Comparing anti-aging hallmark activities of Metformin and Nano-PSO in a mouse model of genetic Creutzfeldt-Jakob Disease.

Advanced age is the main risk factor for the manifestation of late onset neurodegenerative diseases. Metformin, an anti-diabetic drug, was shown to extend longevity, and to ameliorate the activity of recognized aging hallmarks. Here, we compared the clinical, pathologic and biochemical effects of Metformin to those of Nano-PSO (Granagard), a brain targeted anti-oxidant shown by us to delay disease advance in transgenic mice mimicking for genetic Creutzfeldt Jacob disease (CJD) linked to the E200KPrP mutation. We demonstrate that both Metformin and Nano-PSO reduced aging hallmarks activities such as activated AMPK, the main energy sensor of cells as well as Nrf2 and COX IV1, regulators of oxidation, and mitochondrial activity. Both compounds reduced inflammation and increased stem cells production, however did not decrease PrP accumulation. As opposed to Nano-PSO, Metformin neither delayed clinical disease advance in these mice nor reduced the accumulation of sulfated glycosaminoglycans, a pathologic feature of prion disease. We conclude that elevation of anti-aging markers may not be sufficient to delay the fatal advance of genetic CJD.

Beneficial effects of a nano formulation of pomegranate seed oil, GranaGard, on the cognitive function of multiple sclerosis patients.

BACKGROUND: Though often neglected, cognitive impairment is a common feature of multiple sclerosis in 43-70% of patients. None of the novel MS treatment seems to substantially affect or restore cognitive disability in MS. GranaGard (Granalix Bio Technologies LTD) is a food supplement shown to prevent neuronal death in several animal models of neurological diseases. Capsules of GranaGard comprise a self-emulsion nano formulation of pomegranate seed oil (PSO). This oil contains 80-90% of Punicic Acid (PA), one of the strongest natural antioxidants. In animal experiments, administration of GranaGard results in conjugation with linoleic acid (CLA), the main metabolite of PA, which is a well-known neuroprotective agent. AIMS: To investigate whether GranaGard administration has an effect on the cognitive state of MS patients. METHODS: This is a single center, randomized double blind clinical trial that started in May 2018. The study included 30 MS patients; half of them (Group-A) were given GranaGard for the first three months and then placebo pills containing soybean oil for additional three months. Patients in Group-B received placebo for the first three months, and GranaGard for the following three months. GranaGard was administrated in addition to their immunomodulatory MS-treatments. Subsequently, all patients received GranaGard for additional six months. Patients were required to visit the neurologist at baseline (inclusion, visit 1) and at 3 months after treatment-initiation at each cycle of the trial (visits 2 and 3). During the follow up visits, clinical and cognitive examinations were performed, including Expanded Disability Status Scale (EDSS), Multiple Sclerosis Functional Composite (MSFC: 25 ft walking test, 9 PEG hole test & PASAT). Cognitive tests included The Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) battery: 1) Symbol Digit Modalities Test (SDMT); 2) California Verbal Learning Test - Second Edition (CVLT-II); 3) and Brief Visuospatial Memory Test - Revised (BVMT-R). Cognitive outcomes were normalized to the healthy population and expressed as z-scores, depended on age, gender and education. Short quality of life and fatigue questionnaires (SF-12, MFIS-5) were also provided by the participants. RESULTS: No serious adverse effects, related to the product, were observed during the study period. All patients receiving GranaGard reported a ''positive'' effect in their ADL while using the product. While there were no significant differences in the clinical parameters of disability (EDSS scores) between the treatment groups, there was a trend of beneficial effect of GranaGard, on the verbal testing during the first 3-month period of treatment. The z score for CVLT-II, significantly increased (from 0.891 to 1.415, p = 0.012, Wilcoxon rank test) at 3-months in the group of patients who were treated with GranaGard, as compared to baseline. A similar (but not statistically significant) trend was seen also in the BVMTr testing during the same 3 months-period, whereas there was no change in the SDMT. The overall average z-score of all three cognitive functions was significantly improved in the three months of Granagard treatment (-0.0077 at 3 months vs 0.462 at baseline, p = 0.034, Wilcoxon rank test). During the same 3-months period there were no significant changes in the placebo-treated group. For the patients receiving GranaGard in the initial 3 months, the value of z score of CVLT-II remained high (z = 1.415) also at the following three months (while they received placebo), suggesting a longer lasting effect for at least 3 months after discontinuation of the drug. CONCLUSION: This is the first study in which GranaGard, a brain targeted nano-formulation of PSO, was tested in humans. Our results in this small pilot, controlled trial provide indications that GranaGard administration to MS patients might improve/stabilize cognitive disability. Larger studies with longer duration, are needed to confirm these initial observations.

Delay of gCJD aggravation in sick TgMHu2ME199K mice by combining NPC transplantation and Nano-PSO administration.

gCJD is a fatal late-onset neurodegenerative disease linked to mutations in the PRNP gene. We have previously shown that transplantation of neural precursor cells (NPCs), or administration of a nanoformulation of pomegranate seed oil (Nano-PSO, GranaGard), into newborn asymptomatic TgMHu2ME199K mice modeling for E200K gCJD significantly delayed the advance of clinical disease. In the present study, we tested the individual and combined effects of both treatments in older and sick TgMHu2ME199K mice. We show that while transplantation of NPCs at both initial (140 days) and advance clinical states (230 days) arrested disease progression for about 30 days, after which scores rapidly climbed to those of untreated Tgs, administration of Nano-PSO to transplanted TgMHu2ME199K mice resulted in detention of disease advance for 60-80 days, followed by a slower disease progression thereafter. Pathological examinations demonstrated the combined treatment extended the survival of the transplanted NPCs, and also increased the generation of endogenous stem cells. Our results suggest that administration of Nano-PSO may increase the beneficial effects of NPCs transplantation.

Brain targeting of 9c,11t-Conjugated Linoleic Acid, a natural calpain inhibitor, preserves memory and reduces Aß and P25 accumulation in 5XFAD mice.

Deregulation of Cyclin-dependent kinase 5 (CDK5) by binding to the activated calpain product p25, is associated with the onset of neurodegenerative diseases, such as Alzheimer's disease (AD). Conjugated Linoleic Acid (CLA), a calpain inhibitor, is a metabolite of Punicic Acid (PA), the main component of Pomegranate seed oil (PSO). We have shown recently that long-term administration of Nano-PSO, a nanodroplet formulation of PSO, delays mitochondrial damage and disease advance in a mouse model of genetic Creutzfeldt Jacob disease (CJD). In this project, we first demonstrated that treatment of mice with Nano-PSO, but not with natural PSO, results in the accumulation of CLA in their brains. Next, we tested the cognitive, biochemical and pathological effects of long-term administration of Nano-PSO to 5XFAD mice, modeling for Alzheimer's disease. We show that Nano-PSO treatment prevented age-related cognitive deterioration and mitochondrial oxidative damage in 5XFAD mice. Also, brains of the Nano-PSO treated mice presented reduced accumulation of Aß and of p25, a calpain product, and increased expression of COX IV-1, a key mitochondrial enzyme. We conclude that administration of Nano-PSO results in the brain targeting of CLA, and suggest that this treatment may prevent/delay the onset of neurodegenerative diseases, such as AD and CJD.

Mitochondrial dysfunction in preclinical genetic prion disease: A target for preventive treatment?

Mitochondrial malfunction is a common feature in advanced stages of neurodegenerative conditions, as is the case for the accumulation of aberrantly folded proteins, such as PrP in prion diseases. In this work, we investigated mitochondrial activity and expression of related factors vis a vis PrP accumulation at the subclinical stages of TgMHu2ME199K mice, modeling for genetic prion diseases. While these mice remain healthy until 5-6 months of age, they succumb to fatal disease at 12-14 months. We found that mitochondrial respiratory chain enzymatic activates and ATP/ROS production, were abnormally elevated in asymptomatic mice, concomitant with initial accumulation of disease related PrP. In parallel, the expression of Cytochrome c oxidase (COX) subunit IV isoform 1(Cox IV-1) was reduced and replaced by the activity of Cox IV isoform 2, which operates in oxidative neuronal conditions. At all stages of disease, Cox IV-1 was absent from cells accumulating disease related PrP, suggesting that PrP aggregates may directly compromise normal mitochondrial function. Administration of Nano-PSO, a brain targeted antioxidant, to TgMHu2ME199K mice, reversed functional and biochemical mitochondrial functions to normal conditions regardless of the presence of misfolded PrP. Our results therefore indicate that in genetic prion disease, oxidative damage initiates long before clinical manifestations. These manifest only when aggregated PrP levels are too high for the compensatory mechanisms to sustain mitochondrial activity.

Continues administration of Nano-PSO significantly increased survival of genetic CJD mice.

We have shown previously that Nano-PSO, a nanodroplet formulation of pomegranate seed oil, delayed progression of neurodegeneration signs when administered for a designated period of time to TgMHu2ME199K mice, modeling for genetic prion disease. In the present work, we treated these mice with a self-emulsion formulation of Nano-PSO or a parallel Soybean oil formulation from their day of birth until a terminal disease stage. We found that long term Nano-PSO administration resulted in increased survival of TgMHu2ME199K lines by several months. Interestingly, initiation of treatment at day 1 had no clinical advantage over initiation at day 70, however cessation of treatment at 9months of age resulted in the rapid loss of the beneficial clinical effect. Pathological studies revealed that treatment with Nano-PSO resulted in the reduction of GAG accumulation and lipid oxidation, indicating a strong neuroprotective effect. Contrarily, the clinical effect of Nano-PSO did not correlate with reduction in the levels of disease related PrP, the main prion marker. We conclude that long term administration of Nano-PSO is safe and may be effective in the prevention/delay of onset of neurodegenerative conditions such as genetic CJD.

Treatment of a multiple sclerosis animal model by a novel nanodrop formulation of a natural antioxidant.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative stress. In this work, we administered a nanodroplet formulation of pomegranate seed oil (PSO), denominated Nano-PSO, to mice induced for experimental autoimmune encephalomyelitis (EAE), an established model of MS. PSO comprises high levels of punicic acid, a unique polyunsaturated fatty acid considered as one of the strongest natural antioxidants. We show here that while EAE-induced mice treated with natural PSO presented some reduction in disease burden, this beneficial effect increased significantly when EAE mice were treated with Nano-PSO of specific size nanodroplets at much lower concentrations of the oil. Pathological examinations revealed that Nano-PSO administration dramatically reduced demyelination and oxidation of lipids in the brains of the affected animals, which are hallmarks of this severe neurological disease. We propose that novel formulations of natural antioxidants such as Nano-PSO may be considered for the treatment of patients suffering from demyelinating diseases. On the mechanistic side, our results demonstrate that lipid oxidation may be a seminal feature in both demyelination and neurodegeneration.

Pomegranate seed oil nanoemulsions for the prevention and treatment of neurodegenerative diseases: the case of genetic CJD.

Neurodegenerative diseases generate the accumulation of specific misfolded proteins, such as PrP(Sc) prions or A-beta in Alzheimer's diseases, and share common pathological features, like neuronal death and oxidative damage. To test whether reduced oxidation alters disease manifestation, we treated TgMHu2ME199K mice, modeling for genetic prion disease, with Nano-PSO, a nanodroplet formulation of pomegranate seed oil (PSO). PSO comprises large concentrations of a unique polyunsaturated fatty acid, Punicic acid, among the strongest natural antioxidants. Nano-PSO significantly delayed disease presentation when administered to asymptomatic TgMHu2ME199K mice and postponed disease aggravation in already sick mice. Analysis of brain samples revealed that Nano-PSO treatment did not decrease PrP(Sc) accumulation, but rather reduced lipid oxidation and neuronal loss, indicating a strong neuroprotective effect. We propose that Nano-PSO and alike formulations may be both beneficial and safe enough to be administered for long years to subjects at risk or to those already affected by neurodegenerative conditions. FROM THE CLINICAL EDITOR: This team of authors report that a nanoformulation of pomegranade seed oil, containing high levels of a strong antioxidant, can delay disease onset in a mouse model of genetic prion diseases, and the formulation also indicates a direct neuroprotective effect.