Formulations of Cannabldkd Derivatives and Their Use as Modulators of Caaaahmold Receptor t pe 2 (0¾) CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to ITS, Provisional Application No. 62/801,736, filed February 06, 2019 and U ,S, Provisional Application No. 62/870,546, filed July 03, 2019 which are hereby incorporated by reference herein in their entirety FILED OF THE INVENTION

The present Invention relates to compositions, comprising the cannabidiol derivatives of Formula (I) solubilized: in pharmaceutical vehicle as liquid fbrmnlafions, or a tablet, powder, suspension, nanosuspens n, emulsion, which displa inema ed biuavailahiiity an solubility. The present invention also relates to the use of these cannabidiol qninone derivatives of Formula if) for use to the treatment of diseases heiiefning from the modulation of cannahinold receptor type 2 (CBi) activity. Such compounds have a novel mechanism of action (MOAj by targeting complementar signaling pathways that alleviate nenromllammation and favor neuroproteetion, prevent axonal ama e, preserve and potentially promote the my el In structure, and support vaseulogenesis, which is useful in the Ircalmenl of several autoimmune and inflammation-related disorders, including multi le sclerosis (MS) and systemic sclerosis fSSe).

BACKGROUND OF THE INVE TIO

Multiple sclerosis (MS) is a chronic autoimmune demyelmatiug disease of the it i nervous system (CNS) that represents one of the most commonly acquired neurological diseases in young adults. Disease progression is thought to he composed of two underlying processes: myelin destruction (derayclination) with failure to remyelmate, and progressive axonal damage with little capacity for recovery , A variety of neurological symptoms associated with MS result fro® a weakening ability of the cells to conduct nerve signals, MS can cause disabilit progressively over time, including difficulty with mobility and upper limb function, bladder, bowel, and sexual dysfunction, speech and swallowing difficulties, and problems with vision and cognition Currently, there is no curative treatment for MS, and standar of care mainly works oil reducing symptoms. Since exacerbated innate an adaptive immune responses contribute to the pathophysiology of the disease, therapies that are directed towards modu tlon of the i mmune response and aimed at stimulation of axonal remyehnafion are needed.

Systemic sclerosis (SSe), or scleroderma, is a group of rare diseases associate with early and transient inflammation and vascular injury, followed by fibrosis affecting the skin and multiple internal: organs, Systemic sclerosis is classified into two forms: localized scleroderma CfioSi and SSe. While LeS is confined to the skin and/or underlying tissues and is often benign, SSe is a serious condition characterized by microvascu!ar injury and SSe associated excessive fibrosis, which usually includes internal organ involvement SSe may affect vital organs (heart, kidneys, and lungs), other internal organs (stomach and bowels) as well as blood vessels, muscles and joints. As a result, SSe can lead to chronic debilitation and diminished life expectancy. Currently, there is no cure for SSe. Current therapies are clinically ineffective, and available treatment option ar organ and symptom specific.

Peroxisome prolifemtor-aetivaied receptor gamma (PFARy) and cannabinoid receptor type 2 (0¾) are preclinkally validated therapeutic targets, supported by scientific literature, lor the development of novel drugs lo foe treatment of MS (Docagnc F, ei al 2008, Expert Opie. They Targets., 12:185-195; Drew P.D et al. 2008, PPAR Res., 2008:627463; Szslardy L etal. 2013, Neurosei Lett, 554:131-134). hi addition, an activator of the hypoxia- inducible factor (HIF) pathway may have a beneficial effect in MS patients, as th IIIF pathway modulates the immune response that favors neuroptoiecuo an axonal regeneration and is responsible for postnatal myelmaflon (feavarrete€ et ah 2018, i Neurolnfiamniaflou, 15:64), There are classes of marketed drugs that acti vate one or foe other of these pathways including Ghtexanes that activate PPARy and cannabiftoids that activate CBj.

CBz receptors were first cloned from differentiated human HL-60 myeloid cells, and ate most highly expressed in spleen, and cells of the immune system such: as B cells, T ceils, natetal killer cells, macrophages, monocytes, and nentrophils. Lower levels of CBs receptors are also found in the epidermis (including keratlnoeytes, hair follicles, sebocytes, and sweat glands), osteoblasts, osteoclasis, and osteoeytes, as well as stomach, lung, heart and testis. CBa receptor expression has been reported in dorsal root ganglion (DRG), an evidence for CBs receptor expression in other peripheral neurons such as€· and Adelta-fibers has been reported. Recently CBz receptor expression ithin tire MS has been described, at both the spinal and supraspinal levels, Specifically, C8¾ receptors ar found is lumbar (L3-L4) splaaf cord, and in cerebellar granule neurons, cerebrovascular epithelium, microglia an ueitrorts of the brainste (stmiem, thalamic nuclei, hippocampus, amygdala, substantia nigra, eracpedactal gray, spinal trlgeramai «« ecs etc,), cortex and cerebellum,

€ 2 receptors have been Implicated in a number of physiological processes including inflammation and perception of pain immune system regulation, neurogenesis, and bone physiology, tipregulatiou of C¾ receptors Is associated with certain pathophysiological states. Increased CBc receptor expression has been detected in dorsal horn of the spinal cord as well as primary afferent, C-flber neurons in chronic constriction injury (CO), spinal nerve ligation (SNL), complete sciatic nerve section, and saphenous nerve partial ligation models of neuropathic pain,€¾ receptors are uprcgulated in microglia and astrocytes from neuritic plaques found in Alzheimer's disease brains (Benito et at 2003, 1. Neurosei, 23: 11136-1 1 141 ), or by interferon gamma (Carlisle et al 2002, Int, Immunopharmaeol., 2:69-82) or

lipopoiy saccharide (Cabral et al. 2005, J, Leukoc, Biol., 78: 192-197), and in T-lym hocytes from. simian immunodeficiency virus-infected macaques (Benito et at 2005, J, Neurosei,, 25:2530-2536), CBs receptors are found in T-lymphocytes, astrocytes and perivascular an reactive microglia in multiple sclerosis plaques (Benito et ai 2007, 1, Neurosei,, 27; 2396-2402),

Myelin sheaths, which cover many nerve fibers, are composed of lipoprotein layers formed in, arly life. Myelin formed b the ohgodeftdrogba in the central nervous system (CMS) differs chemically and Itnffiuaoioglcally from that formed by the Schwann cells peripherally, hut both types have the same function: to: promote transmission of a neural impulse along an axon. Man congenital metabolic disorders (e.g., phen lketonuria and othe

aminoacidurias; Tay-Sacbs, Niemann- Pick, and Gaucher’s diseases; Buriers syndrome; Krabbe’s disease and other leukodystrophies) affect the developing myelin sheath, mainly i the CMS. Unless the biochemical defect can be corrected or compensated for, permanent, often

widespread, neurologic deficits result.

Demyeiinaiion in later life is a feature of many neurologic disorders; it can result from damage to nerves or myelin due to local injury, ischemia, toxic agents, or metabolic disorders. Extensive myelin loss is usually followed by axonal degeneration and often by cell body degeneration, both of which may be irreversible. However, remyeiination occnrs in many instances, and repair, regeneration, and complete recovery of neural function can be rapid. Recovery often occurs after the segmental demyelinattoo that characterizes many peripheral netutipailues; this rocess may account l r the exacerbations a «ft remissions of S, Central demyelmatlon (i.e., of the spinal cord, brain, or optic nerves) is the p edominant findin in primary tlemyeiinating diseases, whose etiology is unknown. The most well-known is MB, Other diseases include, for example, acute disseminated encephalomyelitis (postinfeetions

encephaiomyelitis , udrenoleifeodystropby, adrenon efoneujmpaihy, Leber's hereditary optic atrophy and related mitochondrial disorders and human T-cetl lymphotropfe vims (HTLY) infection -associated myelopathy ,

femyeli nation is generally accepted as a regular event in MS lesions; however. It is insufficient for myelin repair and axons remain ilemyelinated in MB patients. Possible explanations for this include failure of recruitment or survival of oligodendrocyte progenitor cells (OPCs), disturbance of differentiatiou/maturaiion of OPCs, and loss of capability of myelinforming. Therefore, effective interventions for MS should not only prevent disease progression, but also promote remyelimition.

Them is a need in die art for a disease-moditying drug, an a formulation thereof _* with increased bfoava!labiiiiy and solubility to effort a more efficient drug delivery. There is also a need in the ar for a disease~modiiyiug drug, and a formulation thereof with a novel mechanism of action (MQA) that targets complementary signaling pathways that alleviate neuroiofiarnmadon and favor both neuroprotection and myelin regeneration for management and treatment of various autoimmune diseases, demyeiinatipg diseases, inflammatory-telaied disorders, and diseases of the central nervous system (CMS), such as MS and SSe,

SUMMARY OF THE INVENTION

The invention provides compositions comprising at least one cannahidiol derivative solubilized hi a pharmaceutical vehicle. in cate aspect the compositions have

Increased bioavaiiahility. In another aspect, the compositions have Increased solubility _:

in one aspect, the cannahidiol derivatives, disclosed in the invention, are compounds of Formula (f).

¾ one embodiment, R is the nitrogen atom of a group independently selecte from a linear nr branched alkyiamine, an arylamine, an atyiatkykmimg a imieroatylarnlfte, a heteroaryialkylamine, a linear or branched alkenyl atnine, a linear or branched alkyn l amine, or Midi.

In one embodiment, the composition is a d y powder formulation. In one embodiment, th composition Is a tablet in one embodiment, the composition is a suspension. in one embodiment, die composition is a nanosnspension. In: one embodiment, the composition is an emulsion in one embodiment, the composition is a solution.

In one embodiment, the pharmaceutical vehicle is selected fern the group consisting of aqueous butlers solvents, co-solvents, cyelodexirin complexes, lipid vehicles, and any combination thereof, and optionally farther comprises at least one stabilizer, emulsifier, polymer, antioxidants, and any combination thereof

In one aspect, the composition comprising at least one cannabidiol derivative of the invention, is solubilized m an oil. In so e embodiments, the composition comprising at least one cannahidkd derivative of the in vention, is so bi ed in an oil mixture comprising at least two oils. In some embodiments, the composition comprising at least one cannabidiol derivative of the Invention., is solubilized in a Maisine CC : maize oil mixture.

The Invention also relates, in part, to a method of treating a condition or disease associated with demyeSin.ation in a subject in need thereof The invention further provides a method of treating a condition or disease responsive to a modulation of CBs activity in a subject. In one embodiment the method comprises administering to the st !geet in need thereof a therapeuticall effective amount of ai least one cannabidiol derivative or a formulation thereof hi some aspects, the invention relates t compositions comprising a non-reaetivesynthetic cannabidiol derivative has a novel mechanism of action (MOA) by targeting complementary signaling pathways that alleviate neuromflarnmatioB and iavor neuroprotec don, prevent axonal damage, preserve myelin structure, and potentially promote: femyelination. the compositions comprise a non-tsactive synt etic camiabldml derivative that odulates CBr receptor signaling, in some examples, the compositions comprise a non-reactive synthetic camiahidiol derivative that modulates both PPARy and C& receptor signaling, In some embodiments, the compositions comprise a non-reactive synthetic carmabidlol derivative that modulates PPABy and CBz receptor signaling, and stabilizes HiF- 1«, tfeus uptegulating the expression of several associated taetors that include Erythropoietin (EFO) and Vascular Endothelial Growth Factor A (VEGFA), As a result, such compositions can have a strong potential as diseasemiodifying agents in SSc

The invention farther relates, in pari, to a method of remyelination in a subject i need thereof In one aspect of the invention, the method comprises administering to the subject iherapeuikally effective amount of at least one eannabidiol derivative or a formulation thereof In one embodiment, the subject has a condition or disease associated with dem e!mation. In one embodiment, the subject has a condition or disease responsive to a modulation of CBz activity. In one embodiment, the subject as a condition or disease associated with demyelinaiion. and condition or disease responsive to a modnlation of CBc activity.

In one aspect, the condition or disease responsive to the modnlation of the CBr receptor activity or the condition or disease associated with demyelinaiion is selected fro the group consisting of autoimmune disease, demyelinating disease. I uflammatory -related disorder, and any combination thereof in one embodiment, the condition or disease responsive to the modulation of the CBr recept or activity or the eondi on or disease associated with demyelinaiion Is selecte from the group consisting of SSc, myellnoclastic disorder, analgesia, acute and chronic pain, inflammator pain, post-operative pain, neuropathic pain, muscle relaxation, immunosuppression, allergies, glaucoma, bronebodiiatton, osteoporosis and disorders of the skeletal system, cancer, neurodegenerstive disorders Including but not limited to Alzheimers disease, Parkinson’s disease (FD), and Huntington's disease, MS, muscle spasticity, tremor, fibromyalgia, lupus, rheumatoid arthritis, myasthenia gravis, other autoimmune disorders.

Irritable bowel syndrome, interstitial cystitis, migraine, pmritis, eczema, seborrhea, psoriasis, shingles, cerebral ischemia, cerebral apoplexy, craniocerebral trauma stroke, spinal cord injury, liver cirrhosis, atherosclerosis, coughing, asthma, nausea, emesis, gastric ulcers, ueuromyellti optica, central nervous system neuropathy, central pontine myelinolysis, myelopathy. !eakoeneephalopafhy,, leukodystrophy, peripheral neuropathy, GnillaimBarre syndrome, anti- MAG peri heral neuropathy, Ghareot-Mhriefrooth: disease, progressive inflammatoryneuropathy, amyotrophic lateral sclerosis (ALS), and any combination thereof

BRIEF DESCRIPTION OF THE DRA WINGS

The follow ing detailed desc ption o f various embo iments of the invention wi ll be better understood when read la coiqimetion with th appended drawings. For the purpose of illustrating the invention, there are shown in the drawings illustrative embodiments. R should be understood, however, that the invention is not limited to the precise arrangements; and i immeniallries of the embodiments shown in the drawings.

Figur 1 , comprising Figures 1 A and Figure I B, depicts synthetic schemes for the generation of eannabidioi derivatives. Figure 1 A represents the overall synthesis of amino fitnetionaiized eannabidioi derivative products produced from CBD starring material. Figure I B depicts the generation of VCH-004.8 (Compound of Formula (VIII)} via an ammation of VCE~ 004.

Figure 2 depicts a revised synthetic procedure fur the generation of eannabidioi: derivatives.

Figure 3, comprising Figures 3A and Figure 3B. depicts optimization studies of various liquid formulation mixtures. Figure 3A depicts differem liquid formulation mixtures. Figure 3B depicts a liquid formulation comprising SO 5 v/v of maize oil and Maisine€C mixture.

Figure 4 depicts hloavailahility of different liquid formulations.

Figure 5, comprising Figures 5A and SB, depicts manufacturing flow charts of EHF-I0! liquid and placebo. Figure 5A depicts a manufacturing flow chart of EMP- iO! liquid. Figure SB depicts a manufacturing flow chart of placebo.

Figure 6 depicts kinetic solubility screening of VCE-004 8.

Figure 7 depicts an equation used to calculate log D (distribution coefficient) used as a measure of lipophiiicity.

Figure E depicts a stability of VCE-004.8 during phytosom Nation , at reflux In eflry l acetate at different times (45 min, 6 hr and 24 hr). flgotef depicts an overlay of tire SPEC profiles of ¥GE-004 8 s. tire two phytoso es comple , obtained in the solubility trials at pH 7.4

Figure 10 depicts a dissofeion profiles of FonnulUifons A, B and C of VCB-O04B using Alitru.

Figure 1 1 depicts solvent shift results in Simulated Gastric Fluid for various oral formulations.

Figure 12 depicts solvent shift results in Simulated Irttestinai Fluid for various oral formulations

Figure 13 depicts a graphical representation of t!m Amorphous Solid Dispersion Screening and stabilit results.

Figure 14 depicts a characterization of VCE-004,8 and EHR40Ί,

Figure 15, comprising Figure ISA through Figure 15H, depicts the exemplary results that demonstrate that EHP- 101 attenuates the clinical severity and neuropathology in EAE model figure I SA depicts that EHP-101 significantly ameliorated the clinical signs an progression of EAE, Results are expresse as mean SEM (n 6 animals per group), <

0,01 , ***p < 0 001 EAE · EHP- 101 vs EAE T VEH (one-way ANOVA followed Tnkey's test). Figure 15B depicts the results of clinical activity that was quantified by measuring the area under curve. Results are expressed as ± SEM (e ^:::: 6 to animals per group). * ^ < 0.01 _s * < 0.00

EAE + EHP- 101 vs EAE T Vehicle (one-way ANOVA followed Tukey’s test) figure ISC depicts die cross-sectfonai images of thoracic spinal cord cross-sections of SO pm thick. In whic innuunofiuoreseence with and-ibal was performed. Figure 15D depicts the eross-seetiona! images of thoracic spinal cord cross-sections of 50 urn thick, in which immunofluorescence with GFAP was performed. Figure 15E depicts the cross-sectional images of thoracic spinal cord cross-sections of 50 p thick, in which im nttofltsereseence with myelin staining MBP was performed . Figure 15F depic ts the results of quanti fication of 1MI marker shown as mean ± SEM, and significance was determined fey one-way ANOVA followed Tukey’s test ***p < 0,001 EAE t Vehicle vs CFA; ##p < 0.0 i . ###p < 0 001 EAE 4 EHP-101 vs EAE t Vehicle. Figure 15G depicts tire results of quantification of GFAP marker shown as mean ± SEM, and significance was determined by one-way ANOVA followed Tukey’s test < 0.901 EAE F Vehicle vs CFA; #Fp < 0.0E ###p < 0.001 BAF T EHP-101 vs EAE wVehide. Fignre 15H depicts foe resul ts of quantificatio n of MBP marker Shown as mean ±· SEM:, and significance was s determined by one way ANOVA followed T «key ^' test ** < 0,00! EAE Vehicle vs€FA; ##p < 0.01 , ##% 0.001 EAB 4· EHP-iOI vs EAE wVehkle.

Figure 16, composing Figure 16A through Figure I6H, depicts the exemplary results that demonstrat that de el aiion with persistent activation of microglia and loss of Olig2 expression was prevented by EHP-IOI treatment The quantifications of each marker are shown as mean & SBM, and significance was determined fey one way ANOVA followed Tukey’ test *p < 0.05, < 0,001 EAE < Vehicle vs CPA #p < O S, fe#p < 0.01 , ###p <

0.001 BAB < EHP-iOI vs EAE HfoMele. Figure 16A depicts representative eonfoeal microscopy images of cemhral corpus callosum immuttolabekd for i¼l. Figure 16.8 depicts representative eon focal microscopy Images of cerebral cortex showing that a reduced MBP reactivity was restored by EHP-I01 treatment. Figure 16C depicts representative confbeaf microscopy images that show that loss of 0!|g2 positive cells was preven ted in EHP- 101 treated mice. Figure I6D depicts re rese tairve eeufoeal microscopy images that show that EHPHOI treatment increased the expression of OSTpi in. corpus callosum. Figure 16E depicts the quantifications of Ifeal that is shown as mea ± SBM, and significance was determined by oneway ANOVA followed by Tukcy's test. * < , 5 _» ***p < O.OOI EAE < Vehicle vs CPA; # < 0.05 _» #p < 0.01 , ##p < 0.00:1 EAE < EHP- 101 vs EAE < Vehicle. Figure S 6F depicts the quantifications of MBP Chat is shown as mean ±. SBM, and significance was determined by oneway ANOVA followed fey Tukey ^' s test, *p < 0.05, < 0.001 EAE < Vehicle vs CEA; #p <

0.05, ## < 0.01* #?fop < 0.001 EAE + EHP-I01 vs EAE + Vehicle, Figure 160 depicts the quantifications of OIig2 that: is shown as meaa ± SEM, an significance was determined fey Oneway ANOVA followed by Tukey A test. *p < 0.03 _» ***p< 0.001 EAE < Vehicle vs CEA; # < 0.05, ##p < 0.01 , # #p < 0,001 EAE 4- EMP-101 vs EAE -f Vehicle. Figure 1613 depicts the quantifications of OSTpi that is shown as mean SEM, and significance was determined by onewa ANOVA followed by Ttikey ^' s test, *p < 0,05, ***p < 0,001 EAE < Vehicle v CPA; #p < 0.05, Vehicle.

Figure 17, comprising Figure !7A through Figure 17B, depicts the exemplary results of gene expression profiling of the effect of EHP-IOI in EAE model. Figure 17A depict MA plots (MA plot is an appl ication of a Blsnd-Altman plot for visual representation of genomic data) of the EAB or BAB + EHP- 101 vs control comparisons. The X axis represents the averaged expression as the mean of normalized counts while the Y axis Indicates the magnitude of (he cft&age as the k*g2 transformed fold change. The color indicates genes that surpassed the cutoff of aitjusiad P < 0,05 and fold change < -2 (blue or > 2 (red). Figure i?B depicts functional analysis results for genes that surpasses the previously mentioned cutoff in EAE vs Control aid EAE · EHf-101 (20 mg/ ^' kg) vs EAE comparisons. The presence of a point indicates a significant over-representation (adjusted P < 0.05} of Gene Ontology (Biological Process) terra (¥ axis) in a set of up or down regulates gene (X-axis). Figure l?C depicts heatmap depicting the expression levels for selected genes include in the Aytofcme-mediaced signaling pathway A Figaro 17D depicts heatmap showing the proteome profile of cytokines in CFA, EAE 4- ehicle and EAE < EHF-101 (20 mg/kg). Figure I?E depicts the mRMA expression for inflammatory marker in spinal cord that was quantified by qPCR and normalized versus GAPDM, Data represent the mean ± §EM, and significance was determined by one-way ANOVA followed Tukey's test p < 0.05, ** < 0,01, ***p < 0.001 EAE 4- Vehicle vs CFA; #p < 0.05, # p < 0.01, d##p < 0,001 EAE v E HP-101 vs EAE -f-Vehfc!e,

Figure 18, comprising Figure ISA through Figure 18E, depicts foe exemplary results that demonstra te that EHP-!Ol treatment normalized the expression of genes associated with oligodendrocyte function. Figure ISA depicts Venn Diagram indicating the overlap between foe sets of down regulated genes at EAE vs Control comparison and up regulated genes at EAE 4 EH!M 01 (20 mg/kg) vs EAE comparison. Figure; 18B depicts functional analysis results for the set of 193 overlapping genes. The scatter plot represents the significance of the e richment for the top 15 overrepresented Gene Ontology (BiologieaFFroeessHefms as the - fog 10 transformed adjusted P value. Figure I EC depicts heatmap depleting the expression levels for genes annotatedwith the An eiination” GO term included in the set of 193 overlapping features. Figure 18D depicts the mSRNA expression for myelinaiioa related genes that was quantified by qPCR an normalized versus GAPDH. Figure 1 BE depicts the results of immnnohistoehemistry labelling of spinal cord for Teneurin-4. The quantification o f expressi on o f Teneurin-4 ia White/Grey matter (bottom panel). Data represents the mean 4 8EM, and significance was determined by one-way ANOVA followed TukeyX test ** < 0.01 , ***p < 0.001 EAE 4 Vehicle vs CFA; % < 0.05, ##p < 0.01 , ###p <0.001 EAE 4 EHP-101 vs EAE AVehiele.

Figure 19, comprising Figure 19A through Figure 19E, depicts foe effect qf therapeutic EHF-101 treatment on remyelinatios I» a Guprizone (C ^: PZ)-indueed demyelmation model. Figure 19 A de icts the experimental procedure used to evaluate the effect of therapeutic EFiP-l Oi treatment on remyelination in a CPZ-iodu.ced demyeliaatioo model. Figure 19B depicts the resuits of histological study of myelin fay Cryoniyelin staining in eorpas callosum, Figure 19C depicts the results that demonstrated a significant recover in myelin staining, which was shown by immunofluorescence studies of M B P in cortex. Figure I9D depicts the mean intensity quantification results of histological stud of myelin by Cryomyelin staining in corpus callosum (n ~ 5 animals per group), Figure if 15 depicts the quantification of MBP irmnnnorcaetiviiy that demonstrated a significant recover in myelin staining, which was shown by immunofluorescence studies of MBP in cortex. Data represents the mean ± SEM, and significance was etermined by one-way ANOVA followed Tukcy ^' s test ***p < 0.001 CPZ 6W or CPZ 6W ÷ i or CPZ 6W 4 2 vs Control;

EHP-101 vs€P 6W> 2.

Figure 20, comprising Figure 20A through Figure 20D, depicts the impact of therapeutic EHP-101 treatment on microglia and astrocytes activation in a CFZ-mduced dc yelination model. Figure 20A depicts a decrease on euprixone- induced microgliosis that was detected by immunofluorescence studies of Jbal in corpus callosum. Figure 20B depicts astrogiiosis that was determined by immunofluorescence studies ofQPAP in corpus callosum. Figure 2QC depicts a quantified decrease on eupozone-indueed microgliosis that was detected by iuummofltiorescence studies of Ibal in corpus callosum. Figure 20D depict quantified intensity of asirogiiosis that was determine fay immunotluorescenee studies of GFAP in corpus callosum. Data represents the mean 4 SEM, and significance was determined b one-wa AD OVA followed Tuke A test < 0.001 CPZ 6 W or CPZ 6W 4 1 or CPZ 6W 4 2 vs Control; <

0.01 CPZ 6W 4 2 vs Control ; ##p < 0.01 CPZ 6W i 1 4 EHF- 101 vs CPZ 6 W ÷ I .

Figure 1 depicts representative primers used In real-time PCR analysis.

Figure 22, comprising Figure 22A sad Figure 22B _» depicts representative results demonstrating that EHF-101 reduces axonal degeneration and plasma levels of neutofiament light polypeptide (NEEL). Figure 22 A depicts representative images of immunostaining of SMI- 324 cells jfl the Corpus callosum of different groups of animals. Figure 22B depicts NEEL plasma levels were detected by ELISA in the different groups of animals. Values wer normalized versos control group an correspond: to mean 4 SEM and significance was determine by one-way ANOVA followed by Take A test p < 0.05 CP 6 W or CPZ 6W i vs Control; #p < 0.05 CPZ 6W - ! 4 BMP- 101 vs CPZ 6W 4]

U Figu e 23 depicts t e experimental procedure used to evaluate: the effect of therapeutic: oral LBP-101 t eatment on retoyelmation in a CPA-induced demy elination el

Figure 24, comprising Figure 24A through Figure 24D, depicts grey matter (hippocampus) remyeliimfloa results, Figure 24A depicts PL? staining in the hippocampus. Figure 24B depicts quantification results of FLP in the hippocampus, EEP- i01 -treated animals showed no change in the area of PL P staining ip the hippocampus compared to vehicle control. Figure 24C depicts quantification results of FLP in the hippocampus. Outliers were identified using ChauveneFs criterion. No outliers were excluded f om statistical analysis. Figure 24D depicts hippocampal statistics for PLP stain.

Figure 25, comprising Figure 25A through figure 2SD, depicts: grey matter (cortex) remyef iuation results. Figure 25A depicts FLP stainin in the cortex, figur 25B depicts quantification results of PLP in the cortex. EHP~101 -treated animals at all dose strengths showed to change in the area of PLP staking in the cortical region compared to vehicle control Figure 2SG depicts quantification of PLP in the cortex. Outliers were identified using Oiauvenef s criterion. No outliers were excluded from statistical analysis. Figure 2SO depicts the statistics for PLP stain.

Figure 26, comprising Figure 26A through Figure 26D, depicts white matter (corpus callosum) remyelmafion results. Figure 26A depicts FED staining In the corpus callosum. Figure 26B depicts quantification results of PPD k the corpus callosu (without age matched (AM) sample): the myelinated axons in corpus callosum. Although EFD Ol treatments did not shove a significant: i crease i myelinated axons compared to control, there was a significant difference between the two higher groups when compared to the lowest tested group of the test article. Figure 26C depicts quantification of PPD in the corpus callosum. Outliers were identified using Q rveuef s criterion. Sample 44 was excluded from statistical analysis. Figure 26D depicts number of myelinated axons in corpus callosum statistics (without AM sample).

Figure: 27, comprising Figure 27 A and Figure 27B, depicts white matter (corpus callosum) remyelination results (with AM sample). Figure 27 A depicts quantification results of PPD hi corpus callosum: the myelinated axons in corpus callosum ( with AM sample). Although BMP-] 01 treatments did not show a significant increase hi myelinated axons compared to control, there was a significant difference between the two higher groups when compared to the lowest tested group of the test article, Figure 27B depicts ratmber of myelinate axons in corpu eaiiosn statistics (with AM sample),

Figure 28, comp g Figure 28 A and Figure 28B, depicts white mater (corpus callosum) remyidlnation results (without AM sample). Figure 28A depicts the density of myelinated axons (PFD density) in corpus callosu (without AM sample). The higher doses tested of EHF- T01 treatments showed a significant increase in th density of niyei ated axon compared to control, there was also a significant dif&reaee between the two higher groups when compared to the lowest teste group of the test article. Figure 288 depicts the statistics tor the density of myelinated axons in corpus callosum (without AM sample).

figure 29, comprising Figure 29A and 29B, depicts white matter (corpus callosum} remyeliuati.on results (with AM sample). Figure 29A depicts the density of myelinated axons (PFD density) in corpus callosu (with AM sample). The higher doses tested of EilP-101 treatments showed a significant increase in the density of myelinated axons compared to control, there was also a significant difference between the two higher groups when compared to the lo west tested group of the test article, Figure 2913 depicts the statistics tor the density of myelinated axons in corpus callosum (with AM sample).

D FT AILED DESCRIPTION

It is to be understood that the Figures a descriptions of the present invention: have been simplified to illustrate elements that are relevant tor a clear understanding of the present Invention, while eliminating, for the purpose of clarity , many other elements found in. the method of treating a condition or disease responsi ve to a modulation of€!¾ acti vity or a condition or disease associated with de yel i ation using the compound of Formula (!) as well as methods of making and using such compounds, pharmaceutical compositions, and liquid formulations thereof Those of ordinary shill in the art may recognize that other elements and/or step are desirable and/or required 1» implementing the present invention. However, because such elements and steps are well known in the art, and because they do trot facilitate a better understanding of the present in vention, a discussi on of suck elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the ait. Definitions

As used herein, each of the in! lo wing terms lias the meaning associated with it I» litis section Unless efined elsewhere, ail technical. and scientific terms used herein have the sa e meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used i the practice or testing of the present inven tion, the preferred methods and materials are described.

The articles and“an” are used herein to refer to one or to more than one (ie to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element

The term“about” will be understood by persons of ordinary skill in the art and will vary to some exten t depending on the context in which it is used. As used herein when referring to a measurable value such as an amount a temporal duration, and the like, the term “about' ⁵ is meant to encompass variation of ±20% or ±10%, more: preferabl ±5%, even more preferably ±1 % _» and still more preferably -.rf) J% ftom the specified value, as such variations are appropriate to perform the disclosed methods,

A disease or disorder is“alleviated” If the severit of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

A“disease” is a state of health of an animal wherein the animal cannot maintain homeostasi , and wherein if the disease is nor ameliorated then life animal/ s health continues to deteriorate in contrast, a“disorder in an animal is a stale of healt in which the animal is able to maintain homeostasi s, but in which the n ars state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does amt necessarily cause further decrease in the animaf s state of health.

The terra“In ibits as used herein, means to suppress or block an activity or function b at least about ten percent relative to a control value. Preferably, the activit is suppressed or blocked by 50% compared to a control value, more preferably by 73%, and even more preferably by 95%.

In the context of the present disclosure, a“modulator is defined as a compoun that is an agonist, a partial agonist, an inverse agonist or an antagonist of CBr A modulator may Increase the activity of the CBi receptor, or may decreas the activity of the (¾ receptor la the context of the present disclosure, a¾>“agonist” is defined a s a compotmd fbst increase the basal activity of a receptor (le , signal transduction mediated by the receptor). An“antagonist” i defined as a compound, which blocks the action of an agonist on a receptor. A“partial agonist” is defined as an agonist that displays limited, or less than complete, activity such that it fails to activate a receptor in vitro, functioning as an aniagonist in vivo. An“inverse agonist' ⁵ i define as a compound that decreases the basal activi ty of a receptor.

The terms‘'treatment”,“treating ⁵⁵ an the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may he prophylactic In terms Of completely or partially preventing a disease or symptom thereof and/or may he therapeutic in terms of partially o completely curing a disease and/or adverse effect attributed to the disease. The term Areaimenfi ⁵ as used herein cover any treatment of a disease in a subject an includes; (a) preventing a disease related to an ««desired immune response front occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, be,, arresting its development: or (e) relieving the disease, i.e., causing regression of the disease.

The term“derivative” refer to a small moieeide: that differs in structure front the reference molecule, but may retain or enhance the essential properties of the reference molecule and may have additional properties. A derivative may change its interaction with certain other molecules relative to the reference molecule, A derivative molecule may also include a salt, an adduct, tautomer, isomer, or other variant of the reference molecule.

The term“tautomers ⁵⁵ are constitutional Isomers of organic compounds tha readily infercouvert by a chemical process {tautomerfeaiiouj.

The term“isomers ⁵⁵ o “stereoisomers ⁵⁵ refers to compounds, which have identical chemical eOnsfitoifen, hut differ with regard to the arrangement of the atoms or groups in space.

As used herein“polymorph refers to crystalline forms having the same chemical composition but different spatial arrangements of the molecules, atoms, and/or ion forming the crystal

As used herein,“alkyf refers to a linear or branched chain folly saturated (no double or triple bonds) hydrocarbon (all carbon) group. An alkyl group of this in vention may comprise irons 1 - 20 carbon atoms, that is,“m” ^:S: 1 and“rf’— 20, designated as a ^iv Ci to C¾o alkyl ⁵ In one embodiment“m” I and“n ⁵ 12 (Cs to the alkyl). In other embodiments that I &ad *¾ 0 {€¾ I© C¾ alkyl). Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyi, isopropyl, n-botyk iso-butyl, sec-batyL tert-butyl, amyl, tert-ainyi, hexyl, hepty!, octyl, nonyl, deeyl, undecyL and dodeeyl.

An alky l group of this invention may be substituted or nnsub tiiuted, When substituted, die substituent gmup(s) is(are) one or more group(s) independently selected from cyeionikyL aryl, heteroand, heterealieyclYl, hydrox , alkoxy, aryfoxy, mereaptb, aiky!ifdo, arylthio, cyano, halo, oxo, carbonyl, thiocarfjonyl, O-carfea yl, bbcarhsmyl Odhiocatharayl, - thioearbamyl, C-armdo, N-ar do, S-seilonamido:, N-salfonamido, C-carboxy, 0--cafhoxy, isoeyanatO, thiocyanate, isothlocyanato, nitre, silyl, tribalo etbanesnlfbnyi -NRaRh, protected hydroxyl, protected amino, protected cnrbo.xy, and protected amide groups.

Examples of substituted alkyl groups include, witliout limitation, :2-oxo^prop-l ~ I, 3-oxo~hut~I-yl, eyanomethyl, mteometliyL ehloromethyl, hydroxymethyl,

teirabydropyranyioxymethyi m-tritylexyniethyi, propiot loxy meth l, amiaomeibyi,

oarboxymethyi, allyfoxyearbonylmeth i, aliyloxycarbonylatniuomethyl, methoxymethyi, ethoxymethyl, tteirtoxy meth l, acetoxyrneihyl, ohlototneth L, hrmaome hyi iqdomeihvl, trill ooromethyi, 6~hydroxyhexyi, lA-dieMorobuiyi, 2-aminopropyi, 1 -eh!oroetbyi d-chioroethy!, 1 _“ bremoethyi, 2-ebioroeihyk 1 -ffuoroeihyi, 2 fiuoroeihyl, 1 -iodoethyi, 2-iodoethyi, 1 - cbteropropyl, 2-ciiioropropyl, 3-cbtoropropyl, 1 -hromoptopyi 2-bromopropyb 3-hfomopropyl

.1 -fiuoropropyl 2-fluoropropyl, S-flaoropropyl, 1 -iodopropyl, 2-iodoprepyi, 3-iodopropyl, 2- aminoethyi aniInoediyL M-ben2oyh2-atnlooethyL aceiyl-2-aniiBoethyi, N-benzoyi-l - aPuuoethyl, and M-acetyl-l -aminoethyL

As used .herein,“alkenyr refers to an alk l group that contains in a linear or branched hydrocarbon chain one or more double bonds. Examples of alkenyl groups include, without limitation, vinyl (CBr-Cll·), ally! (CfbCH-CHi-), 1 _“ property!, 2-firepenyL tebuteny!, 2-bntenyl; l-perrtenyl, 2~penienyi, 3~penienyL 4-pentenyk 3-aiethyl~l~h«tenyi and the various Isomers of bexenyl, heptenyl, octenyl, aoaen l, decenyh undecenyl, and dodeceayl.

An alkenyl group of this invention may be unsubstitnted or substituted. When substituted, the sisbstiteeni(s) may be selected from the same groups disclose above with regard to alkyl group substitution. Examples of substituted alkenyl groups include, without limitation, styrenyl, d-chtoro-propen-byl, 3 hloro-bute» -yi, 3-rneth.oxy-propen-2-yl, 3-phenyl buten-2-yl, and l -cyar -buten3-yl. As nsedhereiu,“alkynyfe refers to an alky! group that contains in a linear or b anched hydrocarbon chain one or ote triple bonds.

A» alkynyl group of this invention ay be uasubstitute or substituted When substitute the subsdtuemis) may be selected from the sa e groups disclosed above with regard, to alkyl group substitution.

As used herein,“aryl refers to a earboeyelie fall carbon) ring or two or more fused ring {rings that share two adjacent carbon atoms) that have a fully delocalised pi-electron system. Examples of aryl groups include, but are not limited to, benzene, and substituted benzene, such as toluene, aniline, xylene, and the like, naphthalene and substituted naphthalene, and azidene.

The term“pharmaceutically acceptable salt ^· refers to any pharmaceutic lly acceptable salt, which upon administration to the patient; is capable of providing (directly or indirectly) a compoun as described herein, Such salts preferably are acid addition salts with physiologically acceptable organic or inorganic acids. Examples of the acid addition salts include mineral add addition salts such as, for example, hydrochloride, bydrobromkle, hydroiodide, sulphate nitrate, phosphate, and organic acid addition salts such as, for example, acetate, triffuofoaceiate, makate, funiarate, citrate, oxalate, succinate, tartrate, malate, mandelate, urethane sul honaie and pdoiuenesulphooaie. Exaurpl.es of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, an organic alkali salts such as, for example, etkyienediannne, ethanolaniine, K,N- diaikylenet!mrso!arnme, triethanolamine and basic amino adds salts. However, it will be appreciated that non-pharmaceniieally acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. Procedures for salt formation are conventional in the art.

The term“solvate” in accordance with this invention should be understood as meaning any form of the acti ve compound in accordance with the invention in which said compound is bonded by a non-eovaleni bond to another molecule (normally a polar solvent), deluding especially hydrates and alcoho lutes.

The terms“effective amount” and“pharmaceutically effective amounf refer to a sufficient amount of an agent to provide the desired biological result. That result can he reduction andfor alleviation of a sign, symptom, or cause of a disease or disorder or any other desired alteration of a biological system. A appropriate effective amount in any individual case may fee determined fe one of ordinary skill I the art using routine experimentation.

A“therapeutically effective amount” refers to that amount which provides a therapeutic effect for a given condition and administration regimen. In particular,

‘therapeutically effective amount” means an amount that is effective to prevent, alleviate or ameliorate symptoms of the disease or prolong the survival of the subject feeing treated _* whic may be a human or non hnraan animal Determination of a therapeutically effective _: amount is within the skill of the person skilled in the art.

As used herein, the term“phamiacootical composition” refers to a mixture of at least one compound of the invention with other chemical components and entities, such a carriers, stabilisers, diluents, dispersin agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical compositio facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including* hut no limited to, intravenous, oral, aerosol, parenteral, ophthalmic, ulmonary' and topical

administration.

‘"Pharmaceutically acceptable ⁵ ' refers to those properties and/or substances which are acceptable to the patient from a phannacological/toxieoiogical point of view and to the manufacturing pharmaceutical chemist from a physical/chemical point of view regarding compost lion, formulation, stability _* patient acceptance amt bioavail ability.“Pharmaceutically acceptable carrier refers to a medium that does not interfere with the effectiveness of the biological activity of the active htgredieutfs) and is not toxic to the host to whic it is

administered.

As used herein, the ter “pharmaceutically acceptable carrier' means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material Involved in carrying or transporting a compound useful within the invention within or to the patient such tha it may perform its intended function. Ί ypicall , such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be 'acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the pat ient . So e examples of materials that may serve as pharmaceutically acceptable earners include; sugars, such as lactose, glucose mi sucrose; starches, such as co starch and potato stared· cellulose, and its deri cats ves, such as sodium c iibxymethy! cellulose,ethyl cellulose and cellulose acetate; powdered tfagacaath; malt; gelatin; talc; excipients, such as cocoa butter end suppository waxes; oils, such as peanut oil, cottonseed oil, saSIowet oil, sesame oil olive oil, eoru oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as edtyl oleate and ethyl lanrate; agar; buifering agents, such as m agneslum hydroxide and aluminum liydroxtde; surface active agents; alginic acid; pyrogen-fee water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate bolder solutions; and other non-toxic compatible substances employed in

pharmaceutical formulations. As used herein, ^pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the invention, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The“pharmaceutically acceptable earner' may further include a pharmaceutically acceptable salt of the compound useful within tile invention, Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the invention are known In the art and described, for example in Remington's Fhanoaceodeal Sciences (Genar», Ed., Mack Publishing Co,, 1985, Easton, PA), which is incorporated herein by reference.

the terns "ouiriilonat composition" may be a food product intended for hmoau consumption, for example _* a beverage, a drink, a bar, snack, an ice cream, a d iry product, lor example a chilled or a shelf-stable dairy product, fermented dairy product, a drink, for example a milk-based drink, an infant formula, a growing-up milk, a confectionery product, a chocolate, a cereal product such as a breakfast cereal, a sauce, a soup, an instant drink, a frozen product Intended for consumption after heating in a microwave or an oven, a ready-to-eat product, a fast food or a aotriiioaal formula

The terms“patient,"“subject,"“individual," and the like are used interchangeably herein, and refer to any animal, or eel Is thereof whether in vitro or in situ, amenable to the methods described herein, in certain non- limiting embodiments, the patient, subject or individual is a human . lliroughout m disclosure, various aspect of the in ention eaa be pr sen ed Ih a range format It should he understood that hie description in range format is merely for convenience and brevity and should not be construed as an indexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed at! the possible sub-ranges as well as individual numerical values within tha range. For example, de scription of a range s uch as from 1 to 6 should fee considered to have specifically disclosed sub-ranges such as from 1 to 3,: from 1 to 4, from ^" l to 5, from 2 to 4, from 2 to foirom 3 to 6 etc,, as well as individual numbers within that range, for example, 1, 2, ,7, 3, 4, 5, 5.3, and 6 , This app lies regardless of the breadth of the range.

Description

The invention provides a composition Comprising at feast one cannabidlol derivative soiubiliced in a pharmaceutical vehicle. In one embodiment, the composition has Increased bioavailability. In one embodiment, the composition has Increased bioavaiiabiiity when compared o the bioavaiiabiiity of the same eaomthidioi derivative in a non-fotraulaied mixture. In one embodiment, the composition has increased solubility. In one embodiment, the composition has unproved solubility when compared to the solubility of the same cannabidlol derivati ve in a noo-fommlaied mi ture

In one embodiment, the composition is a dry powder formulation. In one embodiment, the composition is a tablet, wherein the tablets, comprising the: cannabidlol derivatives, are prepared through two manufacturing steps: a granulation step and a tablet preparation step. In one embodiment, the granulation ste is a preparation of the intermediate product (IP), In one embodiment, the granulation step comprises a granulating fluid containing excipients in ethanol that is added to primary powder particles and followed by solvent evaporation. I one embodiment the particle size of the resulting material is reduced by milling in one embodiment, the tablet prepara tion step is a preparation of the Drug Product (BP). In one embodiment, a intermediate product (IP), wherein the Intermediate product (IP) Is obtained from the granulation step, is blended with excipients. In one embodiment, the Drug Product (DP) is tablet compressed by direct compression on a tablet p ess. la one e ¾odMie»L the eompesitioh is a suspension I» one embodiment, the eBiapo t!on is a oanosuspensiom la one embodiment, the composition Is an emulsion, In one embodiment the composition is a solution, la ©a© embodiment, the composition is a liquid formulation in one embodiment:, die composition is a cream. In one embodiment, the composition Is a gel. In oae embodiment, the composition is a lotion, la one embodiment, the composition is a paste, la one embodiment, the composition: is an ointment In one embodiment, the composition is an emollient. la one embodiment, the composition is a liposome la one embodiment, the compositio a naaosphere, la one embodiment, the composition is skip tonic, in one embodiment, the composition is a month wash in one embodiment, the composition is an oral rinse. In one embodiment, the composition is a mousse. In one embodiment, the composition is a spray. In one embodiment, the composition is a pack la one embodiment, the composition is a capsule hi one embodiment, the composition i a tablet. In one embodiment, the composition is powder hi one embodiment, the composition is a granaie. In one embodiment, the composition is a patch. In one embodiment, the composition an occlusive skin agent.

In one embodiment, the composition comprises new drag candidates comprising chemically stable nonpsychotropic aminogninoi chemically derive from synthetic or natural cannabidiol (CR ) through oxidation and amination, I one embodiment, the cannabidloi derivative is a synthetic cannabidiol derivative, lb one embodiment, the synthetic cannabidloi derivative comprises chemically stable, nonpsychotropic aminoqninold chemically derived from synthetic caanabidiol (CBD) through oxidation and amination. In one embodiment, the synthetic cannabidiol derivative comprises chemically stable, nonpsychoiropfc a inoqninoid chemically derived from natural cannabidiol (CBD) through oxidation and amination. In one embodiment, the synthetic cannabidiol derivative is a nou-reactive synthetic cannabidiol derivative. In one embodiment, the non-reaciive synthetic cannabidiol derivative Is a: chemically stable synthetic cannabidiol derivative, in one embodiment, the non~reaetive synthetic cannabidiol derivative Is a synthetic cannabidiol derivative that does not have a detectable affinity for the€81 receptor.

In one embodiment, the composition comprising a non-reaetive synthetic cannabidiol derivative has a novel mechanism of action (MCA) by targeting complementary signaling pathways that alleviate ncuroiiiflam adoii and favor neuroprotection, prevent axonal damage, preserve myelin structure, and potentially promote remyelinatiom In one embodiment, the composition comprising a oon-reaerive synthetic cannabidiol deri ative is a modulator of

2! CBi receptor sigs ing. In. die e o i ent the composition comprising a non-reactive synthetic eaitnaMdiol derivative is a modulator of PPAR id CBs receptor signaling. In ose embodiment the composition comprising a non-reaetive synthetic easnabi ioi derivative is a modulator of PPARy and CBs receptor signaling, and stabilizes HlF-la thus upregukumg the expression of several associated factors that include Erythropoietin (EPO) and Vascular Endothelial Growth Factor A (VEGF A), In one embodiment, the eomposiiiowcomprlsing 8 oohneaetiye synthetic eannabidlo! derivative reduces neotoinBamnMiion presumably by acting on PPARy/CBa receptors, in conjunction with enhanced nenroproieetion and potential remyellnation through the HIP pathway.

In one embodiment, the composition comprising a non-reactive syntlietie eaaaabidioi derivative binds the CBa, In one embodiment, the non-reaetive synthetic eannabidir derivative preferentially binds to CBa receptor as compared to eannabinoid receptor type 1 (CBi)- Therefore, in these embodiments, the non-reactive synthetic eaimahldfol derivative is selective for CBa. In one embodiment, the amine group of ndn-reactive synthetic eannabidiol derivative binds the CBi, In one embodiment, the amine group of non-reactive synthetic canned dfo I derivative selectively binds the CBi receptor over the CBi receptor. In one embodiment, the CBs receptor activity is modulated i vitro, whereas in other embodiments, the Qfc receptor acti vity is modulated in vivo.

In one embodiment the eannabidiol derivative is a compound of Formula (I),

0)

In one embodiment, R is the nitrogen atom of a group independentl selected from a linear or branched afkylamine, an arylamine, an ar ialky!amine, a heieroarylamine, a heteroarylaikylamine, a linear or branched aikenylamine, a linear or branched alkynyiaxnlne, or NKi, la one erobodlnieot, the casaab W deri ati e is selected from the group. consisting pf;

(1:'R _? 6 ^! R)-3-(Etby iaii)-6-hydfoxy-3'-ffie¾y!-4-peaSyl~6 ^, -(pKi|>~l-eri-2-yl)[Lr- bl(cy cloe aae)]~2J ,6-lri eae-2 ~dione.

( i¾. _i 6'R)-3-CPeiiiylam:ii }-6~i ^: iy X) ^; _“ 3 ^, _“ «ieif3yi~4 _“ pe5ityI-0'-(pop-i.-en2-yI}-

[.l,X¾i(cyc > sxane)}-23,6-t«eae- ^' 2,5- ioBe _»

(l’Ry5 ^< R)~3Rlsobuty|:8mi e)~6 _' IIyd sy--3 ^! ~metliy1~4pe«iy!-{>'-(prop _“ |-en~2~yl) l _;] - b!(cyclohexaii.e)j-2 ⁱ _! 3 _y 6~Rieiie _” 2 - ione _;

(1; ₅ 6 ^, R.}-3-(B¾i fe irse)~6~hydroii _: y-3 ^, -i ^: B:eil5yi-4-pe yI-'6'-(pf p~l~eii-2-yi) !, -

(l¾ _, ^< R -3-CMePiyIamiae)-6-Hydroxy-3 ^, ~ ethyi-4-peiify!-6 ^, ~(prop~!-ers~2-yj)[l *- biCc clohexatieJJ'S’JP-tri se-SS-doHe,

(I 'R,6¾}3-(¾C!piOpyisK«fte) _“ 6 _“ Hy<lroX¥"3 ⁱ _“ -ffiei hy R4-p¾nty!6--{prop- 1 _“ »h--2-n1)-·( I , ! - biCcyclob sHte^^yS^- eBe-S.S-dione,

ii'R ₅ h¾)-3~(Ben¾yiam e}~b~hy iOxy~3feneyiyl~4-pentyi-6H rgp~l~em2-yI)[Lr-

(rR,6 ^, R)-3-(Ne0peniy!amine)-6-liydroxy~3 ^, -meihyI-)-4-pentyH '-(prop I -e«~2y!)-|IJ - bi(cyc J bl)ex iie)]~2\3 ₅ 6 _“ t ^> ne~:2 5~dioiie, ami

( i ¾,i>¾)3 -I I sopentyiamhi e)~ 6-IΊ ydroxy-amine-jhffietbyS-d-pentyl-b ^* -(pro - I -enS2-y 1) 1 , 1 fe)(cyclohexane)]~2\3,6-meoe-2,J ^; ~di0te. In one embodiment the pharmaceutical vehicle is selected from the group consisting of aqueous buffers, solvents, co-solvents, cyelodextrhi complexes, lipid vehicles, and any combination thereof, sad optionally further comprising at least one siabiHaer, emulsifier, polymer, antioxidant, and any Oomfeiaation thereefl

In one embodiment the aqueons ba ter Is selected from the group Consisting of aqueous HO, aqueous cit te-HCl bi er, aqueous NaOH, aqueous eitrate-NaOH buffer, aqueou phosphate buffer, aqueous KCI, aqueous borate-KCbNaOH buffer, PBS buffer, and any co mbs n adon thereof^

In one embodiment the aqueous buffer has pH range of pH ~ 0, - 10. In one embodiment, the aqueous buffer has pH range of pH ^~ 0,5, In one embodiment the aqueous buffer has pH 1 ,0 In one embodiment the aqueous buffer has pH ~ 2.0, In one embodiment, the aqueous buffer has pH - 3 0. In one embodiment the aqueous buffer has pH 4.0, In one embodiment the aqueous buffer bu pH 5.0 In one embodiment the aqueous buffer has pH ~ 5,5. hi one embodiment, die aque us buffer has pH *» 6 0. in one embodiment, the aqueous buffer has pH ? 0, in one embodiment, the aqueous buffer has pH ^~ 7.4, In one embodiment, the aqueous buffer has pH ~ 8,0. In one embodiment, the aqueous buffer has pH ~ 9.0, In one embodiment, the aqueous buffer has pH ^~ 9.5, In one embodiment, the aqueous buffer has pH ^

10.0.

In one embodiment the aqueous buffer has a concentration range of 0.05 N— 1.0 N. hi one embodiment die aqueous buffer has a concentration of 0.05 N, In one embodiment, the aqueous buffer has a concentration of 0.1 N, in one embodiment, the aqueous buffer has a concentration of 0. I S N. In on embodiment, the aqueous buffer has a concentration of 0,2 N. In oiie embodiment the aqueous buffer has a coneentratlon of 0,3 N. ha one embodiment, the aqueous buffer has a concentration of 04 N. In one embodiment he aqueous buffer has a concentratlot of 0.5 M. In one embodiment, the aqueous buffer ha a concentration of 0.6 N. In one embodiment the aqueous buffer has a concentration of 0,7 N. In one embodiment, the aqueous buffer has a concentration of 0.8 N In one embodiment, the aqueous buffer has a concentration of 0, N lu one embodiment, the aqueous buffer has a concentration of 0 N.

In oue embodiment, the solvent is selected from the group consisting of acetone, ethyl acetate, acetonitrile, pentane, hexane, heptane, methanol, ethanol, isopropyl alcohol, dimethyl sulfoxide (DMS0), wafer, chloroform, diehioromcthanc, diethyl ether, PBG400, Transcutol (diethylene glycomouoetbyl ether), MCI 70, Labraso! (PEG·· 8 caprylie/eapric glycerides), Labmfii M1944CS (PEG 5 Oleafe), propylene glycol, T nsentol P, PEG400, propylene glycol, glycerol, Capex 300, Tween 85, Gremophor EL, Maisiae 354, Ma e CC, Capmel MCM, maize ml, and any combination '.hereof.

la one embodiment, the co-solvent is selected from the group consisting of acetone, ethyl acetate, acetonitrile, pentane, hexane, heptane, methanol, ethanol, Copropyl alcohol, dimethyl sulfoxide (DMSO), water, chloroform, diehloromethane, diethyl ether, FEG4O0, Transcutol (diethyleoe glyeomonneihyl ether), M€T 70, Labrasol (PEG-8;

eapryiic/caprie glycerides), Eabrafil 1944CS (PEG 5 Oleate), propylene glycol, Trauscntol P, EG400, propylene glycol, glycerol, Capiex 300, Tween 85, Gremophor EL, Maisine 35-1, Maisine CC, Capmul MCM, aize oil, and any combination thereof

In one embodiment, the eyelodexirin complexes is selected front the rou _:

consisting of methyl-p-cyelodextrin, nie hyl-y-cydodextrin, HF-p-eyclodextnn, HP-y- cyolodextrin, SBE-p-eycIodextrin, a~eyclodextrin, y-eyelodexiiiipO-O-ghieosy!-p-eyciodextria, and an combination thereof,

In one embodiment, the lipid vehicle is selected from the group consisting of Capiex 300, Tween 85, Cre ophor EL, Massine 35-1 , Maisiae CC, Capmul MCM, maize oil, and any combination thereof In one embodiment, the lipid ve icle is an oil. In one embodiment, the lipid vehicle Is an oil mixture. In one embodiment, the oil mixture comprises at least two oils. In one embodiment, the oil is selected from the group consisting of Capiex 300, Tween 85, Cremophor EL, Maisine 35 -i , Maisiae CC, Capmnl MCM, maize oil, and any combination thereof

In one embodiment, the oil mixture is 10 : 90 v/v oil mixture. In one embodiment, the oil mixture i 20 : 80 v/v mixture. In one embodiment, the oil mixture is 30 ; 70 v/v oil mixture. In one embodiment, the oil mixture is 40 ; 60 v/v: oil mixture In one embodiment, the oil mixture is 42 : SB v/v oil mixture in one embodiment, the oil mixture is 5 : 50 v/v oil mixture. In one embodiment, the oil mixture Is 55 ; 45 v/v oil mixture, in one embodiment, the oil mixture is 60 : 40 v/v oil mixture, in one embodiment, the oil mixture i 7 : 30 v/v oil mixture. In one embodiment, the oil mixture is 80 20 v/v oil mixture. In one embodiment, the oil mixture is 90 * 10 v/v oil mix ture.

In one embedimeni, the stabilizer is selected from the group consisting of Pharmacoat 603, SIS. Nisso HPC-SSL, Ko!Iiphor, PVP fC30, FVP V,\ 64, and any combination thereof In one embodiment, the stabilizer is an aqueous solution. In one embo i ent, the polymer s selected from the group consisting of f!PMG- AS-MG, HPMC-AS-I .G. HPMC-AS IG, HPMC BPMC-P~5SS, HPMC-P-Sil methyl cellulose, MECLMPC, Eudmgit U0O, Eud git El 00, PEC) 1L, PEG 6000, PVF VA64, EVP 30,

TPGS, Kollieoat IR, Catbopol 980NF, Povoeoaf: ME, Sokplus, Soreteric, Plutonic f ~6§, and any combination thereof

in one embo i ent the antioxidant is selected tram the group consisting of Vitamin A, Vitamin€, Vitamin E, Coenzyme Q} manganese, iodide, meiaionin, alpha- carotene, astaxantbin, beta-carotene, eanfoa anth , eryptojiatithin, lutein, lycopene, zeaxantbin polyphenol antioxidant, ilavonoid, Savones, apigemn, luteolin, iangeritin, ffavonol,

isofoammodm haompferol, myrieedii, promUhoeyafodm, quercetin, ffavanone, eriodletyol, hesperebu, nsringenim flavanol, eateehin, gailoeatedifo, gallatc esters, epicateebin,

epigailocatechln, theafiavin ihearaMgln, isofiavoue phytoestrogen, daidxein, genistein, glychein, sillhenoid, resveratroL pterostllbene, authocyanio, cyanMin, delphinidio, maividm, pelargonklin, peonidin, petonidim chieorie acid, eaffeie acid, dilorogeoie acid, ferulic acid, cinnamic acid, eliagic acid, dlagl annin, gallic acid, gallotannin, rosmatmie acid, salicylic acid, eurcumm, fiavonolignan, silym rin, xanthome, eogettoL capsaicin, bilirubin, citric acid, oxalic acid, phytic acid, n-acetylcysteine, R-alpha-lipoie acid, and any combination thereof

hi one embodiment, die eannabidiol derivative or formulation thereof solubilized in a pharmaceutical vehicle has a solubility range of 0.001 mg/mL ~ 10.0 g/rnL. In one embodiment, the eannabidiol derivative or formulation thereof has a solubility of 0,001 mg/mL. in one embodiment the eannabidiol deri vative or formulation thereof has a solubilit of 0,005 mg/ L In one embodiment the eaunabidiol derivative or formulation thereof has a solubility of 0.006 g/ L. In one embodimen t, the eannabidiol derivative or formulation thereof has a solubility of 0.008 rag mL. la one embodiment, the eannabidiol derivative or formulation thereof has a solubility of 0.01 mg/mL. In one embodiment:, the eannabidiol deri vative or formulation thereof has a solubility of 0 03 mg/mL. in one embodiment, foe canaabi&iol deri vati ve or formulation thereof has a solubility of 0.06 mg/mL, In one embodiment, the eannabidiol derivative or formulation thereof has a solubility of i .0 mg/mL. In one embodiment, the caunahidiol derivative or formulation thereof has a solubility of 2.0 mg/mL. In one embodiment, foe eannabidiol derivative or formulation thereof has a solubility of 2.5 mg/mL. In one e bodi ent, the eannabidiol derivative or formulation thereof has a solubility of 6.1 mg mL, In ©he embodiment, the casuabidfel erivat ve or formulation thereof :has a sefefellity of 10,0 mg/mf. la one embodiment:, the eannabidiol derivative or formidatlon thereof baa a solubility of 10.2 ihg/ihL. In one eniboditneni, the eannabidiol derivative or for u i©.» thereof has a soInfeiHty of 100,0 mg/mL, In one embo iment* the eannabidiol derivative or formulation thereof has a solubility of 250 0 mg/mL Is one embodiment, the eannabidiol derivative or formulation thereof has a solubility of 500 0 mg/mL. i oue embodiniefiL the e ima dioI derivative or formulation thereof has a solubility o 7500 mg mL la one embodiment, the eannabidiol derivative or fommlation thereof has a solubility of 1 , g mL In one embodiment, the eannabidiol derivative or formulation thereof has a solubility of 1.5 g/mL In one embodiment, the eannabidiol derivative or formulation thereof has a solubilit of 5 0 g/mL in one

embodiment, the eannabidiol derivative f formulation thereof has a solubility of 8,0 g/mL. In one embodiment, the eannab idiol derivative or formulation thereof bus a solubility of 10.0 g/mL,

While the compounds of Formula I-X are GB2 receptor ligands, they also have ueuroprotective properties. Thus, the compositions and formulations comprising a compound of Formula I-X are useful in treating neurological disorders including but not limited to stroke, migraine, cluster headaches. The compositions and formulations disclosed herein are also effective in treating certain chronic degenerative diseases that are characterized by gradual selective neuronal loss. In this connection, the present compositions and formulation arc effective in the treatment of Farkinsoris disease, Aliheimeris disease, amyotrophic lateral sclerosis. Huntington's chorea, and prison-associated neurodegenetaddn. Menroprotcction conferred by 0¾ receptor agonists could also he effective in protection and or treatment of nearotoxic agents, such as nerve gas, as well as other insults to brain or nervous tissue by way of chemical or biological agents.

By virtue of their analgesic properties it will he recognized that the compositions and fdramlations according to the present invention will fee useful in treating pain including peripheral, visceral, neuropathic, inflammatory and referred pain. The compositions and formulations disclosed herein are also effective in the treatment of muscle spas and tremor.

Tire pharmaceutical compositions and formulation describe herein can be administered to a subject per se, or in pharmaceutical composition s where they are mixed with other active ingredients, as in combination therapy, or suitable carriers or exeipient(s), techniques for formulation a» administration of the compounds of the Instant application may fee found i« Mfendngtmifolfoatrnaeeutlcal Sciences” Mack Publishing Go., Easton, PA, 18 th edition, 1990.

Suitable routes of administration may, for example, include topical, oral, tecta!, transmueosal, or intestinal administrations parenteral delivery , ineloding intramuscular, subcutaneous, intravenous, intramedullary injections, as well a! intrathecal direct

intraventricular, i raperiteneal, intranasal, or intraocular injjsctio .

Alternatively, one may administer the compound in a local rather than systemic manner, tor exam le via injection of the compoun directly into the area of pain, often in depot or sustained release formylaftom Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coate with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.

The pharmaceutical compositions and fonnui&iions disclosed herein may bemanufactured in a manner that is itself known, e,g., by means of con ventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tahletting processes.

Pharmaceutical compositions and formulations for use in accordance with the present disclosure thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, whic facilitate processing of the active compounds into preparations, which can be used pharmaceutically . Proper formulation is dependent upon the route of administration chosen.. A»y Of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g,, in Eeminglot's Pharmaceutical Sciences, above.

For injection, the agents disclosed herein may be formulated In aqueous solutions, preferably in physiologically compatible buffers such as flank's solution, Rlngeds solution, or physiological saline buffer. For transmueosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, either solid or fluid unit dosage forms can be prepared.

For preparing solid compositions such as tablets, the compound of Formula (I) or derivatives thereof, disclosed above herein, is mixed Into formulations with convent Iona! ingredients soch as talc, magnesium stearate, diealeihm phosphate, magnesium aluminu silicate, calcium: sulfate. starch, lactose, acacia, methyleelleiose, aad functionally similar materials as pharmaceutical diluents or carriers. For oral adufi sf iion _* . the compounds cm be also formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds disclosed herein to be ibrmitlated as tablets, pills _* dragees, capsules, liquids _* g ls, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated _* Fftamaceudcal preparations t r oral use can be obtained by mixing one or more solid excipient with pharmaceutical combination disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, Including lactose, sucrose _* mannitol, or sorbifoi: cellulose preparations such as, for example _* maize starch, wheat starch, rice starch, potato starch, gelatin _* gum tragaeanth, methyl cellulose, hydroxypropylmethyi-celiulose, sodium earhoxymethylceliulose, and/or

polyvinylpyrrolidone (PVP), If desired, disintegrating agents may be added, such as the efOss- linked polyvinyl pyrrolidone, agar, or alginie acid or a salt thereof such as sodiu alginate..

Capsules are prepared by mixing the compound with an Inert pharmaceutical diluent, and filling the mixture into a hard gelatin capsule of appropriate size. Soft gelatin capsules are prepared by machine encapsulation of slurr of die compound with an acceptable vegetable oil _* light liquid petrolatum or other inert oil Fluid unit dosage form for oral administration such as syrups, elixirs and suspensions can be prepared. The water-soluble forms can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents an preservatives to form syrup. An elixir is prepared by usin a hydro alcoholic (e _* g . ethanol) vehicle wi h suitable sweeteners such as sugar and saccharin, together with an aromatic Savoring agent. Suspensions can he prepared with an aqueou vehicle with the aid of a suspen ing agent such as acacia, tragaeanth _* ethy!ce!&!ose and the like.

Dragee cores are provided with suitabl coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arable _* talc, polyvinyl pyrrolidone, caiftopol gel, polyethy lene glycol, and/or titanium dioxide, lacquer solutions _* and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to die tablets or dragee coatings for identiftcarion or in characterize different combinations of active compound doses _*

3! Starch mictospkeies can be prepared by adding a warm aqueous starch solution _* e; g., of potato starch, to a heated Alien of poly ethylene glycol I» water with: stirring in form an emulsion. When the two-phase system has formed (with the starch solution as the inner phase) the mixture is then cooled to roo temperature under continue stirring whereupon the inner phase is converted into gel particles. These particles are then filtered off at room temperature and slurred in a solvent such as ethanol, alter which the particles are again filtered off and laid to dry in air. The micro spheres can be hardened by well-known erossdinkiag procedures such as heat treatment or by using chemical cross- linking agents. Suitable agents include dialdehydes, including glyoxal, malondialdehyde, succinic aldehyde, adipaldehvde, g!utaraldehydc and phtha!aidehyde, diketoucs such as butadiene, epiehlorohydrin, polyphosphate, and borate, Diaidehydes arc used to crosslink proteins such as albumin by interaction with amino groups, and diketones fono sehiffbases with amino groups, Epiehlorohydrin activates compounds with nucleophiles such as amino or hydroxyl to an epoxide derivative.

Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soil, sealed capsules made of gelatin and plasticizer, such as glycerol of sorhitoL The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as tale or magnesium stearate and, optionally, stabilizers in soft capsules, the active compounds may he dissolved or suspended in suitable liquids, sued as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers and/or antioxidants ma be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

The compounds may be formulated for parenteral administration by injection, e.g , by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e g., In ampoules or In multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueou vehicles, and may contain formulatory agents such as suspending, sfabihzing and/or dispersing agents.

Slow or extended-release deliver systems, including an of a number hiopoiymers (biological -based systems), systems employing liposomes, colloids _* resins and other polymeric delivery systems or coofoarterentaUzed reservoirs, can be utilize with the compositions described herein to provide a contlnuons or Song term source of therapeutic compound Such slow release systems are applicable to formulations for deliver via topical, intraocular, oral, add parenteral routes.

Pharmaceutical formulations for parenteral administration include aqueous solutions _· of the active compounds in water-soluble form. Additionally _» suspensions of th active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils snob as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspension may contain substances, which increase the viscosity of the suspension, such as sodium earboxymethyl cellulose, sorbitol, or dextran. Optionally, foe suspension may also contain suitable stabilizers or agents, wh ch increase foe solubility of foe compounds to allow for the preparation of highly, concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g,, sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compounds ma also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds ma he formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt,

A pharmaceutical carrier for the hydrophobic compounds disclosed herein is a co- solvent system comprising benzyl alcohol, a nonpolar surf actant, a water-miscible organic polymer, and an aqueous phase. A common co-solvent system used is a co-solvent system, comprising a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. Naturally, the proportions of a co-solven system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied; for example, other low-toxicity nonpolar surfactants may be used instead of Polysorbate 80; the fraction size of polyethy lene glycol may be varied; other bioeompatlble polymers ma replace polyethylene glyeo!, e.g _* , polyvinyl pyrroiidone; and other sugars or polysaccharides ma he used

Atteruativfeiy, other delivery systems for hydrophobic pharmaceutical co pounds ay he employed Liposomes and emulsions are well known exa les of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as diraefoylsulfoxide also may be employed, although usuall at the cost of greater toxicity, Additionally, th compounds may be delivered using a sustained-release sy stem, sack as semipermeable matrice of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for stabilization may he employed.

Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts -with pharmaceutically compatible counterions. Fharmaeeutieaii compatible salts may be formed with man acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free acids or base forms.

Pharmaceutical compositions suitable for use In the methods disclosed herein include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifieally a therapeutically effective amount means an amount of compound effective to prevent:, alleviate or ameliorat symptoms of disease or prolong foe survival of the subject being treated. Determination of a therapeuticall effective amount is well within the capabilit of those skilled in the art, especially in light of the detailed disclosure provided herein.

The exact formulation, route of administration and dosage for the pharmaceutical compositions disclosed herein can be chosen by the Individual physician In view of foe patient's condition (See e.g., Fingl et ai. 1975, infThe Pharmacologies! Basis of Therapeutics's Ch. 1 p. 1) Typically, the dose about the composition administered to the patient can be from about 0.5 fo 1000 mg/kg of the pa tien t's body wei ght, or 1 to 500 mg/kg, or i0 to 500 mg/kg, or 50 to 100 mg/kg of the patient's body weight. The dosage may be a single one or a series of two or more given in the course of one or more days, as is needed by the patient , N ote that for almost all of the specific compounds mentioned in the present di closure, human dosages for tr t ent of at least some condition have been established Thus, in most instances _» the methods disclosed betein will use those same dosages, or dosages that are between about 0.1% and 500%, or between about 2554 and 250%, or between 50% and 100% of die established human dosage, Where no huma dosage Is established, as will be the case for newly discovered pharmaceutical compounds, a suitable human dosage can be inferred from EIMO or ΪR50 values, or other appropriate values derived fro in vitro or in vivo studies, as qualified by toxicity studies and efficacy studies in animals.

Although the exact dosage will be determined on a drug-hy-drug basis, in most eases, some generalisations regarding the dosage can be made. The daily dosage regimen for an adult human patient may be, for example, an oral dose of between 0.1 nig and 2000 mg of each ingredient, preferably between 1 mg and 250 mg, e _* g., 5 to 200 mg or an intravenous _* subcutaneous, or intramuscular dose of each ingredient between 0.01 g and 500 mg, preferabl between 0.1 mg and 60 mg, e.g,, 0.1 to 40 mg of each ingredient of the pharmaceutical compositions disclosed herein or a phannaeeuPca!ly acceptable salt thereof calculated as the five base, the composition being administered I to 4 times per day . Alternatively, the compositions disclosed herein ma be administered by continuou intravenous Infusion, preferably at a dose of each ingredient up to 400.mg per day. Thus, the total daily dosage by ora! admin Isi tioo of each ingredient wit! typically be in the range I to 2000 mg and the total dall dosage by parenteral administration will typically be in the range ø, 1 to 500 mg, Suitably the compounds will be administered for a period of continuous therapy , for example for a week or more, or for mouths or years.

Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety, which are sufficient to maintain the modulating effects, or minimal effecti ve concentration (MEC) The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual

characteristics and route of administration, However, HPLC assays or b assays cun be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositions should be administered using a regimen, which maintains plasma levels above the MEC for lf]-90 of the time, preferabl between 30-90% and most preferably between 50-90% I» cases of local administration or selective uptake, the- effective Ideal concentration of the drug may not fee related to plasma concentration.

Tile amount o composition administered will, of coarse, he dependent on thesubject being treated, on tite subject's weight, fee severity of tite affliction, the manner of administration anti fee j udgment of fee prescri bing physician.

The phamaeeutica l compositions and formulations may be prepared with pharmaceutically acceptable excipients, which may be a carrier or a diluent, as a way of example. Such compositions can he in fee for of a capsule, sachet, paper or other container, In making the compositions, conventional techniques for fee preparation of pharmaceutical compositions may he used. For example, the compounds of Form la (!) disclosed above herein may be mixed wife a carrier, or diluted by a carrier, or enclosed within a carrier that may he in the form of an ampoule, capsule, sachet, paper, or other container. When the carrier semes as a diluent, it may be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for fee active compound. The compounds of Formula (!) and compositions comprising the same, for use as described above herein can be adsorbed on a granular solid container for example in sachet Some examples of suitable carriers are water, salt solutions, aieohois, polyethylene glycols, polyhydroxycdioxylated castor oil, peanut oil, olive oil, lactose, terra alba, sucrose, cyclodextrin, amylase, magnesium stearate, tale, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, laity acids, fatty acid amines, fatly acid mono glycerides and digiycerides, peutaarythritoi -fatty add esters, poly oxyethyfeue,

hydroxymethykelluiose, and po! viny!pywelidorie. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl tuonosiearate of glyceryl distearate, alone or mixed with a wax. Said compositions may also Include wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.

The compositions for use in the trea tment of conditions or diseases responsive to the modulatio of the CBJ receptor activity, described iu present invention may be formulated so as to provide quick, sustained, of delayed release of the compounds of Formula (I) disclosed herein after administration to the patient by employing procedures welt known in the art.

The pharmaceutical compositions and formulations can be sterilised and mixed, if desired, with auxiliary agents, emulsifiers, salt for influencing osmotic pressure, buffers and/or coloring su stances sa d t he like, which o not deJeterisusly react with the co poun s disclosed above herein.

The pharmaceutical compositions and formulations may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution, This sus ension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredient such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile Injectable formulations may he prepared using a non-toxic parenteraily acceptable diluent or solvent, such as water or I ,3 butane iol, for example. Other acceptable diluents and sol vents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono or di-glycerides. Other parentaily-admimstrab!e formulations which are useful include those which comprise the active ingredient in microci stalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation ma comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

The compositions of the Invention may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active Ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for ad ninislration. The pack or dispenser may also be accompanied with a notice associated with the container i form prescribed by a governmental agenc regulatin the n nuiacPtte, use. or sale of

pharmaceuticals, which notice is reflective of approv al by the agency of the form of the drug for human or veterinary administration. Such notice, for example, may be the labeling approved by the U . S. Food and Drug Administration for prescription drugs, or the approve product insert. Compositions composing a compound disclosed herein formulated in a compatible

pharmaceutical carrier ma also be prepared, placed in an appropriate container, and labeled for treatment of an Indicated condition,

Treatment

The invention also relates, in part, to a method of treating a condition or disease associated with demyeiioation m a subject in need thereof In cate embodiment, the method comprises administering to the subject in Peed thereof a therapeutically effective a ount o at least one caonahidiol derivative or a formulation thereof in one aspect of the invention, the method of treating a conditio» or disease associated with demyelmation comprises

remyelluatiom The invention farther relates, in part to a method of Femyelination in a subject in need thereof In one aspect of the invention, the method comprises administering to the subject a therapeutically effective amount of at least otic eSinnabidiol deri vative or a form lation thereof in one embodiment, the subject has a condition or disease associated with demyelmation. In one embodiment, the subject has a condition or disease responsive to the modulation of the€l¾ receptor activity. In one embodiment, the subject has a condition or disease associated with demyelmation and a condition or disease responsive to the modulation of the CBz receptor activity. The present invention also relates, in part, to: a method of treating demyeiioation diseases.

In some embodiments, the condition or disease associated with demyeimaiion is selected from the group consisting of autoimmune disease, demyelmatmg disease, infiafnmatofy- related disorder, anti any combination thereof In one embodiment, the condition or disease associated wit demyelina ion Is selected front the group consisting S$e myelinoclastic disorder analgesia, acute and chronic pain, inflammatory pain, post-operative pain, neuropathic pain, muscle relaxation, immunosuppression, as anti-inflammatory agents, for allergies, glaucoma, bronelioddation, nearoproteeilon, osteoporosis and disorders of the skeletal system, cancer, neurodegeaerafive disorders Including but not limited to Alriteimer's: disease, Parkinson's disease (PD), and Htmbngtorfs disease, MS, muscle spasticity, tremor, fibromyalgia. lupus, rheumatoid arthritis, myasthenia gravis, other autoimmune disorders, irritable bowel syndrome, interstitial cystitis, migraine, prufitis, eczema, seborrhea, psoriasis, shingles, cerebral ischemia, cerebral apoplexy, craniocerebral trauma, stroke, spinal cord injury, liver cirrhosis, atherosclerosis, as an ami-tussive, asthma, nausea, emesis, gastric ulcers, neuromyelitis optica, central nervous system neuropathy, central pontine myelino!ysis, myelopathy, lenkoeneephalopaihy, leukodystrophy, peripheral neuropathy, Gni!laimBarre syndrome, anti-MAG peripheral neuropathy, Charcot- Marie-Tooth disease, progressive inflammatory neuropathy, amyotrophic lateral sclerosis ( ALS), and any combination thereof.

in one embodiment, the nomreactive synthetic eannabidiol derivative modulates remyelinatlon. In one embodiment, the Uon-reactive synthetic eannabidlol derivative induces remyellnation I» one: embodiment, the non-reactive synthetic esnnahidiol derivative enhances fe-myelintniom In ©tie ©tnbo tmeftL the non-reactive synthetic eannabidioi deri vative modulates demyellnatlon In one embodiment, the non-reactive synthetic cannabidioi derivative prevents demyelmatlotL in one embodiment, the non-reactive synthetic eannabidiol derivative reduces demyellnation, In one embodiment, the «on-reactive synthetic eannabidiol derivative accelerates deniyeliftatlon. I« one embodiment, the fion-teaetlve synthetic eatmabidiol derivative terminates demyellnation, In one embodiment, fee non-re active synthetic eannabidiol derivative modulates nenroinfiammation. in one embodiment, the non-reaetive synthetic cannabidioi derivative alleviates nsuroinflate aiion. in one embodiment, the non-reactive synthetic eannabidiol derivative mo ulates mierogllosis. In one embodiment, fee non-reactive synthetic eannabidiol derivative prevents mkrogliosis. In one embodiment, fee non-reactive synthetic eannabidiol derivative alleviates mierogllosis. In one embodiment, fee non-reactive synthetic cannabidioi derivative modulates asiroghosis. & one embodiment, the non-reactive synthetic cannabidioi derivative prevents astTogiiosk. in one embodiment, the non-reactive synthetic cannabidiol derivati ve alleviates asiroghosis.

in one embodiment, fee non-reactive synthetic oanuahkhol derivative m ulates a gene expression in one embodiment, the non-reaetive synthetic cannabidioi derivative prevents a gene expression. In one embodiment, fee non-reactive synthetic eannabidiol derivative reduce a gene expression in one embodiment, the non-reactive synthetic cannabidioi derivati e enhances a gene expression.

in some embodiments, the nOn-reaeiive synthetic e nnah!did! derivativemo lates a gene expression selected .from fee group consisting of a gene associated with MS pat ophysiology, a gene associated wit oligodendrocyte function, a gene associated with downregul alien in EAE, a gene associated wit expression of 01tg2, and any combinatiothereof. In one embodiment, fee non-reactive synthetic eannabidiol derivative modulates an expression of Teneyrin. In one embodiment, the non-reactive synthetic cannabidioi derivativeModulates an expression of Tenenrin 4 { Ten 4). in one embodiment, the non-reactive synthetic eannabidiol derivative enhances an expression of Ten 4 In one embodiment, the non-reaetive synthetic cannabidioi derivati ve normalizes an expressi on of Tenm 4. In one embodiment, fee non-reactive synthetic cannabidioi derivative modulates a expression of 01ig2 In Oneembodiment, the non-reactive synthetic cannabidioi derivative restores an expression of 0ilg2. ¾ one embodiment, the aan-reaciiv'e s tithei eansabidiol derivative en ances as expression of Obg2. In one embodiment, th non-reactive synthetic eannahidiol derivative modulates a expression of glutathione S-- transferase pi (GSTpi). is one embodiment, the non-reactive synthetic cannabidiol derivative enhances an expression of GSTpi . I one embodiment, the non~ reaetive synthetic cannabidiot derivative restores an expression of GSTpi

In one embodiment, the non-reactive synthetic eaanabidiol deri ative is effective for the attenuation of demyeiination i a sabjept By“attenuation of demyeiination’ ^' ' it is meant that the amount of demyeiination in the subject as a result of the disease or as a symptom of the disease is reduced when compared to otherwise same conditions and/or the amount of remyelination in the subject is increased when compare to otherwise same conditions. By “reduced” it is meant any measurable or detectable reduction in the amount of detnyeimaiioo or in any symptom of the demyeiination disease that is attributable to demyeiination, Likewise, the term“Increased” means any measurable or detectable increase is the amount of remyelination which will also manifest as a reduction in any symptom of the demyeHnatios disease that is attrsbutafale to demyeiination. In an embodiment of the invention, attenuation of demy el motion in a subjeet is; as compared to a control. Symptoms attributable to demyeiination will vary depending on the disease but may include, for example but not limited to, neurological deficits, such as cognitive impairment (me! tiding memory, attention, conceptualization and problemsolving skills) and information processing; paresthesias in one or more extremities, in the trunk, or on one side of the thee; weakness or clumsiness of a leg or hand; or visual disturbances, e.g„ partial blindness and pain in one eye (retrobulbar optic neuritis), dinmess of vision, or scotomas. The ability of a com pound to attenuate demyeiination may be defected or measured using assays known in the art, tor example, the euprizone-indueed demyeiination models described herein, lu one embodiment, the demyeiination disease is any disease or condition that results m damage to the protective covering (myelin sheath) that surrounds nerves in the brain and spinal cord. In a further embodiment of the invention, the demyeiination disease is selected from multiple sclerosis, transverse myelitis, Guiilain Barre syndrome, progressive multifocal ienkoencephaiopathy, transverse myelitis, phenylketonuria and other aminoacidurias, Tay-Saehs disease, Niemann-Piek disease. Gaucher's diseases, Hurler's syndrome, BrahheT disease an other leukodystrophies, acute disseminated encephalomyelitis (postinieetious encephalomyelitis, adrenoienkodysbrophy, adrenomyeloueisropathy, optic neuritis. De.vie disease (neimnnyeiitis optica), Lebeds hereditary optic atrophy an related mitochondrial disorders and BXLV- associated myelopath or the detnyelinatian disease is a result of local injury, i schemia, tele agents* or metabolic disorders Io one embodiment* the demyeiination disease is multiple sclerosis.

CBs modulators (ie , agonists, partial agonists, antagonists, or inverse agonists) have therapeutic utility hat analgesia, acute and chronic pain, inflammatory pain, post-operative pain, neuropathic pain, muscle relaxation, immunosuppression, as anii-inilanunstory agents, for allergies, glaucoma, bronchodi!aiion, neuroproteetion, osteoporosis and disorders of the skeletal system, dancer, neurodegenerarive disorders ncluding but not limited to Alzheimer's disease, Parkinson's disease (PD), and Huntington's disease, multiple sclerosis (MS), muscle spasticity, tremor, fibromyalgia, lupus, rheumatoid arthritis, myastheni gravis, other autoimmune disorders, irritable bowel syndrome, interstitial cystitis, migraine, pruritis, eczema, sebhorea, psoriasis, shingi.es, cerebral ischemia, cerebral apoplexy, craniocerebral dauma, stroke, spinal cord injury, liver cirrhosis, atherosclerosis, as an anti-tussive, asthma, nausea, emesis, gastric ulcers, systemic sclerosis, myelinoclastie disorder, oeuromyelitls optica, centra! nervous system neuropathy, central pontine rnyelinolysis, myelopathy, leukoeneephalopathy, leukodystrophy, peripheral neuropathy, Guillain-Barre syndrome, anti-MAG peripheral neuropathy, Chareot- Marie-Tooth disease, progressive inflammator neuropathy, amyotrophic lateral sclerosis (ALS), and diarrhea.

Thus, in one aspect, the present invention fitrther relates to a method of treating a disease or condition responsive to a modulation of CBj receptor activity in a subject, the method comprising identifying a subject in need thereof and administering to the subject a

therapeutically effective amount of a cnnnabidiol derivati ve or formulation thereof In one aspect, the present invention relates t new drug candidates comprising chemically stable, nonpsychotfopic a inocjumold chemically derived from synthetic or natural eanoabibloi (CBD) through oxidation and aroination. In one embodiment a nom-reactive synthetic cannahidioi derivative has a novel MOΆ by targeting complementar signaling pathways that alleviate nenromflammation and favor neuroproteetion, prevent axonal damage, preserve myelin structure, and potentially promote remyehnution, In one embodiment, the non-reaetive synthetic cannabidiol derivative is a modulator ofCBa receptor signaling. I one embodiment, the nonreactive synthetic cannabidiol derivative is a modulator of PBA&y and€82 receptor signaling. In

4J oue e bodi ent, the nomreaetlve synthetic cannsbidlol derivative is a ct al modulator of FPAfoy and GBe receptor signaling, sad it activates the H!F pathway by stabilMng MIF-i « and upregulates the expression of several associated factor that include Erythropoietin <EPO) an Vascular Endothelial Growth Factor A CVEGPA). I» one embodiment, the non-reactive synthetic cannabidiol derivative reduces neuroinfla matinn presumably by acting on FFARy/CBs receptors, in conjunction with enhanced nenreproteetfon and potential teBsyeiinaiion. through the H! pathway.

In one embodiment, the non-reactive synthetic eannahidsol derivative modulates the activit of a GBs in one e odi ent, the non-reactive synthetic cannabidiol derivative preferentially binds to CBi receptor as compared to CBs. Therefore, in these embodiments, the non-reactive synthetic eanBahkfiol derivat e is selective for C%. In one embodiment, the amine group of BOB -reactive synthetic cannabidiol derivative enhances its binding to th e C B n In one embodiment, the amine group of non-reactive synthetic cannabidiol derivative selectively binds the CBs receptor over the CBi receptor. In one embodiment, the C z receptor activity is modulated in vitro, whereas in other embodiments, the CBa receptor activity is modulated in vivo.

In one embodiment, the cannabidiol derivative or formulation thereof Is administered in combination with another therapeutic agent in one embodiment the cannabidiol derivative or formulation thereof is administered orally. In one embodiment, the earmabidiol derivative or formulation thereof is administered topically. In one embodiment, the cannabidiol derivative or fennnlation thereof is administered using rectal administration. In one embodiment, the cannabidiol deri vative or .formulation thereof is administered using transmncosal

administration. in one embodiment, the cannabidiol derivative or formulation thereof is administered using intestinal administration. In one embodiment, the caunabldio! derivative or formulation thereof is administered using parenteral delivery, In one embodiment, the cannabidiol deri vative or formulation thereof is administered using intramuscular Injection. In one embodiment, the cannabidiol derivative or formulation thereof is administered using subcutaneous injection. In one embodiment, the eannabldkrl derivative or formulation thereof is administered using intravenous injection. In one embodiment the eanuahidiol derivative or formulation thereof Is administered using intramedullary injection, in one embodiment, the cannabidiol derivative or formulation thereof is administered using int iheeal injection. In cam embodiment the cannihidiol derivati ve or formulation thereof is administered using direct intraventricular injection, In one embodiment, the cannabMIoi derivative or fotmnlaiion thereof is administered using iniraperiloneai injection. in one embodiment, the eannabkfiof derivative or formulation thereof is administered using intranasai infection. In one e bodi ent, the cannahidiol derivative or formulation thereof is administered «sing intraocular injection.

In one emhodimenh the canoahklioi derivative or fermnlation thereof is administered whit food or drink.

In one embodiment, the condition or disease responsive to the odulation of the C&2 receptor activity is selected from the group consisting of autoimmune disease*

demy elinating disease, inflammatory-rei ted disorder, and any combination thereof lit one embodiment, the condition or disease responsive to the modulation of the C1¾ receptor activity i selected from the group consisting S&, niyelmoelasiie disorder, analgesia, acute and chronic pain. Inflammatory pain, post-operative pain, neuropathic paitt, muscle relaxation,

immunosuppression, as anti-inflammatory agents, for allergies, glaucoma, bronehodilaiion, neuroprotection, osteoporosis and disorders of the skeletal system, cancer. neumdegenerative disorders including but not limited to Alflieimeds disease, Parkinsmfs disease (FD), and

Huntington's disease, MS, muscle spasticity, tremor, fibromyalgia, lupus, rheumatoid arthritis, myasthenia gravis, other autoimmune disorders, irritable bowel syndrome, interstitial cystitis, migraine, pmrii , eczema, sehhorea, psoriasis, shingles, cerebral ischemia, cerebral apoplexy, craniocerebral trauma, stroke, spinal cord injury, liver cirrhosis, atherosclerosis, as an aniL tussive asthma, nausea, emesis, gastric ulcers, neoro yef ills optica, central nervous syste nenropathy, central pontine myellnoiysls, myelopathy, leukoencephalopaihy, leukodystrophy, peripheral neuropathy, Guiilain-Barre syndrome, anti-MAG peripheral neuropathy, Charcot- Marie-Tooth disease, progressive inflammatory neuropathy, amyotrophic lateral sclerosis (ALS), and any combination thereof,

it will be understood by those of skill In the art drat numerous and variou modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure.

EXPERIMENTAL EXAMPLES The invention Is further described in. detail by reference to die following experimental exam les- These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, bin rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herei ,

Without further description, it Is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention aad practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present inven tion, and are not to be construed as limiting In any way the remainder of the disclosure.

Example 1 : Synthesis of the Compounds

The current manufacturing process of VCB-OOd comprises three steps as shown in Figures I A-1.B and 2, hi short, these steps are:

Step I : CBD Is oxidized by the addition of stabilized d-iodox benzoie acid (SIBX) to a solution of CBD in ethyl acetate (EiOAc) The heterogenic mixture is stirred at elevated temperature and alter completion the mixture is filtered. The filtrate is washed twice with potassium carbonate (K2CO3) solution and once with hydrochloric acid (HQ) solution. Sodium Chloride (NaO) (aq, sat) is added to the last washing to facilitate layer separation. The organic layer Is concentrated to give VCE-004,

Step 2: A peroxide solution in water is added to a solution of VC ^' E-004 in EtOAe. The mix ture is cooled an benzytamlne Is added slowly After completion of tire reaction, aqueous HCI (15%) Is added sod the organic layer is washed several times with water. The organic layer is concentrated, and the product is precipitated from a solution of methanol and water (MeOB/Eb€>), filtered and dried to produce VCE-004.8.

Step 3: VCE-004, 8 is further purified by suspension in MeOE/HiO 85:15 at elevated temperature. The resulting mixture is cooled, and the product Is filtered. The solid is dried and sieve to produce VCE-004 ,8 purified.

The final Drug Substance is sieved, packaged In a double low-density polyethylene bag and Kraft drum, then labelled. ^" arimts pharmaceutical salts were then synthesized is high yields.

Example 2: Substance

VCE-004,8 is a ne w chemical entity described in PCT-EE2014-05776?, The activity of the compound is also described in PCT EP20! 7fo5?3S9. E€T EP2014fo57767 and PCT-EP2# 174157389 are incorporate by re erence herei in their entirety _>

The arulnoqu osd VCE4504 j is a sew chemical entity derived from synthetic cannabidioi (CBD), Characterisation studies showed that VCE4104.8 is an anhydrous and non- sol vated crystalline solid with a molecular weight of 433.6 g/moL The melting point is 90.7 *€, Structural elucidation of VCE-004 J was performed by infrared Spectroscopy (ATR-IR),

Elemental analysis (CBN), High Resolution Electrospray Ionization Mass Spectrometry (ESI - MS), Proton Nuclear Magnetic Resonance (1 H-NMR), Carbon Nuclear Magnetic Resonance (13C-NMR,), other NMR techniques be., Distortionless Enhancement by Polarization Transfer (DEPT135), fleteronuelear Single Quantum Correlation (HSQC), Heteronuelear Multiple Bond Correlation (HMBC) and 2D studies. These structural elucidation studies arc completed, and structure of the molecule has been conformed.

Analytical test methods for release and stability testing of VCE-004.8 Drag Substance were developed for identity, individual and total iniptaides, chromatographic· purit and assay (Tabie 1 ), Potential chiral impurities were also evaluated. Since foe raw material CBD Is highly pure and during synthesis hardly to no enantiomeric form is obtained, the chance of chiral impurity formation dotin Drug Substance manufacturing is considere to be very low. Nevertheless, a chiral method was developed to evaluate the Drug Substance lots.

Table I. Proposed Specifications for VCE-00 Drug Substance.

,

A stress test was performed concluding that VCE-0Q4.S drag substance is stable fbr 3 days at 65 °CTA 5°C m glass vials. Degradation of the product was observed at temperatures above 65“C. A short-term stabilit study at 40 °C was completed ith indecisive residts doe to the early development stage of the impurity method.

A solubilit screening study sh wed that VCS-CKMr8, the active ingredient of EHP-101 Lt¾uid (in predmieal devefopfiieat also known as YC’E-004,8 formulation), is practically insoluble In aqueous solutions at different pH sod hi cyclodextrin complexes (Figures 3 A, 3B, and 4). it is also practically insoluble in co-solvents, such as glycerol, and sparingly soluble !« a eo-solvent like FEG40O. VCE-004.8. is slightly soluble in organic solvents like a-· heptane and methanol to freely soluble In organic solvents like DMSO and DCM, Based on solubility studies, short-term stability studies, and an in vivo bioavai lability study in rats and mice, the composition of E!-EE 01 as hown in Table 2 was selected l r the oral formulation

Table 2, Composition of Drug Product EBF-101 Liquid In the as &ctMiing flow chart of Figure 5 A thecinTefo manufacturing process for he GMP DP batch (20 mg/g) is described based on ike experience to date for the form illation EEP-IOI Liquid and Placeho/Ychicle The process comprises the following steps:

1. Mixing Maisine CC and. Ma e Oil in a ratio of 50:50 v/v

2, Solubilize VCE-004.8 in the Maisine€€;Maize Oil mixture

3. Filling the DP m bulk containers with N2 blanketing.

The bulk mixture of Maisine CC and Maize Oil (50:50 v/v) vehicle will be used for Placebo i the clinical studies (Figure SB).

Analytical test methods developed and used for tbe control of LftlMOl Liquid and Placebo are summarized in Table 3 In the course of development, analytical test methods will continue to be optimized and revised.

Table 3. Tentative Release Specifications for Bulk EίIRMqI and Balk: Placebo

Data of a (Lmontk stability study are available, in which formulations were lad acted at three different concentrations, he . 20 mg/g, 25 mg/g and 30 mg/g. The oil formulation composition is identical to the selected composi tion of the formulation to be used In the clinical studies, Therefore, these formulations are representative of the formulation to be used in the ciimeai studies. The stability study was conducted at the following conditions: 5 °C A 3 °C, 25 °C 2 € m¾ RH ± 5% RH and 40 °( 2 ^a C / 75% R.H ± 5% i

The results of this study showed that the product is chemically stable for at least 6 months at 5 *C, 25 *€00% RTf in amber glass bottles,, without nitrogen blanketing.

Example 4: Formulations for Phase Studies

Different concentrations of Drug Substance were tested in this lipid formulation he,, 20 mg/g, 25 mg/g and 30 mg/g. Because the concentration of 20 mg/g remains solubilized at room temperature without additional heating or swirling, this concentration was selected to he used in the clinical studies.

EBP- 101 Liquid and Placebo ar filled, stored and shipped in bulk bottles.

The liquid formulation, EHP-101 Liquid, disclosed in this invention , consists of a 20 mg/g solution of VCE-004 8 in a mixture of maize oil / MaSsine€€ (50/50 v/v), A similar formulation (up to a concentration of 30 mg/g) has been used for in vivo uonclinicai studies. The selecti on of the liquid oily formulation was based on the solubilization efficiency of VCB-004 and in vivo screening studies of the hioavai lability of >20 formulation prototypes,

Manufacturing of single dose formulations will be prepared by diluting the bulk EHP-lOl with the bulk vehicle. Matching placebos will be prepared b addition of a colorant to the bulk placebo. Analytical methods will be transferred in order to release the single dose formulations and matching placebo and to conduct stability studies on these formulations,

Solubility Screening and Manufacturing of Formula ion Concepts

in order to select the best formulation of V ^: €B-004.8 for oral administration (EHP-101), two main parameters were considered; solubility and oral bioavail ability. The solubility of a compound Is an important factor in determining its absorption front the gastroimesdnal tract and ultimately its oral Osvaiia lity. First i was determined the solubility of YQ 004 8 in a collection of different solvents (e g., aqueous, lipld , organic, etc.). Additionally, a test of stability of YCE-004.8 in selected solvents was also used as a criterion for selection of the best sol vents. Based on solubility studies in lipidic solvents, VCE-404J was shown to be more soluble in a mixture of Maisine CC : make oil than in indi vidual corn oil or Maisiae CC alone (Table 4 and Figure 6),

Table 4. Solubility studies solvents at 25 ¾ and 3? "€.

Selected solvents were used to manufacture several fbrmulatkm concepts of VCE- 004,3 which pharmacokinetic (PfC) profile y oral intake was assessed

Bioavailahilify is One of the principal PKL properties of drugs. It is used to describe the fraction of an ad inistered dose of unchanged drug that reaches the systemic circulation. The measurement of the amount of the drug in the plasma at periodic time Intervals indirectly indicates the rate and extent at which the active pharmaceutical ingredient is absorbed from th drug product and becomes available at the site of action,

Example 5: Turbidtmetrie Aqueous Solubility

An aqueous solubility assessment fo VC -404.8 was performed: at physiological temperature. VCE-004 ,8 (dissolved at 10 mM in DMSO) was mixed with PBS buffer pH 7 4 at 37 °C to achieve a final YCB-004,3 concentration of I mM and a final DMSO concentration of 0,33 % v/v. Incubations were performed In PTFE (Teflon*)· A parallel ineuhatiou was also performed in a polypropylene plate to assess any differences in non-specific binding between PTFE and polypropylene. For the incubations In PTFE; serial samples were then taken over a 2 hr period at 5, I S, 30, 45 and 120 min. For the incubation In polypropylene, samples were removed at 0 min and 120 min only. All samples were added immediately to two volumes of methanol in a mierother plate cooled In dry-ice to halt chemical degradation. When sampling was complete, the sampling plate was allowed to reach room temperature. Samples wet® then removed for § uaniitatlve an lysis of parent com pound by LOMS/ SL An internal standard was included to correct for analytical variation (nicardipine and pyrene). The percentage of parent compound remaining a t each time po int relative to the O min sam ple and the percentage of parentcompound bound to polypropylene compared to PTFE was then calculated from LC-MS/ S peak area ratios (compound peak srea/mternai standard peak area). The percent of parent compound present at 0, 5, 15, 30, 45 and 1213 min alter initiating incubations at 37 ¾ w¾s reported for the PTFE incubations. In addition, the percentage of test compound bound to polypropylene compared to PTEE was calculated. Estimated solubility range (lower and upper bound and calculated mid-mage in mM) are shown in Table 5, indicating a low aqueous solubility of VCE-004.8 ,

Table 5, Estimate Preciphatfoo Range (pM) of YCE-004 J in Aqueous Solubility test at 37 T2 compared to nicardipine and pyrene.

Example 6: Log D Determination

LipophiUcity is a key determinant of the PK behavior of drugs. It can influence distribution into tissues, absorption and die binding characteristics of a drug, as well as being an important factor in determining the solubility of a compound. Log D (distribution co-efficient:) is used as a measure of llpophilieity. Determining the partition of a compound be tween an organic sol vent ( typically oetauol) and aqueous buffer is one of the most common methods for determining this parameter.

To determine log D, tU M phosphate buffer pH 7,4 (saturated with octane!) was added to the vial containing VCE-004J and the solution mixed and sonicated lor approximatel 15 tnin. The solutio was transferred to tubes, centrifuged and the supernatant is drawn oft ^' the top, leaving any solid compound in the botom. This supernatant was then syringe filtered through 0,2 pm filters to produce the initial solution. Three vials were prepared containing different ratios of octanol and compound in phosphate buffer in order to cover a range of log D values KMocom le and caasdMdiol (CBD) were used as control. The vials were -di ed to equilibrium, then centrifuged to ensure the two phases were fully separated foeibre the octanol was removed and the buffer samples analyzed. For the quantitative analysts, the aqueous solutions were analyzed by LC MS/MS, The amount of VCE-004 in each vial was quantified against a 6 points standard curve which was produced by serially diluting the initial solution. Log B was calculated using the equation, shown in Figure 7, wherein ConelMTlAL is a

concentration of compound in the initial aqueous solution, ConeF AI, is a concentration of compound in final aqueous phase, Vaq is a volume of aqueous phase, and Voet is a volume of octanol phase.

Results showed in Table 6 indicate that VCB-004 J is a highly lipophilic compound, in the same range than the parent molecule cannahidio! (CBD).

Table 6. LogX>7,4 Octanol of VCE-OCN· .8 compared to CBD ami ketoconazoie.

^: Example 7: Solubility Screening

A quantitative thermodynamic solubility determination on VGL-004,S was performed. Suspensions of VCB-004.S were prepared in diSsrent pharmaceutical vehicles and organ ic sol vents. T he organic solvents, llpkl and co-solvent vehic les consisted o f pure solvent or lipid, while the cyckxiextnn solutions were prepared in phosphate buffer pH 7 0. After stirring the suspensions tor 24 hr at 25 °C, a small aliquot of the mother liquor wa taken from the suspensions for a solubility determination. The concentration of VCE 004.8 in solution was determined by HFLC analysis. The results of this solubility determination are presented in Table 7 Table 7 Solubility results for VCB-004, 8. ((I) As defined in Fit, Bur,: 1) Fraetieaily insoluble: solubility <0.1 mg/mLi 2} Very slightly soluble solubility between 0, 14 nig/ lgA) Slightly soluble: solubility between 1-10 rng/tnL; 4) Sparingly soluble: solubility between i(F33tag/nsL;

5 ! 5) Soluble: solubility between 33-100 tsg/ia ; 6| Freely soluble: solubility between MKMOO & Lt 7) Ver soluble:: solubility >1000 mg/n L; and (2) The solubility of Maisine 35-1 slid Capraul y CM was determined at 37 ^S C)

These results confirm that YCB-004,8 exhibits a ver low, pH independent, solubility is aqueous buffers, however, in all lipid vehicles and in most of the co-solvents the compound was found to be sparingly soluble. Moreover, the cyclodextrio solubility results 5 indicate that for roeihybflwyelodextrin eomplexatiop with VCE-004.8 cueists, however, the use of cyelodextrius does not significantly improve solubility.

Example 8; Ad itional Assessment of Solubility and Stability in Lipidfe Solvents

Ap addi tional test of solubility IP iipldic solven s was perS¾¾ped. Accordingly, Id: YCEA>04j was dissolved at room temperature an stirre during a maximu of 16 hr IP 6 different tipkic solvents as depleted in Table 7. Assay of the different solubility trials Wa performed by F1PLC using the following parameters: column CI 50724 G0047; IDnetex, CIS; 150 mm, 4,6 m, 2 6 gM; isocratlc acetonitrile; 0 2% formic acid (90:10); flow 0.35 mt/m wavelength 314 urn; column temperature 23‘T¾ run time 20 min injection volume 10 pL.

15 Conceiitnuiae 0.1 mg/fnl, was considered the theoretical 100% of the technique, Results ate shown in Table 8.

Table , First assessment of VCE-004 8 solubility is lipldle solvents by F1BLC (o.d. not determined).

Based on Assay and Impurities percentages, concepts P03, PCM and P05 were selected tor preliminary stability studies. VCE-004 a also found to fee s luble at foe concentration of 2 g/niL in R07 _» RQ8, raid P09. None of the solutions presented any precipitate or visible solid particles. The stability studie conditions and Assay results are sbown 1» Table 9, indicating that P03 was tSie best formulation base on bofo solubility an stability for 31 days. Consequently, ffo!ilsolv ^® CT 70 -Medium chain triglycerides (also known as MigiyoP 812 or Myntofo 3lS} was selected to assess VCE-004.8 PK profile by oral administration in rats. A formulation of 10 rng/mL of VCB-004,8 was prepared for the PK analysis (FofMid&iiop rfl)

Tabic 9. Stability studies of VCEfoOd 8 formulated iu KolMv (P04) _f Labrasol (MS) and Labralil (F05) at 0.4 mg/iui (n.d. uei determined).

Based on results showed In Table 7, tea different prototype lipid fonmilaiion concepts were developed. The composition of the lipid vehicles was chosen as snob to ensnr that all classes from the lipid classification system are represented, Preparation was done as follows, 75 g YCE~004,8 was weighed into a statable container to which 6.75 g of excipient was added while stirring. If necessary, the excipient was heated to 45 "C in order t become liqui » Table 10 gives an overview of the ten different lipid formulation concepts that were developed.

For each developed concept, a sample was stored at 5 *C and 25 °C/60% RH for 4 weeks. Afterwards, stability was assessed by ffPLC (Table 1 1). All concepts, except concept 8 arid 10, showed an acceptable ass at Time Ci (TO). After 4 weeks of storage (T4W) at 25 £/60% foil, concepts 2* 3, 5, 6, 7 and 9 show a sig fieani decrease in assay (5-10%)

Table 1 1 , Stability results for assay {% label claim) of VCB~004 1» different li idic formulations ((1) TO is » approximately 2.5 weeks after preparation, stored at 5 *C; and (2) Not tested at T4W, as already ling at TO).

For ldT supplies, lipid formulation concepts I, 3, 4 and 6 were selected for PK assessment (Formulations to 2, 3, 4, 5 respectively). Formulation to 2 and 3 were freshly prepared as follo ws, 350 .mg VCE-004,8 was weighed info a suitable container to which 34.65 g S of excipient was added while stirring to obtain a concentration of 10 mg/g, if necessary, the excipient was heated to 45 ^¾ G in order to become liquid, Condeattotion was adjusted: from 10 nig/g t 4 mg/g Therefore, three vials of each Formulations to 2 and 3 wet¾ pooled by magnetic stirring, after which each formulation was diluted 2,5 times with the respective excipient mixture. If necessary, the excipients were heated to 45 *€ to order to become Ikfuid On th other:0 hand, Formulation to 4 and 5 were freshly prepared as follows, 140 mg VCE-004.8 was eighed into a suitable eonrafoer to which 34,86 g of excipient was added while stirring to obtai a concentration of 4 mg/g.

Example Kf; S ame Oil

5 the newly approved drug Safivmto, CBO has been for ulated at a

concentration of 100 mg/mL to an oral solution that inefedes dehydrated alcohol, sesame seed oil, strawberry flavor and sucralose. Since GBD is the parent molecule of VCE-004.8, sesame oil was selected to evaluate the PK profile of VCE-004.8 when orally administrated t rats A formulation of 4 rng/toL of VCB-004 J to Sesame Oil (Formulation n ^¾ 6), and another with 40 mg/mL of VCH-004 J with Sesame Oil (97.5%)~Etbanoi (2,5%) {Formulation to ) were

prepared for the PK analysis in rats.

Example 1 1 : INDENT PE YTOSOMBS

Plrytdsonvto is a patented technology developed b Indena Spa (Italy), a leading5 manu&ctoter of drug and nntraeetiiieals. Phytosomes are little cell-like structures that contain the active ingredients bound to phospholipids, mainly phosphaiidylehoime, The phospholipid molecular structure includes a water-soluble head and two firt-solub!e tails. Because of this dual solubi lity, the phospholipids act as an effective emulsifier which produces a lipi compatible molecular complex. This phytosdme technology is a breakthrough model for marked enhancement of hioavaiiabihty, significantly greater cllnkal benefit: assured delivery o the tissues* and withou eo protnis g nutrient safety,

A laboratory process for the preparation of VCErfK)4.8 phytosorne starting form VCE-004 8 was developed, First a solvent screening was performed, selecting ethyl acetate for comparison with ethanol ethanol acetone an dichloromethane and ethyl acetate. Two Fhytosome- VC ^' B~O04 prototypes were prepared.;

I) Fhytosome I 2 ratio, wherein E riphur/S (2 g), VCF-004 (I g) and altodexirin MD05 (0,92 g) were suspended in 40 ml of ethyl acetate. The suspension was reSuxed with stirring for 1 hr, The solvent was removed under reduced pressure (300-400 mhar, external hath at 60 °C} until a soft mass was obtained, The soft residue was dried under vacuum at SO *€ for 16 hr. To the dried solid 2% W/W of Sy!oid 244 PP was added. The solid was coarsely ground and sieved at. 600 pm to yield VCE-004.8 phospho!ipid/SF. Tire weight yield vs. sum of starting powder was about 98% W/W

2} Phyt ome 1 :! ratio, wherein Emulphur SP (1 g), VCE~004.S (1 g) and Malfodexirin MD05 (1.92 g) were suspended In 40 mFof ethyl acetate, The suspension was refluxed with stirring lor ! iu\ The solvent was removed under reduced pressure (300-400 mhar, external bath at 60 °C) until a soft mass was obtained. The soft residue was dried under vacuum at 50 ^* C for 16 hr. To the dried solid 2% W/W of Syloid 244 FP was added. The solid wascoarsely ground and sieved at 600 pm to yield VCE-004.8 pbospliolipid/SFl The weight yield vs. sum of sfariing powders was about 08% W/W,

A preli inary investigation of the compound stability gave indication that the active pr inciple is stable in the process conditions, although an imparity peak not detected in the starting material and almost neglectable at 45 min, increased aft r 6 hr ( 1.7% in area %) au grew after 24 hr (6.2% in area %% as shown in Figure 8.

The solubility of Phytosooie~V€E-004.8 was tested in buffer medium at various pH ( 1.2, 4 5, 6.8 and 8.0}. For each pH, independent supersaturated solutions of VCE-004.8 aud its phytosornes were prepared. The suspensions were sonicate few 10 min and kept in a water bath at 37 C for 2 hr. Then the final suspensions were filtered (with 0.45 PTEB disposable filter) and the solutions were injected for HPLC analysis, The results, shown in Table 12 and Figure % indicated that the hydrophil icily (expressed as aqueous solubility) of VCE-004.8 is practically nil. However, the phytosomieation process Increase significantly foe solubility of foe compound. The 1:2 ratio resulted to he slightly more soluble than the 1 : 1 ration, and the best behavior wa shown at neutral and basic plT Accordingly, Phytoxome 1 2 was selected for following assessment of PK profile its rats (Formulation xf B) Phytosome ϊ :2 contained 24% of V€ ^: E~O04.8 and was prepared in Methyl cellulose 1% in water for oral administration.

Table 12, Concentration of VCIIT004,8 found in the aqueous solutions of VCE-004.8, Fhytosom 1: 1 and Fhytosome 1:2 at the considered pH.

Exam le 12: Echo Pharmaceuticals

Alitra* i a drug delivery technology patented by Beho Pharmaceuticals BY.

Alittafo is an etnulsi! ing technolog that was soeeessfidly developed and used by Echo to Improve release ofcannabmoids in aqueous solutions.

For the formulation of VCE-004 8, ECP0I2A was used, a mixture of excipients designed for oral use as base formulation. This renders a dry powder formulation of VCE-004,8 that was tab!ete to assess its consistency. _: For further investigational purposes, the foree final

YCE-004 8 formulations were delivered as powder,

YCE4104.8 ECP01 A tablets ere prepared through two manufacturing steps bo the active Ingredient UϋE-004,8: a granulation step and a tablet preparation step. The first step was preparation of the intermediate product (IP): a granuiathig fluid containing excipients in ethanol was added to primary powder particles followed by solvent evaporation, The particle size of the resulting material was reduced by milling. This yielded the IF, a granulate ready for tabietlng. The second manufacturing step was preparation of the Drug Product (DP), The IF was blended with excipients an tablets were compressed by direct compression on a tablet press. Three different formulations were prepared as described in Table 13.

Table 13 , Main features of foree formulations of VCE-004V8 using Alitrai; technology.

VCE0O4,S cohfeti of the DP as theasnrad by ffPLC analysis in duplicate,

Dionex Ultimate 3 CKK1 system operating under f hromeleon soil ware. The BPLC method used Is based on the United Slates Pharmacopoeia (USF) method for Dronabinol (de!ta-9- ietrahYdrocatuabmo!, THG) and was developed for measuring of 03:0 and otter cannabhioids.

lie dissolution test was used to indirectly determine the hioavailaMlity of the API and to measure possible differences i bioa vad ability of the API in the different formulations. Dissolution was measured according to section 2.9,3 of the British Pharmacopoeia (BP). li selected dissolution medium coasisied of 2% SDS in water, pH 7. A beaker was placed on a controlled beating mantle with stirring and a temperature between 35 °C an 40 ^¾ C, Once the temperature of the dissolution medium reached 37 {>*0) the experiment was started by dropping one tablet into the dissolutio beaker with: a stainless-steel screen to create a physicalbar ier between the tablets an fee stirrer bar. Samples wer taken at various time points wife a disposable syringe and -were transferred to a vial lor HPLC analysis. The dissolution is expresse as a percentage of the active substance that is dissolved in a specified time frame. Samples were taken at various time points: trif 5, 10, 15, 30, 60, 90 and 120 min, The results of the tests for the three formulations are shown in Figure .10.

Result of the for ulation test showed that f orainlatian A has the highestdissolution: rate (readied 42%) followed by Formulation B and C, The order of dissolution rate is in tine with expected effects of the API ratio to emulsifier: higher emulsifier to API ratio, beter solubility. Although Formulation A showed better dissolution rate, fee three formulations A, B and C were selected for assessing the PK. profile in rats (Fortnnladons t 9 _S 10, 11 respectively). Far the pre aration of those fortmdaiions,, it was taken in to account that fornmiaifon n% contained 13 13% of VCE-004,8, Fortnui&liati i 10 contained 13.75% of VCE- 004.8 and Fornailatkitrif 1 1 contained 12.93% of VCF3KI4.8 It was prepared a 15 mg/ud suspension in water.

Ten diftsrent prototypes aqueous nanosaspension concepts were prepared as follows; 250 g VCE -004,8 was weighed info a suitable container, to which 4,750 g of stabilizer solution was added. Bach concept was stirred using a magnetic stirring bar until a homogenous suspension was formed. Next, to each container, 30 g heads (2YP size I mm) were added, after which the container was sealed and placed on a roller mill at 80 rpm. After 2 days and 5 days, the particle size distribution (PSD) of each concept was measured by laser diffraction. A fter 5 days of milling, all concepts were harvested and diluted to 10 rng/g, ensuring sufficient rinsing of the milling containers and heads. AM ten. concepts were placed on 25 *0/6(1% RH stability conditions for 2 weeks, after which PSD was again evaluated. Results are shown in Table 14. From these results it is concluded that concept 2:. containing 1% Pharmaeoai 603 w 0,1% SLS as stabilizer and concept 4. with 1 % HPC-SSL ÷ 0,1 % SLS, are to be con idered for PK. testing, since for these formulation concepts, the obtained i 0~d50~d:9(l particle size results are all « 1 pm. Table 14. Different nanosuspeusfon concept with PSD result*.

f%nQ8uspe»siiDm coacejji and concept 4 were selected for PK s pplies

(Permoiations n 12 and 13 respectively) and therefore fteshly pepare as follows; 500 mg YCE-CKM.S was weighed into a suitable container, to which 9 J g of respective stabilizer was added. Each concept was stirred «sing a magnetic stirring bar anti! a homogenous suspension

5 was formed. Next, to each container, 30 g beads (ZYP size I m) were added, after which the container was sealed and placed o a roller mill at SO rpni. After 24 hr and 45 hr, the particle size distribution (PSD) of each eoucept was measured by laser diffraction, After 45 hr of milling,, ail concepts were harvested and diluted to I ft mg/g, ensuring sufficient rinsing of the milling containers. Dose was adjusted from 10 rag/g to 4 mg/g. Therefore, three vials of each concept ift were pooled by magnetic stirring, after which each formulation was diluted 2.5 times with the respective stabilizer.

Example 19: Solid Dispersions

The development of solid dispersion formation started wit the selection of

1 polymers for stabilization of amorphous APT Therefore, multiple polymers were screened using the solvent shift method (Table 15).

Table I S, List of polymers used for the solvent shift in SI and SGF based on the solubility of polymers in these solutions, marks polymers that" were dissolved and therefore, the solvent ft shift experiments wem performed; and "X/ ^' mark polymers that were not soluble and thus the solvent shift experiments could not he performed.

l!iese experintfnts applied to a 5 mg/mt solution of YCB-0043 in DMSO, of which: 80 pL was adde to 4 polymer soialicms prepared in simulated intestinal fluid (SIF) an simulated gastric fluid (SGF l Subsequently the samples were incubated at 25 °C under continuous stirring, and after (K5 _< 1 , 2 and 4 hr, an aliquot was taken, filtered and analysed by HPl,C to determine the VCE-004 concentration in solution. Results are presented in Figure 1 1 (SGF) and Figure 12 (SIF)

In SGF, most polymers were not able to maintain a sustaine supersaturated state, except for the TOPS solution, in which alter 4 hr a concentration of about 0.03 mg/mL VCE- 004.8 could he measured. However, the experiments performed in SIF showed several polymers with promising anti-precipitant properties. In general, all the HPMC detivates (except lor HPMC as Is) exhibit high API concentrations (approximatel 60 gg/mL) from 03 to I hr. Moreover, Eudragit L1 O0 and PVP O also maintain supersaturaiJan for at least 1 hr (approximately 60 pg/rnL) , Therefore, these polymers are to be considered in the preparation of amorphous solid dispersions.

The principle behind a successful amorphous dispersion is to prepare a homogenous dispersion of the API in a polymer matrix, such: that the mobility of the API molecules is reduced and nucleaiion is prevented. Drug loading is an important parameter and high drug loads ma result to crystallization of the API, whereas low drag loads could affect the drug product size.

The amorphous solid dispersion screening (AS D) is performed with different drag loads of 10, 25 and 50% Based on the polymer-API interaction observed by tire solvent shift method. MPMCAAS-MCL Eudraglt LI ftft, HFMC~A$ HG and FVP K30 were selected lor further investigation, The homogenous dispersiaus are prepared y ireeze-dfying and placet! on 40 “070% RIT stability conditions. At preparation (TO) and after! days (T2D) and 14 day (T 14 ), samples are analyze by HT-XRPD. Resalts are shown in Figure 13.

ft is concluded that th HPMC-AS-HG dispersion is able to stabilize 10% and

25% drag load for at least 14 days at 40 °C/?5% RR. Eudraglt I.-100 can only stabiliz 10% drug load for 2 days. HFMG-AS-MG: and FVP f 0 do not show stabilization during stability study.

Based on both th solvent-shift results and the amorphous solid dispersion stability screening, the two best performing polymers are HPMC AS HG and Eudragit LI 00 (Foitau liot nf 14). However, as little (or no) release in the stomach is desired, but rapid release In the proximal small intestine is targeted, it was chosen to use HPMC AS LG (Formulation fo 15} Instead of HPMC AS HG. as the latter only dissolves at a rather high pH value of 6,8, while the LG grade already dissolves at pH 5.5,

The two solid dispersions selected were prepared by spray drying on ProSe T

4MJ-TriX equipment. Prior to manufacturing, the optimal spray drying conditions were first determined by spray drying of placebo material (he., without VCE-O04.S), The final settings use for each polymer are summarized in Table 16. After finalization of the spra drying process, the solid dispersion material was dried In a vacuum oven at 25 °C and 20 mbar for 16 hr. As dispersion medium, ft.5% Methoeel E4M + 0.2% Tween 2ft, was prepared.

Table 16. Spray drying conditions for the ProCepT 4M8~Trtx spray dryer module ((.1) As this is a dynamic process, which i constantly being monitored, a dynamic range is gi ven).

n Rats

The PK study s getibtmed in mate Sprague Dawley rals and male Balb fC5?BL/6jRj) mice around 6 weeks old supplied by Janvier Labs, There was entirely artificial lighting the room with a controlled cycle of 12 h light, 12 h dark, It was air conditioned by a system: designed to maintain normal conditions. Each animal was identified by an ear tag.

Animals were examined for general health and elfare before the in visa) test. All animals had free access to food and water during the experiment (ad libitum). Standard process, treatment and euthanasia was conducted. Several iimepoints per formulation were selected (typically 5 min, 20 min, 30 min, 1 h, 3 h, 4 h, 8 b, 24 h for iv; and 30 min, 1 ip 2 It, 4 h, 6 h, 8 h, 10 b, 18 h, 24 ft for oral administration), Usually, at least 3 animals per timepoint were used.

The test immolations were store at 4 ^a C in the dark until the in vivo test was performed (usually in the following 4-6 days after the manufacturing). Formulation containing Maisine 35-1 was warmed to 37 °C ~ 40 °C in a water bath and stirred (magnetic stirring), protected from light, before administration. Formulations were orally ad inistrated to animals and compared with intravenous administration oiVCE-004,8 dissolved 2 g sE hvDMSO and administrated at a dose of 2-10 mg/Kg in a volume of I mL/kg. I mice, selected dose fo oral administration was 20 mg/Kg in a S g/kg volume of administration in rats, selected dose was 20- 50 mg/kg.

For the blood sampling, at prescribed times, blood was collected in the sinus retro-orbital using a capillary tube. Approximately 0.5 ml. per time-point were collected, It was used lithium heparin as anticoagulant Exact sampling times were noted for each blood sampling, Blood samples were centrifuged at 2500 rpm at around 10 °C, the plasma then removed and placed into labelled polypropylene tubes. Individual plasma samples were stored frozen ( -20 °C until analysis.

The analysis of plasma samples, 100 pL of the plasma sample were taken and 300 pE o f acetonitrile were added. After protein precipitation, analysis was performed using EC~ S/MS, For the analytical phase, the substance VCE-004.8 was dissolved at 1 mg/mL wit appropriate solvent DM SO, For the Analytical test, the molecular and daughter Ions were selected for the molecule after direct Infusion into the MS-MS system. The analytical method consisted of a precipitation of the proteins by addition of acetonitrile followed by a LC -MS/MS analysis with CIS column. According to the expected sensitivity., at least 8 calibration standards were used for the preparation of the calibration curve in plasma, The corresponding correlation coefficient w¾s calculated an had to be higher than 0,75 to continue tvith the in vivo test. Th calibratio range to be tested was i to 2000 ng/ L of plasma

Istln tion f PK parameters was performed using inefiea (Version 4 J - Thermo Electron Corporation - Philadelphia - USA) The following parameters were estimated: maximal plasm concentration (Cmax (pg/mL)}, first time ά reach Cmax: (Tmax (h)), area tinder the plasma concentration-time curve fro administration np to the last quantifiable concentration at time t (AfJCt (ngfrafAh)), and absolute

in rats, PE. parameters obtained with the selecte formulations and shown in Table 17 showed that the formulation of VCE-004J with Com oil and Maisme 35-!

(0,4:49.8:49.8} led to the best bioavailability results. This bioavaiiability was confirmed in mice as shown in: Table 18, A similar formulation (with. Maisine CC instead of Maisine 35-1} was selected tor Phase I clinical studies _* and named EfiP-101 Liqui formulation.

Table 17, Phatameol neiic parameters of several formulations of VCE-904.8 orally

administrated an compared to intravenous administration in rats (Note: This table shows a seieetkni of file results obtained for formulations 2 an 7. Co plete results ate shown in (8)), I .

13 S1..S: water i 87.08 I 9.69 I 0 5 j 574.05 I 20.3 j 2 1774

Example 16: Nonelinicai Experience

On the basis of several in vitro biological assays, K was preclioieail conclude dial EHP-101 is: an activator of RRAIΐg signaling; a foneiional ligand agonist for the CBr receptor; and, a nonreactive aininoejumoid that modulates activation of the HIE pathway.

Furthermore, a receptor screening study demonstrated YCE-004.8 specificity; there was no detectable affinity for the CB1 receptor, further supporting the lack of psychotropic effects.

Thus, primary pharmacology studies were conducted to demonstrate the activity of EHP-101 In the treatment of MB using two standard multiple sclerosis (MS) murine models:

i) Experimental Autoimmune Encephalomyelitis (EAE) mode! that mimics human relapsing-remitting MS (R.E. S1; and

2) Theder Murine Encephalomyelitis Virus-induced demye li iting disease model

(TMEV) that mimics progressive forms of MS. EEOVI Ol has demonstrated durable activity in these 2 models whe it was administered both oitraperitooealiy and orally. Primary harmacology studies were also conducted to demonstrate the activity of EfD Ol. in the ireatoien of systemk sclerosis (SSc) using a tnarine model of dermal fibrosis induced by bleomycin. SSc is a chronic tmiidorgan autoiin uae disease of unknown etiology characterized fey v scular and immunological abnormalities. Several lines of evidence have

5 shown that the endocannabinoid system may play a role in die pathophysiology of SSc

Considering that the dital F A y/CB? agonists together with activation of the H1F pathway _* have a strong potential as disease-modifying agents in SSc. BHP-101 was investigated for its activity in those targets.

For assessing Drug Metabolism and fihar aeokineties {idM!fivl and safety of ίq EHP-101 Liquid studies have been performed according to the International Co ned on;

Harmonisation (ICH) M3 guideline, encompassing in vitro and in vivo safety pharmacology studies (cardiovascular, respiratory _* and CMS), in vitro metabolism, plasma protein binding _* in vitro and in vivo geootOMeity studies _* and general repeated-dose toxicity studies in rodent and nonrodent species up to a 28-day duration.

15 The EAE model demonstrated the preetinkal efficacy of VCE-004, 8 showing a highly significant therapeutic effect at doses of 5 mg/kg, ,10 mg kg, and 20 mg/feg VCE-004.8 also significantly reduced microglial reactivit and infiltration of inflammatory cells while preserving; myelin sirpeture in the BAB animals, VCE-004,8 attenuated the clinfcai severity and neuropatholog in TMEV model of MS _* as measured by the aefimeter test The treatment with 0 VCE-004,8 ameliorated the motor deficits in mice infected with; Theiler s virus V€ ^’ E-004

significantly reduced microglial .reactivity att infiltration of infiatumatory cells and preserves myelin structure in TMEV-iufeeted mice VCE-004 M treatment also reduce the number of infiltrated 0)4 ^" T ceils an immune cells in the spina! cord of TMEV mice. An intense dem eiinatfen, which was found in the spinal cord of TMEV mice, was significantly reduced by 5 the treatment with V€B-CK}4.8, It was found that axonal disorganization in TMEV mice was prevohted by the treatment with VCE-004,8

Studies were also conducted to show that the acti ity of EBE-101 is consistent with dual FPARy/Ofc ligand agonist that prevents microglia activation, axonal degeneration, and demyelmation in vivo. Additionally, in vitro studies» performed with EHP-101 demonstrated 0 that the molecule stabilizes the expression of H!F- 1 « and HIP-2o proteins in microglia _*

oligodendrocytes, and endothelial mferovasew!af cell lines. HIE- let stabilization induced the release of erythropoietin (BPO) and vascular endothelial growth factor fVEGF) A, which areknown to fee netrreproieetlve and have the potential for reffiyeiioation,.

EMP-101 capacity to prevent fibrosis related to SSe and recover the vascular morphology was evaluated In the experimental model of SSe. V€E~004.8, the active principle substance of BHP-10L inhibited TGFP-taduced CoflA2 gene transcription and collagen synthesis in vitro. Moreover, YCB-004.8 inhibited TCPP-tnedlated myofibroblast diifematiatkm and impaired wound-healing activity, EffP-iO 1 reduced dermal thickness* Wood vessels collagen accumulation and prevented mast eeli degranulation and macrophage infiltration in the skin. EBP-101 also prevented the reduced expression of vascular CD31 typical of skin fibrosis. In ad tion, EMAseq analysis of skin biopsies showed a clear effect of EHR-Ί0Ί in the

inflammatory and cpitheiiaf-meseuchyrnal transition transcriptomfe signatures, qualifying EHP- 101 as a candidate for the management of SSe. Effects and Abuse Potential

EHP-10I (ie., VCE-004,8} does not bind and activate the CB1 receptor and therefore does not induce psychotropic effects, including sedation an catalepsy. There are no specific abuse-related studies at this time. Abuse-related AEs are AEs of special interest (AESls) for this study and will be monitored for occurrence throughout the study (Section 1(14.1.1),

Several studies were performed in which it was shown that VCE-0O4.8 did not have an affinity for the eannabinoM CBI receptor. It was shown in a screening stu y that the compound did not show affinity for the CBI receptor at a concentration of 10 pM (4336 ag/ L). Considering the high plasma protein binding of VCE-004 (>99%) and conservative free fraction estimate of 1% in plasma, YCE-0G4.8 is highl unlikely to yield any clinicall relevant CBi receptor affinity in vivo at total (unbound > bound) plasma concentration of at least up to 1 ®M (433600 ug/mL). Tins plasma concentration is approximately SfKfold higher than the C values observed at no observed adverse effect level (NOAEL) in rats and in dogs after 4 weeks of treatment. Therefore, no clinically relevant effect on the CB ! receptor Is anticipated in the clinical situation. Moreover, the only intermediate in the synthesis is VCE-004 (als calle HU331 ), which has not been reported to hin to CB 1 or to induce psychoaetive effects in mice _*

Example 17: EHP-101 Therapeutics The therapeutic potential of IBP- 101 in experimental models of MS . EHE-lbl wax shown to reduce neuroin fla matioo by acting on PPARy/CB;? receptors while also providing nenroproteetkm arid potentially inducing te~mydination through the BIT pathway, BMP- 101 treatment reduced both incidence and severity of clinical manifestations of the disease in experimental models of MS, Taken together these data indicate that EBP- 101 may provide clinical benefit to MS patients try potentially being disease-modifying.

In addition, the therapeutic potential of BliP-ϊ til CVGE-004,8) in SSe was also shown, providing evidence of the efficacy to alleviate skin inflammation„ vascular damage and dermal fibrosis in the bleomycin murine model

Example 18: Effects of ERB-I OI on Inflammation au Remvelination in Murine Models of MS

MS is characterized by a combination of inilaminatory and aeorodegenerative processes that are dominant in different stages of the disease. Tints, immanosuppression is the gold standard for addressing the inflammatory stage and novel remyeimation therapies are being umfed to restore lost fenefton. VCE-004-8 Is a mu!tlfargoted synthetic cannabinoid derivative acting as a dual PPAI¾y/GB2 ligand agonis that also activates the RIF pathway. VCE~0O4,8 wa shown to prevent neuroinilammation in two different models of MS (EAE and Theiler’s murine encephalitis virus-induced demyelmating disease). Oral EHP4CH (a lipidic femtulatlou ofVCE- 004.8) showed a dose-dependent efficac profile with prevention of neuroinpanimation In the EAE model (Figure 14).

In EAE, transc ipto le analysis by EMA-Seq and qPCR demonstrated that EHP- 101 prevente the expression of a large number of genes closely associated with MS

pathophysiology in the spinal cord. In addition, ERP-jO! normalized the expression of several genes associated with oligodendrocyte function, such as Teneurln 4 (Tenm4) that was downregulated in EAE. Immunohixtochemixtfy analysis confirme the recovery of Tenm.4 expression in the spinal cord Confocal analysis revealed that ERF- 101 treatment prevented microglia activation f!ba! staining), and demydination (MBP staining) in both the spinal cor and the brain. Moreover, EAE was associated with a loss In the expression of Ohg2 in the corpus callosum, a marker for oligodendrocyte differentiation, which was restored b EBP- 101 treatment. In addition, EHF-101 enhanced the expression of glutathione S -tran ferase pi (GSTpi), a cytosolic isoenzyme used as a marker for mature oligodendrocytes In the brain. These data are indicative of the potential of ESP- 1 1 t prevent demyeiinaiion in an MS murine modei Elgure 15 through Figure 18).

To further evaluate the poteutial of ESP-IOL the effect of EHP-d 01 in a euprixone model of demyeiinaiion was investigated, Mice were fed with a diet containing 0,2% enprirone for 6 weeks and the» the animals were switched to a normal diet and either treated or not treated (control) with EflP-101 ( 10 and 20 mg/kg) for 2 weeks. Cupri zone induced a clear loss of myeli In the feral» measured fey eryomyelm staining and MFB expressi on. Spontaneous recovery from demyeiinaiion was negligible after I and 2 weeks but remyelination was significantly accelerate fey EHP- 101 treatment, Moreover. EHF-IOl also revenied cuprizone- indueed microglial activation and astrogiiosis detected by ifeal and CiFAP staining, respectively (Figure 19 through Figure 26)

In conclusion, EF1P- 101 represents a possible drug candidate for treatment of various diseases and disorders, such as different forms of MS arrd other demyelinaimg diseases.

Example 19: EEP-H)f and Remyelinafion

Methods of Example 19

Compound

EHF-IOl is a lipid-based formulation of VCE-004S [( i ^, R,6 ^, R)-3~(Ben¾ylamhie)- 6-hydroxy-3 -methyl-4-peufyl--6 ^, --(prop-i -en~2~yl) [Li foi(eyclohexane}]-2\3,fe-tfiene-2,S~ dlone)] The chromatographic purity of VQ3-0O4.8 in EHF-IOl was 97.6%,

Cimrixone-lnduced Demyelhiatlon Model

To in uce delay eKtatioti, 8-week old CS7BL/6 male mice were fed -with 0.2% cugfthscme TD.140800 diet (Envigo, Barcelona, Spain) for six weeks. Control group (no

demyelmation) was fed with control mouse TD.0O217 diet (Envigo, Barcelona, Spain) for the entire period. To study the effect on re yeliaation, EHP-lOt was administered daily b oral gavage at 20 mg/kg from week six. For Comparison, animals in the cuprkone control group posfi demyeiinaiion received the same volume of vehicle by oral gavage. To study the dynamic effect of BlfP-lO i ø» remyelinatio», animals in each group were sacrifice at weeks 6, 7 (6+1 W), 8 (6+2 W) post-treatment for further analysis. Tissue Processing

Mice were anesthetized by i.p, administration with: a ketandne-xykxme solution an they were transcard la Sly perfused with saline 0 9%, Brains were fixed, eryoprotected and frozen at ~Z( C for further analysis, Analysis

For antigen retrieval brain sections were boiled for 10 m la sodium citrate _: buffer (10 rn . pH 6.0} or Tris- EDTA buffer (10 o-M Iris Base, 1 mM EOT A 0 05% Tween 20, pH 9.0} (Sigma-Aldrich, St, Louis, MO, USA). The sections were washed three times in PBS, Nonspecific antibody-binding sites were blocked for I h at oom temperature with 3% bovine serum albumin (BSA) (Sigma-Aldrieh, St. Louis, MG, US A in PBS). Next, the sections were incubated overnight at.4 °C in following primary antibodies diluted in PBS with 3% BSA:

microglia cells were stained with a rabbit antUIl -I antibody (1: 1,000; Wako Chemical Pure industry, Osaka, Japan), astrocytes were stained with a mouse anti-GFAP antibody (1 ;500, Santa Cruz Biotechnology, Santa Crux, C , USA), myelin basic protein was marked with a rabbit anti- Myelin Basie Protein antibody (1 ;!000; Abeam, Cambridge, UK). After extensive washing in PBS, slides were incubated with secondary antibodies for 1 b at room temperature in the dark. The im unoreactious were revealed using anti-rabbit Texas Red. (It 100), anli-moase/rabbif A!exa 488 (1 : 100} obtained from Thermo Fischer Scientific, Waltha m, MA, USA, Tbe slides were then mounted using Veeiashield Antifade Mounting Medium with DAP! (Vector

Laboratories, Burlingame, Ca, USA), Myelin integrity was analysed using the Hlto

CryoMyefinStain™ Kit (Gold phosphate complex Myelin Staining Kit) following

manufacturers reemnmendation (Hitobiotech Corp., Kingsport, TiN, USA). All images were acquired using a spectral confocal laser-scanning microscope LSM 710, (Zeiss, Jena, Germany) with a 20x/O _> 8 Plan-Apocbromat lens and quantified in 9-15 randomly chosen fields using Imaged software (rsbweb.nib.gov/ij7).

Data Analysis

All the In vivo data are expressed as the mean & SEM. One-way A OV followed by the Tukey's post hoc test for parametric analysis or Krnskal -Wallis post hoc test in the case of non-pnraraetrie analysis tests were used to determine the statistical significance. The

7 ! level of siguiieanee was set at pfoiOS. Statistical analyses were perform using GraphPad mm version 8.00 Oraphf ad, San Diegp, CA, USA),

Results of B a nie 19; EBP-fOf Accelerates Rgrrweli tfon in a Cunrtxmte-Ch lenged. Mous

§ Model

To evaluate the effect of EHP-101 oft myelin damage m u CPZ-induced demyelination model (Fi gure 19 A), drain coronal sections f om animal alter § weeks of CPE 0.2% diet and 2 weeks of EHPU Ol treatment were analyzed is this model, EHP-101 treatment began alter CPZ diet removal, to more directly evaluate formulation effects oa re yelination.0 First the evaluation of MB P (cortex) was determined both immtmohisioehemis ry and

Cryomyel (corpus callosum) (Figure 19C and Figure 19B, respectively) staining -where myelin was stained using a gold phosphate complex myelin staining kit in stained preparations, and myelin is intensely black. Spontaneou recovery from demy elinafiotrwas insignificant after 1 and 2 weeks but remyelination was significantly accelerated by EHP-101 treatment

5 Interestingly, both studies showed !llP-f 01 to enhance retnyelination in Corpus Callosnm in the ease of staining (Figure I9D p ^~ < 0.0001 GPZ6W, CPZ6M W, CPZfoZ W vs Control;

<0,000:1 CPZ6+1W 4 EHP-101 20 mg/kg vs CPZ6M W; p~ <0.0001 CPZ642W EHP-101 20 mg/kg vs CPZ64-2W) and Cortex throughout immunohisiochemistry studies (Figure 19E p ^::: < 0.0001 CPZ6W, CPZ64 IW, CPZ64-2W vs Control; p- <0.0001 CPZtUl W 4- EHP-101 20 mg/kg0 vs CPZ6+! W), Moreover, the effect of EHP-101 on neerohiil noiation-assoeiated glial

activation was also investigated using iromnnoflnoreseenee staining of iba-I and GFAP in the Corpus Callosum in control mice microglia and astrocytes were detected at low levels. Mice exposed to CPZ showed microglial and astrocytic hypertrophy, which were _. attenuated by EHP- 101 treatment (Figure 20A and Figure 20B) Quantitative assessment also showed a significant5 Increase in the number of Ibaf 4 and OF APE cells in Corpus eallosmti upon CPZ intoxication.

Mi erogliosis and astrocytic reactiva tion was ameliorated after 1 week of EHP-101 treatment (Figure 20C m < 0.000 i CFZ6W. CPZ64I W, CPZ642W vs Control; - 0.0017 CPZfo I A + EFiP-f Of 20 mg/kg vs CPZ6-HW; Figure 200 p- < 0.0001 CPZ6W, CPZCH W vs Control; p- 0,00! 7 CPZb¾W VS Control ).

0 Ex mple 20; Effects of EHF-fOl on inflammation aacl in murine models of

MS is an autoimmune disease that affects the CMS and is characterized by pathological changes, including neuroinfia ation, dom elination and axon intury. The spontaneous repair of damaged .myelin sheaths and axons has been described during the ranilsaion period of classical rel apsing-remi ttitig M S (RRMB), where demye&ated axons could be rewrapped fey the regenerated myelin sheath, thus ameliorating axonal dysfunction. In this sense, the remission period Is also considered the period of remyel matron , which I ImportaBt because it eoidd be a key time point for the treatment of RKMS patients noth drags pre venting inflammation and enhancing remyeUnation.

Small molecules including cannabinokis acting at draggable targets of the endocannabinohi system (ECS) are being explored for the management. ofCNS pathologies including MS. in this sense, several lines of evidence suggested a role for the ECS in

oligodendrocyte function and remyeiination activity i MS, The ECS is composed fey the G- protein couple receptors CB I and€B2, endocannabinolds and the enzymes regulating their synthesis and catabolism:. In addition, eanuahinoids of different nature also target ionotropic receptors of the TRP family and nuclear receptors such as peroxisome proSiferator-aetivated receptors (PPARsi CB1 receptors are expresse mainly in the CMS at neuronal terminals and regulate neurotransmittar release and psychoacti ve processes in contrast, CB2 receptors are located primarily on the peripheral immune system, and during neuroinf!amrnatiou on activated microglia in the CMS. Key considerutioas tor developing CB2 receptor agonists include absence of psychoactive effects, sustained anti-inflammator activity, tissuc/eell protection, lack of cardiovascular adverse effects and efficacy in several disease models ou neurolnfiammatlen Including MS.

PPARs are members of the nuclear hormone receptor superfam!l of !igarid- activated transcriptional factors with well-identified regulatory roles in lipid and glucose homeostasis and adipocyte differentiation. In addition to adipocytes and hepatocytes, PPARy has been shown to be expressed in different CMS cells and in immune cells, Furthermore, PPARy has been described as an important factor in the regulation of the immune response. In this sense, PPARy activation has been shown to suppress the expression of inflammatory cytokines in astrocytes and macrophages/microglia. Furthermore, PP ARy stimulated oligodendrocyt differentiation frOtti neural stem cells, promoted and accelerated tire differentiation of oiigodendroeyto progenitor cells In vitro with air additional increase in aatfexidant defences and increased lipid production and terminal differentiation of cultured oligodendrocytes, thus suggesting an additional possible protective role of PPARy in MS as a mediator of remydinati The neoroprotective effect of PPARs, including PPARy, have also been widely documented in vitro in various experimental paradigms oineurodegeneration, broadening its potential therapeutic perspectives in MS,

Although most current therapies tor MS are directed towards modulation Of the exacerbated immune response, novel therapies aimed to axonal remyeimatio are urgently needed, A novel a proximation to achieve this would be fee hypoxia preconditioning process which, Induced b mild oxygen depletio _; is beneficial in a wide number of neurological disorders, including MS The cellular adaptation to severe or mil hypoxia i very fas ami involves the activation of the hypoxia-inditeible factor-la (RIF), whose activation ma play a role m the inflammator and the remitting phases of MS. In addition, there is evidence suggesting that activation of the I-IIF path way may also be linked to neuroprotection and perhaps remye!ination Fo instance, erythropoietin (EPO), whose gene is dependent on HIE activation, i neimuproteetive in different animal models of MS

It was previously shown that ¥CE4K)4,8 i a promising cannabldtoi derivative acting as a dual agonist of PPARy and CB2 that also activate the BIF pathway. Indeed, VCE- 004.8 prevented neuroinf!ammaiion and demyelination in two different murine models of MS, such as EAE and Theiler’s virus-induced encephalopathy. EHR-.I0I is an oral formulation of VCE-004.8 that showed efficac in a murine model of systemic sclerosis. More importantly, EMP-IOl has co pleted a Phase 1 clinical study ( lnicaitrialgov; NCT03745001 ) and initiation of Phase If studies la SSc and MS patients are being planned. The present example shows the efficacy of EllP-fOl in preventing nenroinilammaiion and demyelination in EAE and to enhance remyelinatlon hi the euprixone model of demyelination.

Methods of Example 20

Compounds ί .ί IP- 10! is a iipidic-based formulation of VCE-D04.8 :[(! 'R.iv R ;-3- (Betxylammej-d-hydroxy-B’-tneihyi-d-peniyifo gfop-d -eB~2-yi} [1,1 foifo c!ohe a elj-Gyfo- tri:ene~2,5~dioBe)l. The chromatographic purify of VCE-004,8 in EBP- 101 was 97,6% Animals

AO experiments were performed in /strict accordance with Ell av d governfoeufnl regulations. Handling of ani als was performed m compliance with the guidelines of anlraa! care set by the European Union guidelines 86/609/EEC, and the Ethics Committees op Animal Experimentation at the€¾a! Institute (CSiC, Madrid) and the University of Cordoba (UOO, Cdrdoba, Spain) approved all the procedures described in this study (for BAB at Cajal Institute protocol number' 96 2013/03 CEBA-IC and for cuprizone model at UCO protocol number; 2018P1/02 (UCO). Measures to improve welfare assistance and clinical status as well as endpoint criteria were established to minimize suffering and ensure animal welfare, Briefly, wet food pellets are placed on the bed-cage when the animals begin to develop clinical signs to facilitate access to food and hydration. For EAE model female C57BL/6 mice were purchased from

Harlan (Barcelona, Spain), in the case of cupri oiie model male C56BL/6 mice were purchased from Janvier Labs (Le Genesi- Saint- Isle, France), All animals were housed in the animal f acilities tinder the fol lowing controlled conditions; 12 h iighPdark cycle; temperature 2( Q (±2 *€) an 46-50% relative humidify with free access to standard food and water,

Induct ion and assessment of EAE

EAE was induced in C37BL/6 female mice at 6-8 weeks of age by subcutaneous immunization with MOG3$~55 (300 tig; peptide synthesis section, CBM, CSIG, Madrid, Spain) and 200 pg of Mycobacterium tuberculosis (H37R& Difco, Franklin Lakes, Mi, USA) in a 1 ; I mix with incomplete Freund’s adjuvant (CBA, Sigma). On the same day and 2 days later, mice were injected intraperiioiieai! with 200 ng of pertussis toxin (Sigma) in 0.1 ml PBS. Control animals (UFA) were inoculated with the same emulsion without MOG and they did not receive pertussis toxin. Treatment started at day 8 post-immunization: when animals showed the first symptoms of the disease and consisted in daily oral BHP-dOi () , 5, id and 20 g/kg) for the followin 21 days. The mice were examined daily for clinical signs of EAE and disease scores were measured as follows;: 0, no disease; i, limb tail; 2, limb tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb and front limb paralysis; 5, Moribund an eath. All animals were sacrificed at 28 days for further analysis,

Ciiprfrone-liKiueed demvcl (nation

To induce detnyelfoation E-week old C57BL/6 male mice were fed wife 0.2% euprfeone TD. 1.40800 diet (Envigo, Barcelona. Spain} for six weeks. Control group (no derayelinafien) was fed with control mouse T .00 17 diet (Emdgo,, Barcelona, Spain) for foe entire period, To study foe effect on remyeifeation, EHfi-401 was administered daily by oral gavage at 20 mg/kg from week six. For comparison, animals in foe euprizone control group (maximal demye!inaiioft} received the same volume of vehicle by gavage. To study the dynamic effect ofEHP-101 on remyelination, animals in each group were sacrifice at weeks 6, 7 (ffr l W), 8 (6f2 W) for further analysis.

Tissue Processing

Mice were anesthetize by i.p, adnonisiratfon with a keia iae-xvlaxine solution and they were transcardialiy perfused wife saline 0.9% The spinal cord was obtained by extrusion with saline. Brain and cervical spinal cord were immediately frozen and kept at -SOT.' for BT-PCR analysis, the remaining brain and spinal cord were fixed in 4% pamfornmldekyde ia 0.1 M PBS, washed in 0.1 M PBS, cryoprotected with a 15% an then a 30% solution of sucrose In 11.1 PBS, and frozen at -80 ^a C. Free-floating brain and thoracic spinal cord sections (50 pm thick; Leica Microsystems CM 1:900 cryostat, Barcelona, Spain) were then processed for Immunohisioebenfestry or inummofluoroscence, in foe ease of euprizoue model whole brains were fixed, cryoprotected and frozen at -8( C for further analysis. Analysis

For THC analysis, free-floating thoracic spinal eord (SO pm) section ere washed with 0 IM PB, Endogenous peroxidase activity was inhibited wife 3.3% hydrogen peroxide in methanol. The sections were blocked with 2.5% normal horse serum and then incubated overnight at 4*C in blocking buffer wife a rabbit anii-Teaenrin 4 antibody (I ;SQ: bfovus

Biological, Colorado, USA). Slides were meitbated wife !mrnPRBSS reagent (Vector

Laboratories; Burlingame,€a, USA) and then developed with dianimobenzidme chromogen (Merck, Darmstadt, Germany), Samples were photographed, digitalized using a Leica DFC420C camera and analyzed using image J software. Myelin integrity _: as analyzed using the Hito CryOMyel nStain ^' ^ Kit (Gold phosphate complex Myelin Staining Kit) following

manufacturers recommendation CHitobioteeh Corp,, Kingsport, TN, USA), Microscopy Analysts

For antigen retrieval, spina! cord or brain sections were boiled tor 10 rain in sodium citrate holler P0 raM, pH 6,0) or Tris-EDTA buffer (10 m Iris Base, I mM EDTA0.05% 1 ween 20, pH 9,0) (Sigma-Aldrich, St Louis, MO, USA), The sections were washed three times in PBS. Nonspecific antibody-binding sites were blocked for 1 h at room temperature with 396 bovine serum albumin (BSA) (Sigraa-Aldrich, St, Louis, MO, USA in PBS). Next, the sections were incubated overnight at 4 °C with the following primary antibodies diluted in PBS with 3% BSA: microglia cells were stained with a rabbit aaii-Iba-l antibody (U 1,000; Wako Chemical Pure Industry, Osaka, Japan), astrocytes were stained with a mouse anii-GFAP antibody (i :SO0 _* Santa Cruz Biotechnology, Sant Cruz, C , USA), myelin basic protein was marked with a rabbit anit-Myelin Basie Protein antibody (f :1000 ^* Abeam, Cambridge, UK), oligodendrocytes were marked with a mouse anti-01ig2 ( 1 : 100, San ta Cruz, CA, USA) an a rabbit anti-GSTPi (1:250, Abeam, Cambridge, UK) axonal damage was determined with a mouse antUNewrofdarosot H (NP-H) Nonphosphorylaled antibody (SMI-32) ( 1:50 Biolegend, CA, USA). After extensive washing in PBS, slides were incubated with secondary antibodies for 1 h at room temperature in tire dark. The imntunoreactions were revealed using anti-rabbit Texas Red (J:!00) _* a ti-mouse/rabbit Alexa 488 (1 : 100) obtained from Thermo Fischer Scientific, Waithamm, M A, USA. The slides were then mounted using Vectaslheld Antilade Mounting Medium with D.AP1 (Vector Laboratories, Burlingame,€a, USA). .411 images were acquired using a spectral eonibeal laser-scanning microscope LSM710, (Zeiss, Jena, Germany) with a 20*70.8 Plan-Apoehromat lens and quantified in 0Ή5 randomly chosen fields using trnageJ software (rsbwebmlltgov/ij/).

Total RNA was Isolated from spinal cord tissue using QIAzol lysis reagent (Qiagen, Hikien, Germany) and purified with RNeasy Lipid Tissue Mini kit (Qiagen), Then, samples were processed for high throughput sequencing using poly- A selection with the TruSeq Stranded tnRNA Library Prep Kit (Cat, Mb. RS-!22:~210i, Hiumiua, San Diego,€A, USA) In biM, 1 pg of total RNA fro each sample was used to construct cDNA library, followed by sequencing on the Illumine HiSeq 2500 system with single end 50 bp reads and -40 millions of reads per sample (h ^::ϊ 3 per group) FASTQ files were pre-processed with Irhnrnomatic (v036) and aligned: to mouse genome assembly mm 10 using HIS ATI (v2, LO). Then, counts per gene matrix were obtained with featareCounts (v Ud J ) using the in-built ReiSeq annotation for m 10 genome assembly and the differential expression analysis was carried out using !>ESeq2

(vl, 20,01, excluding genes with less than 15 counts across ail samples. The functional over- representation analyses were performed using EnrichR and elusterProfiler. AM the P values were adjusted to control the false di cover rate (EDR) using the Benjamini and Hochberg approach, RNA-seq data have been deposited in the Gene Expression Omnibus databank (accession no. GSE13 IS54}-

Quantitative Reverse Transormtase-PCR

TotalUNA (L pg) wa reirot nseribed usiug the iScripi cDNA Synthesis Kit (Bk>Rad, Hercules, CA, USA) and the cDNA analysed by real-time PCR using the IQTMSYBR Green Supermix (Bio-Rad) and a GFX96 Real-time PCR Detection System (Bio-Rad),

G A.PDH gene was used to standardize mEMA expression iir each sample. Gene expression was quantified using the 2-AA€t method and Che percentage of relative expression against controls was represented. The primers used In this study are described in Figure 2L

Determination of light Polypeptide (MEED

Blood samples were taken under general anesthesia* and Lithium-Heparin plasma was collected- Samples were centrifuged for 20 mm at 2000 ^x g within 30 min of collection, and circulating levels of Neuroiilamesit, Light Polypeptide (NEFL), were quantified wit an Enzyme- linked immunosorbent Assay Kit for NeuroEiament Light Polypeptide (NEFL) (Cloud Clone Corp/USCN Life Science, Houston- TX, USA) according to the manufacturer's Instructions. Values were normalised versus control group and correspond to mean ± SEM of 4 to 6 animals per group- Data Analysis

All the in vivo data are expresse as tim mean A SEM One-way AHOV A followed b the Tukey ^' s post hoc test for parametric analysis or SCruskal-WalHs post hoc test ih the ease of nos-parametrie analysis tests were used to determine tire statistical significance. The level of significance was set at p<0.05. Statistical analyses were performed using GraphPad Prism version 8,00 ( ^: i3 pkPa<L San Diego, CA, tiSA).

BEP-lfil Attenuates Clinical Severity an Nenrdmfiammaflon m EAE

The efficacy of EHP-I 01 in MS was first evaluated in BAB, perfor ing the treatments ai an early stage of the disease since mice received increasing doses of EME-TOl at ay 8 p,L (post-immunization). Subcutaneous immunization with MOGSS-SS Induced EAE in all mice that received the vehicle alone, All vehicle-freated mice developed disease that peaked by day 16 pi. and maintained at day 28 pi. B contrast, animaTs score showed therapeutic efficacy of FIfP-101 with all the doses tested, being the higher dose (20 mg/kg) able to prevent the symptoms completely (Figure 1 $A p~ 0.0002 EAB+EliP-lO 1 20 mg/kg; vs EAE+Vehieie; = 0.0046 BAB+EHP-IOI 10 mg/kg vs EAE+VeMele; p* 0,0068 EAE+EFIP-IOI 5 mg/kg vs BAET¥ehic ) Clinical score data from Figure 15 A were used to determine the area under curve and it i showed in Figure 15B (p <0 0001 EABs-EBP-10! 1/5/10 20 mg/kg vs EAF+Vehiele) that EHE~ 0i improved symptomatology in a dose-dependent manner.

To determine whethe EHF-101 was able to target nemwinEam ation in EAE, mierogllosk and astrogliosis were evaluated in the spinal cord, Mistopathoiogical analysis showed that the extensive microglia/macrophage activation (Figure I SC through Figure 15F p 0.0003 BAE+Vehicle vs CM; jwOvOOOb EAE<Effi 0! 20 mg/kg vs EAEWeMele) and astrocyte activation (Figure 15C through Figure 15E, Figure 15G p <0.0001 EAE-rVehlcle vs €FA: p- 0.005 EAE+EMP401 20 mg/kg vs EAE+Vehieie) in the spinal cord of EAE mice evidenced by both lba-I and CiPAP staining was greatly reduced by EBP-IOL MS pathology is characterized by focal demyelinating lesions in the CHS at both spinal cord an brain levels, Therefore, to determine the extent of demyeli nation, myelin was evaluated by MBP

Immunol a belling, A clear derayefination was found in the spinal cord of EAE mice that was significantly prevented by EHP-IOI treatment (Figure 15€ through Figure ^" 15E, Figure !SE p— 0.0001 EAEWehtele vs CFA; p <0.0001 EAE+BBi til vs EAEvVehicfeT

Cerebral cortical demyeSination as well as callosal pathology are widely recogsized features of MS, Is addition, the cerebral cortex plays a central role is

interhe isphetic communication, and callosa! atrophy in MS patients has bees shown to correlate with disability states Therefore _* It was also examined whether these structures ight also be affected in EAE mice. Am increase in inflammatory lesions was seen throoghout the EAE fbrebrain (Figure 16A through Figure I 6D) Specifically, it was observed that microglia! reactivity was Increased In Corpus callosum of EAE ice and the treatment with EFSP-lQl reverted the ntierogtiosis process (Figure !6B p 0.0002 EAETVehlole vs CFA; p 0,0305 EAE+EHP- iO i 20 mg/kg vs EAE+Vehicle}. Furthermore, brain sections from EAE-affeeted mice were also analyzed for the distribution of MBP reactivity, MBP immunoreaetivity appeared significantly reduced in cerebral cortex (Figure I OF p ^~: 0.0159 EAE Vehicle vs CPA; p ^~: 0.0024 BAE-fEHP-10120 mg/kg vs EAE-EVcM e) and this loss of myelin expression was strongly reverted by EMP401 treatment, Moreover, EAE Is associated with a loss in th expression of 01ig2 in tire Corpus eallosnm, a marker for oligodendrocyte differentiation, which was restored by EHP~10f treatment (Figure 160 p <0.0001 EAE< Vehicle vs CFA; ^~ 0,0008 EAE+EFO Ol 20 nig/kg vs EAB+Vehiele), In addition, EHI 01 enhanced the expression of glutathione S- transforase pi (GSTpi), a cytosolic isoenzyme used as a marker for mature oligodendrocytes in the brain (Pignre I6.H p ^:: 0i)222 EAE-FEB!M01 20 mg vs EAB-EVehidef. These data are riieative o the potential of EHPGfil to prevent demyelinatioo in an MS murine model.

EH P- 101 Normalizes EAE Transcripfomle Signature at Spinal Cord

To evaluate the global expression changes produced by the EEO 01 treatment, an RNA-Seq analysis of the spinal cord from mice was petiPnned in the following conditions: Control, EAE and EAE with EBPAO I treatment (20 mg/kg) Sequencing dat for three biological replteates were obtained for each experimental group. Then, the Eanscriptomie profile was compared between the different conditions to get a first Insight into the changes occurring at the model, with or without treatment. As expected, many changes were found, both in magnitude and significance in EAE mice compared to the group treated wit EHP- Ifil (Figure 17A), Then, to evaluate those changes at a biological level, ovewrepresemation analysis was performed usiag genes that surp sse th cutoff of an adjusted P < 0,05 and absolute Ibid change > 2 m the EAE vs control and BAE<EHP-101 vs BAE comparisons. The « re significant enrichments were found in the groups of upregolaied genes by EAE and downregelated genes by the treatment. A complementary signature was observed between those two groups, where terms like ‘"neutrophil mediated im unity”, "‘inflammatory response” or“cytokine-mediated signaling athw y” appeared, highlighting an anti-inllam atory effect of the EHPAOl tfoaltnent at the spinal cord (Figure !7B), The heatmap in Figure 170 represents gene from the‘"cytokine- mediated signaling pathway” that are induced by EAE and dowaregolated by BHF-iOi .

Furthermore, to confirm this anti-milammatory effect of EITR-Ί 1 in spinal cord, the gene expression by RT-PCR. of several genes, such as 116, Timpl , Veam, Mb, Cel4 and Cei.2, was determined:. Figure 17 E shows that EHP-101 treatment downregulatcd the expression of these genes upregulated in EAE mice (116; p ^“ 0.0360 EAE+Veliicle vs CFA; p ^~ 0.0451 EAEFERP-tOI 20 mg/kg vs EAEf Vehicle; Timpl : ^“ <0.0001 EAEAVehiele vs CFA; p ^~ 0,01)01 EAE+EHP- 101 20 mg/kg vs BAEa-Yehide; VC AM: p 0,0058 EAE-fVehicle vs CFA, p~ 0.0381

EABWEHP-IOl 20 rng/kg vs EAE+Vehi e; iL lb: p ^“ 0,0018 EAE< Vehicle vs CFA: 0,0027 EAEvEHP-101 20 mg/kg vs BAEvVelhcle:€cI4: p~ <0.0001 BAE+ Vehicle vs CFA; p

<0,0001 EA.E-fEHP-101. 20 mg/kg vs EAEWebide; CeI2: p^ 00003 EAEWehicie vs CFA; p- 0.0054 EAE+EHJMOl vs EAB+Veht e), thus validating the results found in the 1S.NA-Seq analysis.

Next, a secon analysis was performed to explore changes in the opposite direction to the pattern shown by the pro-infiamn^atory genes. Thus, down-regulated genes were selected at the EAE vs control comparison and up-regulated in EAE+EHP-lOl vs EAE comparison. Both groups of genes were intersected to evaluate the overlap between them, resulting n a total of 193 genes downregnlated in the untreated model that increased their expression in response to the treatment (Figure 18A). Then a second functional analysis was performed, using the list of overlapping genes as Input, to explore the most significantly enriched GO terms. As depicted in Figure 1 SB, several terms related to the metabolic process of sterols an hydroxy compounds were found at the top of the list. However, given the background of the disease, focus was given to the“myelmatron” process. To explore the changes of features belonging to this annotation, the expression levels of genes that produced this result in the heatmap were depicted and are shown in Figure ! 8C, This allowed ns to identify several key

8! gdoes of the myelination process that were restoring their levels with EBP·- i 01 treatment .

Iniere.sv gh _{· ,} these results indicated that IBP- 101 normalized -he expression of several genes associated with oligodendrocyte function, such as Gap junction gain a-3 ( jc3), also called Connexin 29, and Te»enrin-4 (Tenmd) that were downregolated in EAE. These results are relevant since Tenra4 has bee» described as a critical regulator of oligodendrocyte differentiation and CHS myehnadon. To validate the transeriptortne analysis, the expression of C¾c4 and Tenm4 wa studied by RT-PCR (Figure I 8D mn4: p 0.0020 EAE<VeMefe vs CPA; p 0.003 EABAEHP-TOI 20 mg kg vs EAIA-Vehieie; Ci|c3 ; p- 0.0006 EAE-f Vehicle vs CPA; p- 0,0462 EAEAEBP-I O I 20 mg/kg vs EAE-fVehicle) and the protein levels by THCE As depicted in Figure 18E (p~ < 0.0001 EAEAVehieie vs CFA; p~ < 0,0001 EAEAEBE-101 20 mg/kg vs

EAE+Vehiele), a decrease of Tenm4 expression was observed in white matter of spinal cord compared to the CFA group which was prevented b EBP- 101 treatment, Taken together, these results are indicati ve of the potential of EHP-i 01 to prevent deroye!ination in EAE model

EBP- 101 Accelerates RemveiinaEon in€uprixone~€lmllengedA fee

To evaluate the effect of EHP- 101 on remyefinafton during the acute GP2- imiueed demyelmation protocol (Figure 19A), brain coronal sections from animals after 6 weeks of CPZ 0.2% diet and 2 weeks of EBP-! 01 treatment were evaluated. In this model EBP-! 0 f treatment started after removal of the CPZ diet to study the effect of EHP-I 01 on spontaneous remyehnatkm, First, the evaluation of MBP was determined by CryoMyelio an IBC staining (Figure 19® and Figur 19C, respectively), Spontaneous recovery from de yelinatiou was Insignificant after 1 and 2 weeks in untreated mice but remyelina lion was significantly accelerated: by EBP-101 treatment in both the Corpus callosum (Figure I 9D p < 0 0001 EPZ6W, ClPZiAIW, CPZ6<2W vs Control ^ <0,0001 CPZft-H W < EHF-I 0I 20 mg/kg vs CPZ6-HW; p ^::: <0.0001 CPZ6+2W < Ff ifM O i 20 mg/kg vs CPZ6+2W) and the cerebral codes. (Figure 19E - < 0.0001 CPZbW, CPZ64-I W, CPZfepW vs Control: p- <0 0001 CPZ6-<5 W v BMP- 101 20 mg/kg vs CPZ6A I W). Moreover, the effect of EHP- 101 ou neuroiuflammation- associated glial activation was Investigated by staining iba-H· and GFAPA cells in the Corpus callosum. In control mice low level expression of Iha-iA an GPAPA cells was detected but mice exposed to CPZ showed microglial aud astrocytic activation, which wa attenuated by EBP- 101 treatment (Figure 20A and Figure 20B). Quantitative assessment also showed a significant Iterease in the number f fhal v nd GPAtH celts i Corpus callosum upon CFZ intoxication, Mlerog!Iesls and astrocytic activation was ameliorated after 1 week of EBPri O! treatment (Figure 20C p ^:: < 0.0001 CPZftWyCPZfrH W, CFZ6- 2 vs Control; - 0.0017 CTZ6G W + EHP-10I 20 mg/kg vs CPZfH 1 W; Figure 20D p- < 0.0001 CFZ6W, CPZ6+ ! W vs Control; p= 0.0017 CPZ0+2W vs Control). To examine the effects of EMF- !Ol on cuprizonednduced deniyelioafion on axons in the Corpus callosum, the non-phosgharylated form of neurofilaioen proteins (SMI -32 staining) was investigated. Although SMI 32 immunoreaetivity is normally seen In axons, its accumulation In axonal spheroids is a characteristic of axonal pathology. Increased SML32 labeling after 6 and 7 weeks of CPZ intoxication demonstrated that there was a significant effect on axons and this effect was ameliorated after t week of EBP- 101 treatment (Figure 22A). Moreover plasma levels of Neuroftiament Light Polypeptide (NEFL) were determined. As depleted in Figure 22B, an increase of euprizoue-iraioced NBFL plasma levels was detected by ELISA studies after 6 and ? weeks of CPZ exposure compared to effiuro I mice. It was also shown that one week of treatment with BHP-101 reduced the plasmatic levels of NEFL induced by cuprfeone (Figure 22B p ^~ 0.01 13 CPZ 6W vs Control; p ^~ 0.0151 CFZ6+1 W vs Control; p 0.0125 CFZ6GW w EHP-101 20 mg/kg vs CPZ&H W).

Natural proditcis, including phyfoeaanabinoids, have been soecessiully used for the development of synthetic and seunsyntheiie derivatives with Improved bioaeftviies. Tire experiments described herein disclose the development of the eompoundVCE-OOd J, a se i synthetic derivative of eanuabidioL winch is a dnal agonist for FPARy/CBd that als inhibits the activity of HIP prolyl hydroxylases (PBDs), Therefore. VCB-004.S is targeting several pathways that may have a positive effect in nenrohrftammation and remyehnation in EAE and Tfteiler's Murine Encephalomyeliti Virus-induced demyeimafing disease. Herein described studies disclose the effect of EHP401 , an oral iipidie formulation of VCE-004,8, i the two most commonly used models of demyelination that are EAE an ioxicully induced defflyeiiuatiou via euprizone

EAE in C57BI/6 mice has generally been thought to predominantly target the, spinal cord, leading to sensory and motor impairments. Nevertheless, It is also recognized that EAB involves other CNS structures including the cerebellum an the hippocampus The data clearly indicate that EHP~ 101 is effective to alleviate neuroinftammation in tire spinal cord, in tfee cerebral cortex and in the corpus callosum (€€}, In t e L AE model it was not possible to distinguish: w ether the effect of E! ^; IP~101 occurs at the peripheral immune system, at the 0¾S of both. It has been demonstrated that the brain blood barrier (BBB) Is disrupted in BAE allowing the migration of autoimmune cells and molecules to the brain. However, it Is likely that BHI OI may exert and inflammatory effects b acting both at the peripheral immune syste and at the CMS- For instance, EHP-!O! showed aniidnfiammatory activity in another antoim ane disease sued as Systemic Sclerosis where the BBB is not affected and herein it was shown that BMP- 101 also alleviates neuroinflammation in CFZ intoxicated mice, CFZ-mduced demyef mating lesions are characterized by severe oligodendrocyte loss and demyelmation with concomitant activation of microglia: and astrocytes, but it does not induce BBB damage and lacks the characteristic T ceil mflitration and consequently the peripheral autoimmune component of the disease.

The mechanism of action of BfIP~ 101 hi the rem.yelmatfen process Is still unknown hut it can be probably related to the HIF pathway. Extensive experimental Mifeies have revealed that acti vating HIF- 1 by inhibiting the activation of PHDs can provide nenroprotection and perhaps remyelinadon mainly from the increased expression of HlF-1 target genes, which combat oxidative stress, improve blood oxygen and glucose supply, promote glucose

metabolism, regulate iron homeostasis and block cell death signal pathways, increasing HIF-i activit may be an important potential strategy to prevent the onset or to ameliorate the pathogenesis of nemmlegenerative: diseases. Interestingly, the improvement of the myelmatien index was parallele by enhancement of QPC proliferation, P:DGF¾-recep†or expression, and precursor migration flora the CC midline to the lateral parts followed by an induction of the expression of myelin protein in addition, early asiroghosia in the demye!mated areas paralleled with a moderate stimulation of IGF- 1 expression IGF-) synefgtees with P-GF-2 to stimulate oligodendrocyte progenitor entry into the cell cycle. This is of particular interest because IGF- 1 induced HIF- 1 activation that can be mimicked b VCE-004.8 in the brain, and FDGFa and FGF2 are also regulated by VCE-004.S-mediated activation of the HIF pathway.

Demyeiinaiion and partial axonal damage in MS lesions are closel associated with reactive activation of microglial cells which are seen in close contact with axons, that reveal acute axonal injury, such as the formation of axonal spheroids or a disturbance f fast axonal transport. Reactive microglia produce a large array of toxic and proinfiammatory molecules, which: triggers yelin destruction, oligodendrocyte deterioration, mm damage and even neuronal loss. Here It was found that a ! BMP- ! 01 aka prevented microglia activation and demyelination In both spinal cord and brain suggesting that after oral absorption VCE-004 8 penetrates into the brain in EAB mice. Moreover _» it was also found that EHP-101 preserves the axonal structure ameliorating the typical accumulation on spheroids of SMI -32 used as a marker of axonal damage in. CEZ in toxicated mice. Again, ibis result suggests that VG E-004.8 can also cross the BBB t at is not affected in the CFZ model

Oligodendrocyte progeni tor eel is (OPGs) are produced from neuroepithelial stem cells and subsequently proliferate and migrate throughout the entire spinal cord. During differentiation, oligodendrocytes initiate expression of myelin proteins cri tical for the achievement of proper functioning of the CMS. Teneurin-4 (TenmA) is a type II transmemhrane protein that is highly expressed in the CNS and whose expression is induced in response to endoplasmic reticulum stress and has been suggested to he involved in bipolar disorder i humans A mouse mutation, designated fume which results in tremors and severe

hypomyehoatk of s all -diameter axons, reduces oligodendrocyte differentiation especially in the spinal cord of the CMS, and it has been associated with the absence of Teom4 expression:. Thus, Tenm4 is a critical regulator of oligodendrocyte differentiation and CNS myeliuaiiou. Herein it was shown for the first time that in EAE mice the expression of TenrnA i

downregnlated in the spinal cord and the treatment with BHP-iCH reverses this downregalation probably as die result of the aati-inilammatory activity of YCE-O04

In addition, oligodendrocytes are electrically and tnetahofically coupled through intercellular channels called gap junctions (fiJs), composed of conaexins 0x29, Cx32 and€x47, with other oligodendrocytes ns well as with astrocytes. This glia! network of communication play s important roles In the homeostasis of brain function. Several studies have also provided the role of oligodendrocyte connex s in acquired dem elinadng CMS disorders, in particular, MS and related experimental models. They also appear to have a regulatory role in

neuminflammation as their absence further aggravates inflammator dentyelinaiion. Again, the results sho wed that EfiP-I Ol prevented die downregulatlon of€!jc3 f eonnexin 29) expression in EAE mice vs control mice. In the light of the relevance of TenmA and Gjc3 for oligodendrocyte functio and myeli preservation, the results further support the potential tty of EHP-101 to he developed as a novel treatment of MS. In on fusion, th disclosed /studies provide· the protective effect of EM! ³ - 10! against demyeikation and its capability to enhance rernyeikaiion. These results open new strategies for the treatment of tmdtipie sclerosis, since novel therapies aimed to axonal reray el mation are urgently needed. la summary, MS is characterized by a eorofekatlon of inflammatory and neyrmiegenerative processes in the spinal cord and the brain. Natura land synthetic eannahkoids Such as VCE-004 have been studied k preelinieal models of MS and, therefore, -represent promising candidates for drug development VCE~004~8 is a maltiiarget synthetic carm bidioi derivative acting as a dual PPARy/CBS ligand agonist that also activates the H!F pathway. .EHP- 101 is an oral Sipldic formulation ofVCE-004.8 that showed efficacy in other preelkieal models of autoimmune diseases.

The efficacy of El IF- 101 In vivo was ev aluated in two murine models of MS such as experimental autoimmune encephalomyelitis (EAE) and onprizone-indueed demyelinaiion. In EAE the transcriptomic analysis was performed by RNA-Seq and qPCR, and inflammatory and myelination markers were detected by kimunaiiistochemisiry (IHC) and eonfoca! microscopy in both models of MS.

EBP- 101 alleviates clinical symptomatology in EAB and transcriptomic analysis demonstrated that EHP- 101 prevented the expression of many Inflammatory genes closely associated with MS pathophysiology in the spinal cord. Eiff fl! normalized the expression of several genes associated with oligodendrocyte function such as Teoeurm 4 (Tenm4) and Gap _j unction gamma-3 (Gjc ^' ) that were downreguiated in EAE EldP-101 treatment prevented microglia activation and demyeimatioo in both the spinal cord and the brain. Moreover, EAE was associated with a loss in the expression of 01ig2 in the Corpus callosum, a marker for oligodendrocyte differentiation, which was restored by EHE-fOl treatment. In addition, EHP- 101 enhanced the expression of glutathione S-transforase pi CGSTpl), a marker for mature oligodendrocytes in the brain. It was also found that a diet containing 0.2 % of cuprizone for six weeks induced a clear loss of myelin in the brain measured by Cfyomyelm staining and MPB expression. Moreover, EHP- 101 al o prevented euprlzone-mbueed microglial activation and sstrog!iosis, reduced axonal da age and decreased plasma levels of Neofotiament Light Polypeptide (NEFL),

The results disclosed herein provide evidence that EMP~ i01 showed potent anti- inflammatory activity, prevented deniydmation and. enhanced rerayelmation, Therefore, EHP- 501 represents a promising drug candidate for the otential treatment of different forms of MS, 21 : Myelin Assessment in Grey and White Matter

Myelin assessment in grey and white matter was evaluated via: ( 5) PLP staining and density in the hippocampus and cortex; and (2) PPD staining and manual counts in the corpus callosum. The myelin assessment in grey and white matter mode! summary is

demonstrated in Table 19. VCE-004,8 was formulated into BHP-10! and daily PO administration of EH P~ 101 was constructed.

Table 19: Myelin Assessment in Grey and White Matter Mode! Summar

Grey Matter Rcmvelinatlon

As shown in Figure 24, PLP staining in the hippocampu and quantification of PLP in the hippocampus demonstrated that EHP401 treated animals showed n change in the area of PLP staining In the hippocampus compared to vehicle control Furthermore, PLP staining in the cortex and quantification of PLP in the cortex demonstrated that EHF401 treated animals at all dose strengths showed no change m the area of PLP staining in the cortical region compared to vehicle control (Figure 25}. White Matter Reinycilnatlon

As shew» iii Figure 26 th agh Figure 29, PFI3 staining; i» the corpus callosum an the myeiit le axoiis ift the corpus callosum e onstr ted that although EBJ-MOi treatments did not skew a significant increase ja myelinated axons compared to control, there as a significant difference between the two higher groups when compared to tire lowest tested group of the test article. Moreover, Figure 26 , Figure 2SA _V and Figure 2§B demonstrated that the higher doses tested of EMP 101 treatments showed a significant iuef ase _: in the density of myelinated: axons compared to control. There was also a significant difference between the two higher groups when compared to the lowest tested group of the test article.

In summary, animals demydlnaied very well, and as expected, as demonstrated by the lack of myelin at the 1240 time point in the Cup- Rap treatment paradigm. There was no significant increase in myehnaiion in the hippocampal area at any dose of VCiE~004.¾, There appeared to be uo significant increase in cortical myeiimdion with any dose of VCE-O04 . VCB- 004.8 appeared to .have a dose related effect on myeiination in white matter in the observed region of the corpus callosum. Increased levels of myelinated axons were observed at higher doses.

Example 22: Oral Administration of EBP-101 Promotes Remyellnaiion In White Matter In th

As stated above. EHP- 101 is air oral llpidi formulation of VCB-004,8, a novel non-psychotropic aminoquinone derivative of synthetic eafttabidlol that recently completed a Phase I clinical study. VCE-004.8 is a dual agonist of the PPARy and CBi receptors with potent antidnfiammatory activity. YGEdXM.S has also demonstrated activation of the HIF pathway in human microvascular endothelial cells, oligodendrocytes, and microglia. In vi vo, EBP-iOt lias been shown t prevent demyeirnation in different murine models of MS and was also shown to induce remyciinario is brain in a mouse euprmone model with less complete demyefinailon, faster reniyeHnatlon, an only a 2- eek treatment window.

As such, the present example focuses on the evaluation of the potential of oral administration of EHP - 101 to promote mmyelinalton h gray and white matter in the cuprizonc/rapamyeio (C/R) mouse model ©f extensive demyeiinafinn with slower spontaneous tcnryelmaiion and a 6-week Peatmen t window,

Male C57BL/6J ( ^:::: 5 or 12/groujp) were treated for 12 weeks with C/8, to cause demyeSinafioo of hite and gray matter regions of the brain. The mice were then orally administered EBP- 101 at 0, 5, 10, and 20 mg/kg/day for 6 weeks (Figure 23). Thereafter, the brains were harvested and processed for immonohistoehemieal staining and quantification of myelinated axons, in gray matter (hippocampus (HIP), cerebral cortex (C ^' DQ) by proleolipid protein (FTP) staining and white matter (corpus callosum CC)) by paraphenyknedlai me {PH}} staining.

After 12 weeks of C/R administration, there was a near complete axonal tissue demyelination in the cortex and hippocampus as quantified by a decrease in myelin proteolipid protei staining and in (he corpus callosum as quantified by paraphenylenediamine staining when compared to age-raalched controls. There was no significant change in the area of PLP staining in the hippocampus and cerebral cortex after EHP-!Oi treatment. There was no significant Increase in gray matter myelinalion when compared to vehicle control following ora! administration of, EHP-101 at > 5 mg/kg/day. in white matter, there: was a dose-dependent increase in the levels of myelinated axons in the corpus callosum. Statistically significant increases in the density of myelinated axons were observed after administration of lil-D ^s ~ 101 at 10 (p < 0.005) and 20 mg/kg/day (p < 0,001) relative to controls.

In summary, in the augmented ctrprizone model of demyelination, oral administration of EI:IP~101 induced significant remyelin lion of demyehnaied axons in while matter but not gray matter. EHP-101 induced a significant, dose-related increase in the density of PPD staining in the corpus callosum. These data support the advancement of EHP-101 into Phase 2 clinical studies as a therapy for treatin MS patients.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this Invention has been disclosed wit reference to specific embodiments, it is apparent that other embodiments and vatia lions of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are Intended to be construed to include all such embodiments and equivalent variations.