.

C.3.4 cell death

NTI164 does not increase cell death in the case of excitotoxic cell injury.

Fig. 7 shows that CBD is toxic in this example, while NTI164 is non-toxic and has a positive effect on cell number and cell viability.

C.3.5ARG1 expression

NTI164 (NTI) normalizes inflammation-induced (injured cell) Arg 1 expression. It is generally believed that macrophage-specific up-regulation of arginase-1 promotes inflammation. Arginase 1 expression increased due to inflammation, but in inflammatory activated cells, NT1164 normalized expression toward control levels. Figure 9 shows microglial response under inflammatory conditions assessing arginase 1 expression.

Figure 10 outlines arginine metabolism and its effect on the overall balance of anti-inflammatory and pro-inflammatory signals.

Example 4-preclinical study related to biomarkers involved in neuroinflammation

D.1 study objectives

To determine the effect of NTI164 on COX-2, IL2 and TNF- α levels in human derived microglia.

A variety of neurological disorders occur due to inflammation induced by an immune response disorder.

For example, multiple Sclerosis (MS) is a progressive inflammatory disease characterized by the loss of myelin sheath within the central nervous system. Typical symptoms include fatigue, difficulty walking, speech and vision impairment. Cyclooxygenase-2 (COX-2) is considered to be the primary enzyme responsible for inflammation, a common mechanism of disease involved in MS. COX-2 is a powerful clinical biomarker for assessing disease progression and overall treatment management.

IL2 plays an important role in immunomodulation and in MS progression. IL-12 is a cytokine that plays a key role in the pathogenesis of multiple sclerosis. Blocking the cytokine by neutralizing antibodies resulted in significant improvement in animal models and multiple human trials of the disease.

TNF- α plays an important role in dysregulation of acute inflammation involved in MS attacks.

D.2 materials and methods

D.2.1COx-2

Immunohistochemical (protein level) assay: cells were fixed with 4% Paraformaldehyde (PFA) in PBS for 10 min. After washing 3X 5min with PBS, cells were incubated with primary antibody (anti-COX 2) at 1:1000 overnight at 4℃and after washing 3X 5min in PBS, cells were then incubated in the appropriate fluorescent secondary antibody 1:250 (Invitrogen) for 2 hours at room temperature. After the last wash, nuclei were stained with DAPI in fixed media as described previously. A micrograph of the cells was taken in three fields of view per well from duplicate wells and the regional coverage of each marker was analyzed using Fiji.

D.2.2IL-2 and TNF-alpha

Multiplex cytokine/chemokine assay: microglial cell media harvested after the start of treatment was centrifuged briefly to remove particles (300 g,10 min). Cytokine and chemokine levels in microglial media were measured using a Bio-Plex 200 with 96-well magnetic plate assay according to the manufacturer's instructions (Bio-Rad). Measured cytokines and chemokines include IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 (p 70), IL-13, G-CSF, GM-CSF, IFNγ, TNFa, CXCL1 (KC), CCL2 (MCP-1), and CCL5 (RANTES). All samples were run in duplicate and data was analyzed using Bio-Plex Manager software.

D.2.3 sample preparation and dilution

500Mg of dry NTI164 plant material was suspended in 20ml absolute ethanol (using a 50ml blue top Falcon tube suitable for centrifugation), vigorously stirred/shaken for 60 seconds. The tube was then placed in an ultrasonic bath at 35-40C for 10 minutes. After the sonication was completed, the samples were then placed in a disk shaker (200 rpm) at room temperature for 30 minutes. Once completed, the samples were centrifuged at 4400rpm for 5 minutes. The supernatant was collected for testing and development.

Units used to describe the treatment of the test product and the concentration of NTI 164.

C. Dilution of the extract 1/10000-1 UL (stock substance is NTI164-1UL, which is equal to 0.1 μg/ml CBDA)

For CBD samples, pure standards (in powder form) were used. CBD 98% isolate was purchased from LGC STANDARDS (london, uk) (CAS No. 13956-29-1) as a reference standard. A CBD standard reference was prepared at a concentration of 1mg/ml in acetonitrile. CBD dilutions in acetonitrile were prepared as follows: 2. Mu.g/ml; 6 μg/mL; and 0.1 μg/mL.

D.3 results

D.3.1COX-2

Preclinical studies in cells using immunohistochemical analysis demonstrated that NTI164 can suppress and inhibit COX-2 expression in human-derived microglia. NTI164 inhibits COX-2 up to three times as effectively before and after inflammatory injury when compared to CBD alone. See table 7 below.

Table 7: summary COX-2 inhibition in cells when treated with NTI164 versus CBD alone

D.3.2IL2 and TNF-alpha

NTI164 inhibits key biomarkers statistically more effectively when compared to CBD and cbd|thc (1:1) mixtures alone: IL-12 and TNF-alpha.

These results confirm that: NTI164 IL-12 p=0.0011 is highly significant relative to CBD alone; NTI164 TNF- αp=0.0575 is a positive trend relative to CBD alone; NTI164 IL-12 p=0.0069 is highly significant relative to cbd\thc combination; NTI164 TNF- αp= 0.0446 is significant relative to cbd\thc combination.

Table 8 summarizes the significance of NTI164 in inhibiting TNF- α and IL-12 relative to the CBD and CBD\THC combinations alone (1:1 concentration ratio).

D.4 discussion

D.4.1COX-2, IL2 and TNF-alpha

These results, which showed inhibition of COX-2, IL2 and TNF- α, again demonstrate the effective nature of NTI164 in modulating inflammatory processes in neurological conditions in which inflammatory disorders are induced by immune responses.

E example 5 further characterization of NTI164

E.1 research objectives

The NTI164 extracted by oil was subjected to compositional analysis using the UPLC/MS method (as described in the examples above).

E.2 materials and methods

Upc equiconcentration isolation of the main cannabinoid was provided at a cycle time of 10.5 minutes using an ACQUITY upc H-Class system in combination with CORTECS UPLC Shield RP particle chemistry. NTI164 samples were assayed weekly using CBDA as the primary marker of stability indicator. The results presented in table 6 demonstrate that NTI164 is stable in inert oil medium at room temperature for more than 6 weeks. No decarboxylation or product degradation was observed in this time frame.

The device comprises: the following devices were used: 10mL glass scintillation bottle with cover; cobram's Estate olive oil; plant grinders (similar to coffee or food grade grinders) with pore sizes up to 50 μm-80 μm; a watman paper, grade 1; a pipette; weight scales (transfer boats and spoons); eppendorf tubes; 50mL Falcon tube; a tabletop centrifuge (Eppendorf Centrifuge 5702); oz Design Brand 6Litre Fruit,Wine and Cider Press.

Extracting: extrusion and centrifugation: all work was done at standard laboratory temperature (18-22 ℃). The shoots of NTI164 were peeled from the hard stems and the stems were discarded. The mill was cleaned with 70% EtOH and the milling chamber was filled with dry plant material. The material was milled for 10 seconds (1-2 mm particle size) at the finest of the three settings. The grind was then mixed with 100mL olive oil in an autoclaved Schott bottle at a plant/oil ratio of 333 mg/mL. Next, it was placed on a stirrer for 1 hour at room temperature, and stirred with a magnetic stirrer (50 rpm). The oil plus plant mixture was then placed into Oz Design bran 6Litre Fruit,Wine and Cider Press to recover the oil component (mash) from the plants. Next, the recovered oil was placed in a 50mL Falcon tube and spun at 300g for 15 minutes at room temperature (separation 1). The oil was then transferred to a clean Schott bottle and the volume recovered was recorded. The recovery of oil for separation 1 was about 40%. The mash is discarded after each separation. To the recovered oil was added an additional 333mg/mL of ground plant/oil (additional 100 mL) material, mixing was repeated for 1 hour, and the oil was recovered and reused until a total of 999. Mu.g/mL (3X 100 mL) of the plant/oil mixture passed (separation 2). The recovery of oil for separation 2 was about 50%. For the last time we placed in a Falcon tube and rotated as described above (separation 3). Recovery of the oil from separation 3 was about 50%. Then we collect only the oil and place it in Eppendorf tubes for treatment. This triple extraction procedure produced a total volume of 50ml of final product at a concentration of 48mg CBDA in 1ml olive oil, as determined by the UPLC potency test, using the method described below.

E.3 results

The results of the UPLC analysis are presented in table 9 below.

Table 9 shows NTI164 compositions extracted using ethanol extraction and components quantified using UPLC methods described herein

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By expanding the pore size in the mill system from 50 μm to 80 μm we were able to extract 1-3% of the Cannabinol (CBN) in the final oil extraction product.

Example 6-evaluation of anti-inflammatory Properties of combination of NTI164 and diclofenac

F.1 study objectives

To evaluate the anti-inflammatory properties of the combination of NTI164 and diclofenac compared to the combination of CBD and diclofenac.

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID). Diclofenac is used to treat signs and symptoms of mild to moderate pain or osteoarthritis, rheumatoid arthritis and neuropathic pain.

F.2 materials and methods

F.2.1 immunohistochemical (protein level) determination

Human microglial cells were fixed with 4% Paraformaldehyde (PFA) in PBS for 10 min. After washing 3X 5min with PBS, cells were incubated with primary antibody (anti-COX 2) 1:1000 overnight at 4℃and after washing 3X 5min in PBS, cells were then incubated in the appropriate fluorescent secondary antibody 1:250 (Invitrogen) for 2 hours at room temperature. After the last wash, nuclei were stained with DAPI in fixed media as described previously. A micrograph of the cells was taken in three fields of view per well from duplicate wells and the regional coverage of each marker was analyzed using Fiji.

COX-2 was measured using immunohistochemical assay as described above. As part of the positive control, the cells are treated with interleukins and interferons, which cause the cells to enter an "active inflammatory state". In the treatment group, these cells were also treated with various treatments, and then after these treatments (1 hour after treatment) the expression of COX-2 was evaluated.

F.2.2 sample preparation and dilution

The following samples were prepared: a CBD alone, NTI164 alone, diclofenac + NTI164 were prepared.

In this example, ethanol extraction was used. 500mg of dry plant material NTI164 was suspended in 20ml absolute ethanol (using a 50ml blue top Falcon tube suitable for centrifugation), vigorously stirred/shaken for 60 seconds. The tube was then placed in an ultrasonic bath at 35-40C for 10 minutes. After the sonication was completed, the samples were then placed in a disk shaker (200 rpm) at room temperature for 30 minutes. Once completed, the samples were centrifuged at 4400rpm for 5 minutes. The supernatant was collected for testing and development.

Diclofenac (CAS number 15307-86-5) was purchased from MERCK CHEMICALS (purity, 98% HPLC). A reference stock standard of 1mg/ml was established using acetonitrile and diluted as follows: 2. Mu.g/ml; 6. Mu.g/ml; 0.1. Mu.g/ml.

The final concentrations of NTI164 (CBDA equivalent), CBD and diclofenac used in this study were 2 μg/ml.

F.3 results

F.3.1 synergistic action

Synergy was observed with the nti164+diclofenac combination. COX-2 inflammation was reduced to 8% with the NTI 164+diclofenac combination, as opposed to 24% with diclofenac alone and 55% with NTI164 alone.

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

Table 10 gives the inflammatory results of the combined study (n=9), nti164+diclofenac.

Example 7-evaluation of anti-inflammatory Properties of combination of NTI164 and celecoxib

G.1 research objectives

To evaluate the anti-inflammatory properties of the combination of NTI164 and diclofenac compared to the combination of CBD and celecoxib.

Celecoxib is a non-steroidal anti-inflammatory drug (NSAID). Diclofenac is used to treat mild to moderate pain or signs and symptoms of disorders such as osteoarthritis or rheumatoid arthritis.

G.2 materials and methods

G.2.1 microglial cell BV2 cultures

Immortalized microglial cell line BV2 was purchased from american type culture collection (American Tissue Culture collection). BV2 was cultured in RPMI medium containing gentamicin and supplemented with 10% Fetal Bovine Serum (FBS) (for amplification) and 5% FBS (when plated for experiments). All cells were from between passage numbers 39 and 45. Cells were plated at 45,000 cells/mm ², and after plating treated with phosphate buffered saline (PBS as control) or interleukin-1b+interferon-gamma (IL-1b+ifnγ to induce inflammation) for 24 hours. To test the effect of NTI164 in altering inflammatory responses, NTI164 and concurrent therapy were applied 1 hour post-inflammation (see below). The extracted NTI164 was applied at 10uL, 3uL or 1uL from the isolate obtained using the original extraction protocol: range = 1.0-0.1ug CBDA determined from mass spectral data. Treatments were celecoxib 5, 25 and 125 μm.

G.2.2 Multiplexed cytokine/chemokine assays

Microglial media were harvested after the start of the treatment, centrifuged briefly to remove particles (300 g,10 min). Cytokine and chemokine levels in microglial media were measured using a Bio-Plex 200 with 96-well magnetic plate assay according to the manufacturer's instructions (Bio-Rad). Measured cytokines and chemokines include IL-1α, IL-1β, IL-2, IL-6, IL-10, IL-12 (p 70), IL-13, G-CSF, GM-CSF, IFNγ, TNFa, CXCL1 (KC), CCL2 (MCP-1), and CCL5 (RANTES). All samples were run in duplicate, averaged for analysis, and at least three samples were run per group.

G.2.3 immunohistochemical (protein level) assay

Cells were fixed with 4% Paraformaldehyde (PFA) in PBS for 10 min. After washing 3X 5min with PBS, cells were incubated with primary antibody (anti-COX 2) 1:1000 overnight at 4℃and after washing 3X 5min in PBS, cells were then incubated for 2 hours at room temperature in the appropriate fluorescent secondary antibody 1:250 (Invitrogen). After the final wash, nuclei were stained with DAPI in a mounting medium as described previously. A micrograph of the cells was taken in three fields of view per well from duplicate wells and the regional coverage of each marker was analyzed using Fiji.

G.2.4 determination of cell viability (mitochondrial Activity)

G.3 results

G.3.1g.3.1 synergistic action

Synergy was observed with the nti164+celecoxib combination. The nti164+celecoxib combination resulted in decreased inflammation, cytokine release (tnfα, G-CSF, IL1a, IL 6) and COX2 proteins observed by MTT assay compared to celecoxib alone and NTI164 alone.

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

Table 11 gives the inflammatory results of the combination study to demonstrate the synergistic effect of NTI164 and celebrix (Celebrex).

Example 8-evaluation of anti-inflammatory Properties of combinations of NTI164 and prednisone

H.1 research targets

To assess the synergistic inhibition of inflammatory response by combined prednisone and NTI164 treatments in preclinical in vitro studies.

H.2 materials and methods

Cells were fixed with 4% Paraformaldehyde (PFA) in PBS for 10 min. After washing 3X 5min with PBS, cells were incubated overnight with primary antibody (anti-COX 2) 1:1000 at 4℃and after washing 3X 5min in PBS, cells were then incubated in the appropriate fluorescent secondary antibody 1:250 (Invitrogen) for 2 hours at room temperature. After the last wash, nuclei were stained with DAPI in fixed media as described previously. A micrograph of the cells was taken in three fields of view per well from duplicate wells and the regional coverage of each marker was analyzed using Fiji.

The treatment group comprises:

(1) Control: PBS buffer

(2) Positive control: inflammatory stimulation by interferon gamma and interleukin-1B activation, prednisone (PDN) concentration 5uM

(3) Combination therapy: prednisone (PDN) 5uM+NTI164 concentration 7.5ug/mL

H.3 results

Analysis of results: calculated as inflammation reduction, PDN vs combination therapy (prednisone 5um+nti164 concentration 7.5 ug/ml). Statistical analysis was performed using Student's t-test.

Treatment with NTI164 alone is very effective in reducing cellular inflammatory processes, but these anti-inflammatory effects are even stronger when combined with prednisone.

Table 12 gives the inflammatory results of the combination study to demonstrate the synergy of NTI164 and prednisone.

Example 9-evaluation of anti-inflammatory Properties of combinations of NTI164 and Psylocibin

I.1 study objectives

To assess synergistic inhibition of inflammatory response by combined psylocibin and NTI164 treatments in preclinical in vitro studies.

I.2 materials and methods

Microglial cell media harvested after the start of the treatment was centrifuged briefly to remove particles (300 g,10 min). The levels of TNFα in microglial media were measured using Bio-Plex 200 with a 96-well magnetic plate assay according to the manufacturer's instructions (Bio-Rad). Samples were run in duplicate and averaged for analysis. At least three samples were run per group.

I.3 results

Analysis of results:

the TNF- α assessment of psylocibin combination studies is given in Table 13.

Table 13: TNF-alpha assessment

Sample of	TNF-A assessment results

PBS control	1.00
		Positive activation of I & I	10.67

		NTI164 5ug/ml	2.07
NTI164 1.75ug/ml	15.87

SubP(Psylocibin)5ug/ml	9.17
		SubP(Psylocibin)1ug/ml	4.87

		NTI164+SubP 5ug/ml	1.67
NTI164+SubP 1ug/ml	1.73

Treatment with NTI164 alone is very effective in reducing cellular inflammatory processes, but these anti-inflammatory effects are even stronger when combined with psylocibin.

J example 10-NTI164 for treatment of ASD level II/III-4 weeks

J.1ASD study design and methods

Of the 18 patients enrolled in the study, patients receiving NTI164 accounted for 94% (n=17). Active patients account for 78% (n=14), patients who were discontinued after receiving their first NTI164 dose account for 16% (n=3), and patients who were discontinued before receiving NTI164 but after intake account for 6% (n=1). The average age of active patients was 13.4 years, the youngest patient was 10 years, and the oldest patient was 17 years (fig. 11).

All active patients were diagnosed with ASD levels II/III, assessed as "mild illness", "moderate illness", "significant illness" or "severe illness" on the CGI severity scale at baseline (fig. 12).

Patients began 5 mg/kg/day of NTI164 treatment, and increased 5 mg/kg/day weekly for 4 weeks until 20 mg/kg/day or maximum tolerated dose was reached. The daily dose was calculated by multiplying the dose by the patient's body weight and then dividing by the concentration of CBDA in oil (53 mg/mL). This returned to the total daily volume in mL (table 14), which was divided into twice daily (BD) AM and PM doses.

TABLE 14 calculation of daily dose per patient

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The average maximum daily dose for active 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 a maximum daily dose between 6 mg/kg/day and 19 mg/kg/day (fig. 13).

J.2 results of the study

Evaluation was performed using a clinical global impression-severity (CGI-S) scale:

Overall improvement: overall improvement in rating, whether or not at the discretion of the clinician is entirely due to medication;

Severity of disease: comparison of baseline and post-baseline (28 days NTI164 treatment); and

Efficacy index: based only on efficacy ratings. This is a score calculated based on the treatment effect and the extent of side effects.

Overall improvement

93% Of active patients showed improvement after 28 days of daily treatment with NTI 164. Of these patients, 64% had a global improvement of "great improvement", 29% had a global improvement of "minimal improvement", and only one patient (7%) had "no change" (fig. 14). Statistical significance was assessed using the Wilcoxon Signed-Rank Test and paired t-Test:

pairing t test: the average difference in CGI-S between 28 days treatment and baseline was-0.714, 95% confidence interval = -1.332, -0.097, p-value = 0.027. The Wilcoxon Signed-Rank Test is statistically: -15, the corresponding p value being 0.047.

Severity of disease

At baseline, the average rating for disease severity was 4.4 (fig. 12). After 28 days of NTI164 treatment, it was reduced to an average rating of 3.6 (fig. 15, 16).

Therapeutic effects

After 28 days of daily treatment with NTI164, 14% of active patients exhibited the second highest possible efficacy index 2: has remarkable therapeutic effect and side effect of not remarkably interfering with the functions of patients.

72% Of active patients have efficacy index 5 or 6: moderate treatment effect, half of these patients had no side effects, and the other half had side effects that did not significantly interfere with patient function; 7% has a efficacy index of 9: minimal therapeutic effect, no side effects; only one patient, 7%, had an efficacy index based on no observed change in the condition, 13: no change or worse, no side effects (fig. 17).

K example 11-NTI164 for treatment of ASD level II/III-20 weeks

K.1ASD study design and methods

Example 10 above provides the treatment results at the 4 week (28 day) time point (n=14 positive). This example 11 presents the treatment results at the 20 week time point (n=12 positive). As discussed in example 10 above, patients began NTI164 treatment at 5 mg/kg/day, which increased 5 mg/kg/day weekly for 4 weeks until 20 mg/kg/day or maximum tolerated dose was reached and (in this study) continued for 16 weeks (providing a 20 week total daily dosing period).

The general objective of this study was to evaluate the sustained safety and efficacy of NTI164 administered daily over a 20 week period. A secondary objective is to assess the efficacy of NTI164 in treating symptoms associated with the autism spectrum. Utility various doctor-dominated and parent-dominated standard questionnaires used in the art were measured.

Patient (n=12)

At week 20, the average age of active patients was 13.3 years, the youngest patient was 10 years, and the largest patient was 17 years (error | no reference source found). All positive patients were diagnosed with ASD levels II/III and were assessed as "mild illness", "moderate illness", "significant illness" or "severe illness" on the CGI severity scale at baseline (fig. 19).

Dosage of

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

To reduce the risk of side effects, study drugs were dosed progressively over the course of four weeks, starting at 5 mg/kg/day, with 5mg increase per week until the maximum tolerated dose or 20 mg/kg/day was reached. The maximum tolerated dose was then administered over the course of 20 weeks. The daily volume was administered via two doses of AM and PM.

The formula for calculating the dose for each patient is: body weight x dose/NTI 164 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 either his maximum tolerated dose or 20 mg/kg/day of NTI164.

NTI164 was prepared in oil for oral administration. The total concentration of oil was 53mg/ml.

At the end of week 20, participants opted to end their participation and gradually decrease down by 5 mg/kg/week until they stopped studying the drug or continued their maximum tolerated dose until week 52.

Primary endpoint

Security is monitored and measured using standard procedures in the art. In addition to the parent/caregivers and physician questionnaires completed at baseline and every four weeks up to week 20, whole blood tests, liver and kidney function tests and vital signs were used to monitor and measure safety.

Secondary endpoint

Efficacy is monitored and measured using standard procedures in the art.

Clinical global impression scale-disease severity (CGI-S). The clinician's impression of disease severity is reflected on a 7-point scale ranging from 1=no at all until 7=in the most severe disease. Time frame: baseline, week 4, week 8, week 12, week 20 ].

Vineland adaptive behavior Meter, third edition (Vineland-3). For measuring adaptive functions across three core areas (communication, daily life skills and sociality) and two optional areas (motor skills and maladaptive behaviour); each term was rated on a 3-point scale (0=never; 1=sometimes; 2=usually or frequently). The core domain sum is the total adaptive behavior combination. Time frame: baseline, week 20 ].

Social reactivity scale, second edition-School-Age Form (SRS-2). Five areas were evaluated, including: social awareness, social cognition, social communications, social motivation, and limited interests and repetitive behaviors. Each item was rated on a 4-point scale (range from 1=not true until 4=almost always true). Time frame: baseline, week 20 ].

Clinical global impression scale-improvement-caregivers (CGI-I-Ca). Which is a 7-point scale measuring the change in symptoms from baseline. Are provided as baseline and post-baseline caregivers and clinician questionnaires. Time frame: baseline, week 4, week 8, week 12, week 20 ].

Clinical global impression scale-improvement-clinician (CGI-I-Cl). Which is a 7-point scale measuring the change in symptoms from baseline. Are provided as baseline and post-baseline caregivers and clinician questionnaires. Time frame: baseline, week 4, week 8, week 12, week 20 ].

Clinical global impression scale-target behavior change (CGI-C). The clinician's impression of behavior change is reflected on the 7-point scale, ranging from 1=no at all to 7=very serious problem. Are provided as baseline and post-baseline questionnaires. Time frame: baseline, week 4, week 8, week 12, week 20 ].

Clinical global impression scale-attention variance (CGI-CA). The clinician's impression of change in attention was reflected on a 7-point scale ranging from 1=no at all until 7=a very serious problem. Are provided as baseline and post-baseline questionnaires. Time frame: baseline, week 4, week 8, week 12, week 20 ].

Childhood anxiety scale-autism spectrum disorder-parental edition (ASC-ASD-P). A parental/caregivers format developed to detect anxiety symptoms in young people with ASD. Consists of four component scales (presenting anxiety, uncertainty, anxiety arousal and separation anxiety), each item rated on a 4-point scale (0 = never, 3 = always). The sum of the component tables is equal to the total score. Time frame: baseline, week 4, week 8, week 12, week 20 ].

Childhood anxiety scale-autism spectrum disorder-childhood version (ASC-ASD-C). A form of children developed to detect anxiety symptoms in young people with ASD. Consists of four component scales (presenting anxiety, uncertainty, anxiety arousal and separation anxiety), each item rated on a 4-point scale (0 = never, 3 = always). The sum of the component tables is equal to the total score. Time frame: baseline, week 4, week 8, week 12, week 20 ].

The childhood Sleep Disorder Scale (SDSC). The six component table includes initiating and maintaining sleep disorders, sleep breathing disorders, wake disorders, sleep wake transition disorders, excessive somnolence disorders, and sleep hyperhidrosis. Each item was rated on a 5-point scale, where 1 = never, 5 = always (daily). The score sum of the score tables is equal to the total score [ time frame: baseline, week 4, week 8, week 12, week 20 ].

G.2 results of the study

Security results

The security data concludes as follows: in this study population, 5, 10, 15 and 20mg/kg of NTI164 was administered at twice daily doses, which was safe and well tolerated. This conclusion is further supported by laboratory values. No changes were observed in the patient's whole blood examination, liver function, or kidney function tests. Patient vital signs were also not observed to change.

Efficacy results

The statistical significance of the analyzed dataset was assessed using the Wilcoxon Signed-Rank Test and paired t-Test.

Pairing t test: the average difference in CGI-S between 20 weeks treatment and baseline was-1.08, 95% confidence interval = -1.772, -0.3948, p-value = 0.005303.

The Wilcoxon Signed-Rank Test is statistically: -15, the corresponding p-value being 0.009654.

After 20 weeks of daily treatment with NTI164, 100% of patients (n=12) exhibited a "great improvement" in symptoms associated with disease severity.

Table 15 below is a summary of the results of Wilcoxon Signed-Rank Test and paired T-Test performed on the analyzed data set at week 20.

TABLE 15 overview of Wilcoxon Signed-Rank Test and paired T-Test on analyzed dataset at 20 weeks

Overall improvement. 100% of active patients (n=12) showed improvement after 20 weeks of daily treatment with NTI164, with overall improvement of all patients being "2. Of these patients, 3 scored previously as "3. Minimal improvement" after 4 weeks of treatment. See fig. 20 and 21.

Severity of disease. The average rating of disease severity at baseline was 4.3. After 20 weeks of daily NTI164 treatment, it was reduced to an average rating of 3.3. See fig. 22-24.

The treatment effect. After 20 weeks of NTI164 treatment per day, 67% of active patients exhibited the highest possible efficacy indices 1 and 2: significant therapeutic effect-substantial improvement. All symptoms are completely or almost completely relieved. 33% of patients have efficacy index 5, 6 or 7: moderate therapeutic effect-significant improvement. The symptoms are partially relieved. See fig. 25 and 26.

Conclusion(s)

NTI164 showed that doses up to 20/mg/kg/day were safe and well tolerated. NTI164 showed statistically significant efficacy in ameliorating symptoms associated with autism spectrum disorders after 20 weeks of daily treatment.

Claims

1. A composition comprising 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.

2. The composition of claim 1, wherein the cannabinoid is present in an amount selected from the group consisting of:

Composition 1 comprising

w/w％

CBDA 50％；

CBD 2％；

CBG 5％；

CBDP 2％；

CBDB 2％；

CBGA 5％；

CBN 3％；

THC <0.3%; and

Additional active ingredients;

And

Composition 2 comprising

w/w％

CBDA 45％；

CBD 1％；

CBG 4％；

CBDP 1％；

CBDB 2％；

CBGA 4％；

CBN 2％；

THC <0.2%; and

Additional active ingredients.

3. The composition of any of the preceding claims, further comprising an oil selected from the group consisting of: synthetic oil; a plant-based oil; mineral oil; canola oil; and olive oil.

4. The composition of any of the preceding claims, wherein the composition comprises less than 5% w/w terpene.

5. The composition of any of the preceding claims, wherein the composition comprises less than 2% w/w organic plant material.

6. The composition of any of the above claims, wherein the composition comprises less than 2% w/w plant phenol.

7. The composition of any of the preceding claims, wherein the cannabinoid component of the composition is selected from the group consisting of: a concentration of between 1 and 500 mg/ml; between 10 and 100 mg/ml; the concentration was 50mg/ml.

8. The composition of any of the preceding claims, wherein the CBDA component of the composition is selected from the group consisting of: a concentration of between 1 and 500 mg/ml; between 10 and 100 mg/ml; the concentration was 50mg/ml.

9. The composition of any of the preceding claims having a UPLC mass spectrum corresponding to figure 3, determined using the conditions described in example 1.

10. A composition according to any preceding claim, wherein the additional active ingredient is selected from: diclofenac, prednisolone, and celecoxib.

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

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

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

14. The dosage form of any one of claims 12 to 13, wherein the CBDA component of the composition is selected from the group consisting of: 600mg;400mg;300mg;200mg;100mg;50mg;10mg;5mg;2mg;1mg.

15. A method of treating a disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of a dosage form of claims 12 to 14.

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

17. The method of any one of claims 15 to 16, 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. Use of a composition according to claims 1 and 10 in the manufacture of a medicament for the treatment of a condition.

19. A method of extracting the composition of claims 1 to 10 from cannabis plant material, the method comprising the steps of:

(1) Grinding cannabis plant material to a sufficient grind size;

(2) Contacting the grind resulting from step a) with oil;

(3) Mixing the grind and the oil for a time sufficient to form a mixture;

(4) Extruding the mixture to recover oil;

(5) Centrifuging the oil to further refine the oil; and

(6) The oil extract is collected in a suitable container.

20. A method of extracting the composition of claims 1 to 10 from cannabis plant material, the method comprising the steps of:

(1) Grinding cannabis plant material to a sufficient grind size;

(2) Contacting the grind resulting from step a) with an alcohol;

(3) Mixing the grind and alcohol for a time sufficient to form a mixture;

(4) Sonicating the mixture;

(5) Centrifuging the mixture; and

(6) The alcohol extract is collected in a suitable container.

21. A product produced by the method of claim 19 or 20.

22. A kit comprising a dosage form of claims 12 to 15 and instructions for use.

23. The compositions, methods and processes as described in the preceding examples.