Effects of EHP-101 on inflammation and remyelination in murine models of Multiple Sclerosis

Background: Multiple Sclerosis (MS) is characterized by a combination of inflammatory and neurodegenerative processes in the spinal cord and the brain. Natural and synthetic cannabinoids such as VCE-004.8 have been studied in preclinical models of MS and represent promising candidates for drug development. VCE-004.8 is a multitarget synthetic cannabidiol (CBD) derivative acting as a dual Peroxisome proliferator-activated receptor-gamma/ Cannabinoid receptor type 2 (PPAR γ /CB 2) ligand agonist that also activates the Hypoxia-inducible factor (HIF) pathway. EHP-101 is an oral lipidic formulation of VCE-004.8 that has shown efficacy in several preclinical models of autoimmune, inflammatory, fibrotic and neurodegenerative diseases. Methods: The efficacy of EHP-101 in vivo was evaluated in two murine models of MS, the experimental autoimmune encephalomyelitis (EAE) and cuprizone-induced demyelination models. In EAE, transcriptomic analysis was performed by RNA-Seq and qPCR, and inflammatory and myelination markers were detected by immunohistochemistry (IHC) and confocal microscopy in both models of MS. Results: EHP-101 alleviated clinical symptomatology EAE and analysis that EHP-101 prevented the expression of many genes closely MS the spinal cord. EHP-101 normalized the expression of several genes associated with oligodendrocyte such that were activation and demyelination the a in corpus 20 mg/kg vs EAE+Vehicle), a decrease of Tenm4 expression was observed in white matter of spinal cord compared to the CFA group which was prevented by EHP-101 treatment. Taken together, these results are indicative of the potential of EHP-101 to prevent demyelination in EAE model.

3 containing 0.2 % cuprizone for six weeks induced a clear loss of myelin in the brain measured by Cryomyelin staining and Myelin basic protein (MBP) expression. Moreover, EHP-101 also prevented cuprizone-induced microglial activation, astrogliosis and reduced axonal damage. Conclusions: Our results provide evidence that EHP-101 showed potent anti-inflammatory activity, prevented demyelination and enhanced remyelination. Therefore, EHP-101 represents a promising drug candidate for the potential treatment of different forms of MS.

Background
Multiple sclerosis (MS) is an autoimmune disease that affects the central nervous system (CNS) and is characterized by pathological changes, including neuroinflammation, demyelination and axon injury [1][2][3]. The spontaneous repair of damaged myelin sheaths and axons has been described during the remission period of classical relapsing-remitting MS (RRMS), where demyelinated axons could be rewrapped by the regenerated myelin sheath, thus ameliorating axonal dysfunction. In this sense, the remission period is also considered the period of remyelination [4,5], which is important because it could be a key time for the treatment of RRMS patients with drugs preventing inflammation and enhancing remyelination.
Small molecules including cannabinoids acting at druggable targets of the endocannabinoid system (ECS) are being explored for the management of CNS pathologies including MS [6]. In this sense, several lines of evidence suggested a role for the ECS in oligodendrocyte function and remyelination activity in MS [7][8][9]. The ECS is composed by the G-protein coupled receptors Cannabinoid type 1 (CB 1 ) and type 2 (CB 2 ), endocannabinoids and the enzymes regulating their synthesis and catabolism. In addition, cannabinoids of a different nature also target ionotropic receptors of the Transient 4 receptor potential channels (TRP channels) family and nuclear receptors such as peroxisome proliferator-activated receptors (PPARs) [10,11]. CB 1 receptors are expressed mainly in the CNS at neuronal terminals and regulate neurotransmitter release and psychoactive processes. In contrast, CB 2 receptors are located primarily in the peripheral immune system, and its expression is increased during neuroinflammation on activated microglia in the CNS [12,13]. Key considerations for developing CB 2 receptor agonists include absence of psychoactive effects, sustained anti-inflammatory activity, tissue/cell protection, lack of cardiovascular adverse effects and efficacy in several disease models on neuroinflammation including MS [12,[14][15][16][17].
PPARs are members of the nuclear hormone receptor superfamily of ligand-activated transcriptional factors [18] with well-identified regulatory roles in lipid and glucose homeostasis and adipocyte differentiation [19]. In addition to adipocytes and hepatocytes, PPARg has been shown to be expressed in different CNS cells and in immune cells [20].
Furthermore, PPARg has been described as an important factor in the regulation of the immune response [21]. In this sense, PPARg activation has been shown to suppress the expression of inflammatory cytokines in astrocytes and macrophages/microglia [22][23][24]. Although most current therapies for MS are directed towards modulation of the exacerbated immune response [33], novel therapies aimed at axonal remyelination are urgently needed. A novel approach to achieve this would be the hypoxia preconditioning process which, induced by mild oxygen depletion, is beneficial in a wide number of neurological disorders, including MS [34,35]. The cellular adaptation to severe or mild hypoxia is very fast and involves the activation of the hypoxia-inducible factor (HIF)-1α, whose activation may play a role in the inflammatory and the remitting phases of MS (reviewed by [36]). In addition, there is evidence suggesting that activation of the HIF pathway may also be linked to neuroprotection and perhaps remyelination [37]. For instance, erythropoietin (EPO), whose gene is dependent on HIF activation, is neuroprotective in different animal models of MS [38,39].
We have previously shown that VCE-004.8 is a promising cannabidiol (CBD) derivative acting as a dual agonist of PPARg and CB 2 that also activates the HIF pathway [17].
EHP-101 is an oral formulation of VCE-004.8 that also showed efficacy in a murine model of systemic sclerosis (SSc) [40,41]. EHP-101 has completed a Phase I clinical study (clinicaltrial.gov: NCT03745001) and initiation of Phase II studies in SSc and MS patients are being planned. Herein we show the efficacy of EHP-101 in preventing neuroinflammation and demyelination in EAE and enhancing remyelination in the cuprizone model of demyelination.

RNA-Seq and bioinformatic analysis.
Total RNA was isolated from spinal cord tissue using QIAzol lysis reagent (Qiagen, Hilden, 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 mRNA Library Prep Kit (Cat. No. RS-122-2101, Illumina, San Diego, CA, USA). In brief, 1 µg of total RNA from each sample was used to construct a cDNA library, followed by sequencing on the Illumina HiSeq 2500 system with single end 50 bp reads and ~40 millions of reads per sample (n=3 per group). FASTQ files were pre-processed with Trimmomatic (v0.36) [42] and aligned to mouse genome assembly mm10 using HISAT2 (v2.1.0) [43]. Then, counts per gene matrix were obtained with featureCounts (v1.6.1) [44] using the in-built RefSeq annotation for mm10 genome assembly and the differential expression analysis was carried out using DESeq2 (v1.20.0) [45], excluding genes with less than 15 counts across all samples. The functional overrepresentation analyses were performed using EnrichR [46] and clusterProfiler [47]. All the P values were adjusted to control the false discovery rate (FDR) using the Benjamini and Hochberg approach [48]. RNA-seq data have been deposited in the Gene Expression Omnibus databank (accession no. GSE131854).
Quantitative reverse transcriptase-PCR. Total RNA (1 µg) was retrotranscribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and the cDNA analyzed by real-time PCR using the iQTM SYBR Green Supermix (Bio-Rad) and a CFX96 Real-time PCR Detection System (Bio-Rad). Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene was used to standardize mRNA expression in each sample. Gene expression was quantified using the 2-ΔΔCt method and the percentage of relative expression against controls was represented. The primers used in this study are described in Table 1.

Data analysis.
All the in vivo data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) followed by the Tukey´s post-hoc test for parametric analysis or Kruskal-Wallis post-hoc test in the case of non-parametric analysis tests were used to determine the statistical significance. The level of significance was set at p˂0.05.

EHP-101 attenuates clinical severity and neuroinflammation in EAE.
The efficacy of EHP-101 in MS was first evaluated in EAE, performing the treatments at an early stage of the disease since mice received increasing doses of EHP-101 at day 8 post-immunization (p.i.). Subcutaneous immunization with MOG35-55 induced EAE in all mice that received the vehicle alone. All vehicle-treated mice developed a disease that peaked by day 16 p.i. and maintained at day 28 p.i. By contrast, the reduced clinical score showed therapeutic efficacy of EHP-101 with all the dose levels tested, with the highest dose (20 mg/kg) able to prevent the symptoms completely (Figure 1a  Cerebral cortical demyelination as well as callosal pathology are widely recognized features of MS [49][50][51]. In addition, the cerebral cortex plays a central role in interhemispheric communication, and callosal atrophy in MS patients has been shown to correlate with disability status [52][53][54]. Therefore, we also examined whether these structures might also be affected in EAE mice. An increase in inflammatory lesions was seen throughout the EAE forebrain ( Figure 3a). Specifically, we observed that microglial reactivity was increased in corpus callosum of EAE mice and the treatment with EHP-101 reverted the microgliosis process (Figure 3b

EHP-101 normalizes EAE transcriptomic signature in the spinal cord
To evaluate the global expression changes produced by EHP-101 treatment, we performed an RNA-Seq analysis of the spinal cords from mice in the following conditions: Control, EAE and EAE with EHP-101 treatment (20 mg/kg). Sequencing data for three biological replicates were obtained for each experimental group. Then, we compared the transcriptomic profile between the different conditions to get a first insight into the changes occurring in the model, with or without treatment. As expected, we found many 13 changes, both in magnitude and significance in EAE mice compared to the group treated with EHP-101 ( Figure 4a). Then, to evaluate those changes at a biological level, we performed an over-representation analysis using genes that surpassed the cutoff of an adjusted P < 0.05 and absolute fold change > 2 in the EAE vs control and EAE+EHP-101 vs EAE comparisons. The more significant enrichments were found in the groups of upregulated genes by EAE and downregulated genes by the treatment. We observed a complementary signature between those two groups, where terms like "neutrophilmediated immunity", "inflammatory response" or "cytokine-mediated signaling pathway" Next, we performed a second analysis to explore changes in the opposite direction to the 14 pattern shown by the pro-inflammatory genes. Thus, we selected down-regulated genes in the EAE vs control comparison and up-regulated in EAE+EHP-101 vs EAE comparison. We intersected both groups of genes to evaluate the overlap between them, resulting in a total of 193 genes downregulated in the untreated model that increased their expression in response to the treatment (Figure 5a). Then we performed a second functional analysis, using the list of overlapping genes as input, to explore the most significantly enriched Gene Ontology (GO) terms. As depicted in Figure 5b, we found several terms related to the metabolic process of sterols and hydroxy compounds at the top of the list. However, given the background of the disease, we decided to focus on the "myelination" process. To explore the changes of features belonging to this annotation, we depicted the expression levels of genes that produced this result in the heatmap shown in Figure 5c. This allowed us to identify several key genes of the myelination process that were restoring their levels with EHP-101 treatment. Interestingly, these results indicated that EHP-101 normalized the expression of several genes associated with oligodendrocyte function, such as Gap junction gamma-3 (Gjc3), also called Connexin 29, and Teneurin-4 (Tenm4) that were downregulated in EAE. These results are relevant since Tenm4 has been described as a critical regulator of oligodendrocyte differentiation and CNS myelination [55]. To validate the transcriptomic analysis, we studied the expression of Gjc3 and Tenm4 by RT-PCR

EHP-101 accelerates remyelination in cuprizone-challenged mice.
To evaluate the effect of EHP-101 on remyelination during the acute CPZ-induced demyelination protocol (Figure 6a), brain coronal sections from animals after 6 weeks of CPZ 0.2% diet and 2 weeks of EHP-101 treatment were evaluated. In this model, EHP-101 treatment was started after removal of the CPZ diet to study the effect of EHP-101 on spontaneous remyelination. First, the evaluation of MBP was determined by CryoMyelin and IHC staining (Figure 6b and 6c  GFAP + cells in the corpus callosum. In control mice, low expression levels of Iba-1 + and GFAP + cells were detected but mice exposed to CPZ showed microglial and astrocytic activation, which was attenuated by EHP-101 treatment (Figure 7a and 7b). Quantitative assessment also showed a significant increase in the number of Iba1 + and GFAP + cells in corpus callosum upon CPZ intoxication. Microgliosis and astrocytic activation was ameliorated after 1 week of EHP-101 treatment (Figure 7c p= ˂ 0.0001 CPZ6W, CPZ6+1W, CPZ6+2W vs Control; p= 0.0017 CPZ6+1W + EHP-101 20 mg/kg vs CPZ6+1W; Figure 7d p= ˂ 0.0001 CPZ6W, CPZ6+1W vs Control; p= 0.0017 CPZ6+2W vs Control). To examine the effects of EHP-101 on cuprizone-induced demyelination of axons in the corpus callosum, we investigated the non-phosphorylated form of neurofilament proteins (SMI-32 staining). Although SMI-32 immunoreactivity is normally seen in axons, its accumulation in axonal spheroids is a characteristic of axonal pathology. Increased SMI-32 labeling after 6 and 7 weeks of CPZ intoxication demonstrated that there was a significant effect on axons and this effect was ameliorated after 1 week of EHP-101 treatment (Figure 8).

Discussion
Natural products, including phytocannabinoids, have been successfully used for the development of synthetic and semisynthetic derivatives with improved bioactivities [56].
We have developed the compound VCE-004.8, a synthetic derivative of CBD, which is a dual agonist for PPARg/CB 2 that also inhibits the activity of HIF prolyl hydroxylases (PHDs) [17,57]. Therefore, VCE-004.8 is targeting several pathways that may have a positive effect on neuroinflammation and remyelination as observed in EAE and Theiler's Murine Encephalomyelitis Virus-induced demyelinating models [17]. Herein we have studied the effect of EHP-101, an oral lipidic formulation of VCE-004.8, in two of the most commonly used models of demyelination, which are EAE and toxically induced demyelination via cuprizone [58]. EAE in C57Bl/6 mice has generally been thought to predominantly target the spinal cord, leading to sensory and motor impairments. Nevertheless, it is also recognized that EAE involves other CNS structures including the cerebellum and the hippocampus [59,60]. Our data clearly indicate that EHP-101 is effective in alleviating neuroinflammation in the spinal cord, in the cerebral cortex and in the corpus callosum. In the EAE model we cannot distinguish whether the effect of EHP-101 occurs in the peripheral immune system, in the CNS or both. It has been demonstrated that the blood-brain-barrier (BBB) is disrupted in EAE allowing for the migration of autoimmune cells and molecules to the brain [61].
However, it is likely that EHP-101 may exert anti-inflammatory effects by acting both in the peripheral immune system and in the CNS. For instance, EHP-101 showed antiinflammatory activity in another autoimmune disease, systemic sclerosis, where the BBB is not affected [41] and herein we show that EHP-101 also alleviates neuroinflammation in CPZ-intoxicated mice. CPZ-induced demyelinating lesions are characterized by severe oligodendrocyte loss and demyelination with concomitant activation of microglia and astrocytes, but it does not induce BBB damage [62] and lacks the characteristic T-cell infiltration and consequently the peripheral autoimmune component of the disease [2].
The mechanism of action of EHP-101 in the remyelination process is still unknown but it can be probably related to the HIF pathway [17]. Extensive experimental studies have revealed that activating HIF-1a by inhibiting the activation of PHDs can provide neuroprotection and perhaps remyelination mainly from the increased expression of HIF-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-1 activity may be an important potential strategy to prevent the onset or to ameliorate the pathogenesis of neurodegenerative diseases [63]. Interestingly, the improvement of the myelination index was paralleled by enhancement of oligodendrocyte progenitor cell (OPC) proliferation, platelet-derived growth factor (PDGF)a-receptor expression, and precursor migration from the CC midline to the lateral parts followed by an induction of the expression of myelin protein. In addition, early astrogliosis in the demyelinated areas paralleled a moderate stimulation of insulin-like growth factor (IGF)-1 expression [2]. IGF-1 synergizes with fibroblast growth factor (FGF)-2 to stimulate oligodendrocyte progenitor entry into the cell cycle [64]. This is of particular interest because IGF-1 induced HIF-1 activation that can be mimicked by VCE-004.8 in the brain, and PDGFa and FGF2 are also regulated by VCE-004.8-mediated activation of the HIF pathway [17,65,66].
Demyelination and partial axonal damage in MS lesions are closely 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 of fast axonal transport [67,68]. Reactive microglia produce a large array of toxic and proinflammatory molecules, which triggers myelin destruction, oligodendrocyte deterioration, axon damage and even neuronal loss [69] [70]. Here we found that oral EHP-101 also prevented microglia activation and demyelination in both spinal cord and brain suggesting that VCE-004.8 penetrates the brain in EAE mice after oral absorption.
Moreover, we also found that EHP-101 preserves the axonal structure ameliorating the typical accumulation of spheroids of SMI-32 used as a marker of axonal damage in CPZ intoxicated mice [67,71,72]. Again, this result suggests that VCE-004.8 can also cross the BBB, which is not affected in the CPZ model [62].
Oligodendrocyte progenitor cells (OPCs) are produced from neuroepithelial stem cells and subsequently proliferate and migrate throughout the entire spinal cord [73]. During differentiation, oligodendrocytes initiate expression of myelin proteins critical for the achievement of proper functioning of the CNS [74]. Teneurin-4 (Tenm4) is a type II transmembrane protein that is highly expressed in the CNS and whose expression is induced in response to endoplasmic reticulum stress [75] and has been suggested to be involved in bipolar disorder in humans [76]. A mouse mutation, designated furue, which results in tremors and severe hypomyelination of small-diameter axons, reduces oligodendrocyte differentiation especially in the spinal cord of the CNS, and it has been associated with the absence of Tenm4 expression. Thus, Tenm4 is a critical regulator of oligodendrocyte differentiation and CNS myelination [55]. Herein we showed for the first time that in EAE mice the expression of Tenm4 is downregulated in the spinal cord and the treatment with EHP-101 reverses this downregulation probably as a result of the antiinflammatory activity of VCE-004.8.
In addition, oligodendrocytes are electrically and metabolically coupled through intercellular channels called gap junctions (GJs), composed of connexins Cx29, Cx32 and Cx47 [77], with other oligodendrocytes as well as with astrocytes. This glial network of communication plays an important role in the homeostasis of brain function [78,79].      Effect of EHP-101 treatment on remyelination in acute CPZ-induced demyelination. (a) Experimental procedure (b). Histological study of myelin by of GFAP in corpus callosum and quantification of intensity is shown (d). Data represent the mean ± SEM, and significance was determined by one-way ANOVA followed by the Tukey´s post-hoc test. ***p < 0.001 CPZ 6W or CPZ 6W + 1 or CPZ 6W + 2 vs Control; **p˂0.01 CPZ 6W + 2 vs Control; ##p < 0.01 CPZ 6W +1 + EHP-101 vs CPZ 6W +1. 44