Use of a composition comprising 7-hydroxymatairesinol
11590101 · 2023-02-28
Assignee
Inventors
Cpc classification
A61P25/28
HUMAN NECESSITIES
International classification
A61P25/28
HUMAN NECESSITIES
Abstract
7-hydroxymatairesinol and a composition thereof for ameliorating the neurodegenerative disorders, in particular the disorders associated with dopaminergic neurons loss, such as the Parkinson's disease.
Claims
1. A method for alleviating Parkinson's disease in a person in need thereof comprising administering to said person an effective amount of 7-hydroxymatairesinol (HMR).
2. The method according to claim 1, wherein said effective amount is from about 10 to about 500 mg per day per adult person.
3. The method according to claim 1, wherein said effective amount is from about 30 to about 300 mg per day per adult person.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The present invention will be better understood by reading the following examples, given by way of illustration and not of limitation, to be read with the accompanying drawings, wherein:
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EXAMPLES
(9) The effects of natural compound HMR were tested in a rodent model of Parkinson's disease-like nigrostriatal neurodegeneration caused by stereotaxic injection of dopaminergic neurotoxin 6-OHDA.
(10) The experiments were carried out on male Sprague-Dawley rats (Charles River, Calco, LC, Italy), weighing 200-225 g at the beginning of the experiment, housed one per cage at 20-22° C. on a 12-h light/dark cycle, with food and water ad libitum. Animals were left in the housing facilities for at least 1 week before initiating the experiments.
(11) The Parkinson's animal model used in the present experimental protocol is based on the local, unilateral injection of the 6-hydroxydopamine (6-OHDA) neurotoxin into the corpus striatum via stereotactic surgery.
(12) Animals were anaesthetized with Zoletil (50 mg/kg; Virbac) and placed into a stereotactic frame (Stoelting), with the incisor bar positioned 3.3 mm below the interaural line. Animals received a unilateral injection of 6-OHDA (20 μg in 3 μL of saline solution with 0.02% ascorbic acid; Sigma) into the right striatum (1.0 mm anterior, 3.0 mm lateral and 5.0 mm ventral, with respect to bregma and dura) at 1 μL/min using a Hamilton 10 μL syringe with a 26-gauge needle. The needle was left in place for 5 min before being retracted to allow complete diffusion of the medium, and wounds were clipped.
(13) Rats (n=33) were randomly assigned to different treatment groups, in order to evaluate the effects of HMR when this was administered before (PRE group) or after (POST group) induction of the nigrostriatal lesion.
(14) Animals in the PRE group received for 4 weeks before 6-OHDA infusion either 5% sucrose enriched food with HMR (10 mg/Kg per day; n=9) or 5% sucrose enriched food without HMR (vehicle; control n=8). After 4 weeks the animals were subjected to 6-OHDA injection and sacrificed after 4 more weeks.
(15) Animals in the POST group received 5% sucrose enriched food with HMR (10 mg/Kg per day; n=8) or vehicle (control, n=8) for 4 weeks, starting right after the 6-OHDA injection. The animals were sacrificed at the end of the fourth week of treatment.
(16) Food consumption and body weight changes were monitored daily. No differences between treated and untreated rats were detected, either in food intake or weight gain, throughout the experiments, indicating that all animals in the active treatment group received the same amount of HMR.
(17) Twenty-eight days after 6-OHDA infusion, animals were deeply anesthetized with 150 mg/kg of Zoletil, underwent cardiac puncture blood sampling and were perfused with saline and ice-cold 4% paraformaldehyde (Merck).
(18) Brains were rapidly removed, post-fixed for 24 hours in the same fixative and subsequently transferred in solutions of sucrose at increasing concentrations (up to 30%).
(19) Brains were cut in serial coronal sections containing both the striatum and the SNc using a microtome (Leica SM 2000R) and were stained for different markers.
(20) Different tests were run on blood and brain samples.
(21) Statistical analysis results were expressed as mean±SEM (Standard Error of the Mean). Statistical analysis was performed using the GraphPad Prism 3 software (GraphPad software, SanDiego, Calif., USA). Comparisons between groups were made using Student's t-test for unpaired data. Statistical significance was set at p<0.05.
(22) Image analysis was performed using an AxioSkop2 microscope connected to a computerized image analysis system (AxioCam MR5) with dedicated software (AxioVision Rel 4.2).
(23) Extensive experimental data, shown in the following examples 1 to 5, demonstrated that chronic treatment with HMR, when started right after the infusion of 6-OHDA, induces an appreciable reduction of the nigrostriatal damage caused by the neurotoxin. This was associated with reduced microglia activation and a preferential expression of the anti-inflammatory, cytoprotective M2 microglial phenotype. No such effect was observed in animals treated with HMR before 6-OHDA injection.
(24) Test 1—HMR Plasma Levels Assay
(25) Concentration of HMR and its major metabolite enterolactone was measured in plasma samples (50 μL) from rats fed for 4 weeks with 5% sucrose enriched food with HMR, using an LC-MS/MS system (AP14500 Qtrap ABSciex).
(26) Calibration curves in the range from 0.1 ng/mL to 100 ng/mL were obtained from measurements on control samples containing the pure analytes, prepared from stock solutions of HMR and enterolactone in MeOH at the concentration of 1 mg/mL. Said stock solutions were diluted in acetonitrile (ACN) at a concentration of 250 μg/mL, then working solutions were prepared by sequential dilution in ACN.
(27) 45 μL of plasma and 5 μL of working solution were added to 300 μL of an ACN solution containing the internal standard catechin (conc. 200 ng/mL). After mixing, samples were centrifuged for 5 min at 3000 g at 5° C., and injected into LC-MS/MS.
(28) The plasma samples from the in vivo treatment were prepared subjecting 50 μl of sample to centrifugation for 5 min at 3000 g at 5° C., and were injected into LC-MS/MS.
(29) Calibration curves allowed determining, by comparison, the amount of each analytes in the plasma samples obtained from the rats of the study.
(30) HMR was detected in the plasma of all animals in the active treatment groups, with a mean value of 0.92±0.74 ng/mL.
(31) Enterolactone was below the detection limit of 0.1 ng/mL (no peak detected) in all the analysed samples.
(32) Test 2—Nigrostriatal Damage Evaluation
(33) The nigrostriatal lesion was assessed by immunohistochemistry for the dopaminergic marker tyrosine hydroxylase (TH) on coronal sections of both SNc and striatum.
(34) Sections were processed with rabbit anti-TH primary antibody (1:1000, Chemicon) and a biotinylated anti-rabbit IgG secondary antibody (Vector Laboratories), then revealed using a commercial kit based on the avidin-biotin technique (Vectastain ABC Elite kit, Vector Laboratories). Reaction products were developed using nickel-intensified 3′-3′-diaminobenzedine tetra-hydrochloride (DAB Substrate Kit for Peroxidase, Vector Laboratories).
(35) In the SNc, TH-positive (TH+) neurons were counted bilaterally on every four section throughout the entire nucleus using the unbiased stereological optical fractionator method (Stereo Investigator System, Microbrightfield Inc.).
(36) TH+ neurons are those still producing dopamine, therefore the extent of the neurodegeneration, represented by the amount of dead dopaminergic neurons in the lesioned hemisphere, was extrapolated by comparison with the amount of active dopaminergic neurons in the intact hemisphere. Results were expressed as the percentage of TH+ neurons in the lesioned SNc compared to that found in the intact hemisphere, for each group of animals.
(37) On the lesioned striatum, densitometric analysis was performed to evaluate the loss of dopaminergic terminals. In this case, the striatal degeneration was evaluated and expressed as the percentage of striatal volume deprived of TH immunoreactivity, with respect to the entire striatal volume of the lesioned hemisphere.
(38) The intra-striatal injection of 6-OHDA induced marked loss of TH+, dopaminergic, terminals and cell bodies in the ipsilateral nigrostriatal pathway of all vehicle-treated animals; no loss of TH+ terminals or cell bodies was observed in the contralateral, intact, hemisphere.
(39) The comparison between vehicle-treated animals and animals that received HMR before the intra-striatal injection of 6-OHDA showed no differences in nigrostriatal damage, either at the striatal or SNc level.
(40) At day 28 post-surgery, in the lesioned side of the brain it was possible to observe up to 55% TH+ neurons depletion in the SNc and 65% TH+ terminals loss in the striatum, as compared to the contralateral hemisphere, in both HMR-treated and control animals.
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(42) Surprisingly, substantial differences were evident between animals that received HMR immediately after 6-OHDA infusion and the control group.
(43) At day 28 post-surgery, in the lesioned SNc it was possible to observe about 20% reduction of the 6-OHDA induced loss of TH+ neurons in rats treated with HMR after 6-OHDA injection, as compared to the vehicle-treated animals (p=0.055).
(44) Additionally, in the lesioned hemisphere striatum a statistically significant (p<0.05), about 32% reduction of TH+ terminal loss was observed in rats treated with HMR, as compared to the control group.
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(47) Test 3—Neuroinflammation Evaluation Through Microglia Activation Assessment.
(48) The evaluation of the 6-OHDA induced neuroinflammation was carried out via immunofluorescent staining of coronal SNc sections of both HMR-treated and control animals.
(49) Microglia activation was assessed by observing sections of the lesioned SNc stained for CD11 b, under low and medium magnification (20×-40×), both quantitatively, by counting the microglial cells in the lesioned SNc, and qualitatively, analysing the morphological changes of the cells.
(50) Mouse anti CD11b (MCA275R, Serotec, 1:300) primary antibody was used to assess microglia activation and density in the SNc. Anti-TH primary antibody was added for localization and cell count. Goat anti-mouse AlexaFluor488 and goat anti-rabbit AlexaFluor594 (Life Sciences, 1:300) were used as secondary antibodies.
(51) Cell count was performed by analysing three different SNc sections, chosen according to rostro-caudal coordinates.
(52) Morphological changes mirroring the state of activation were scored according to the Colburn scale. Cell density was assessed by counting CD11 b+ cells from a stack of 16 pictures (in a 0.04 mm.sup.2 frame, 1 μm-thick, 40× magnification) taken from three discrete areas of the same SNc section.
(53) Colburn's scale takes into account four activation stages, which differ for the cells morphology and density, and are summarized below.
(54) Resting stage (score 0): microglial cells are ramified, well spaced-out, with a small cell body.
(55) Stage 1 (score +): microglial cells are ramified, with a decreased intercellular space.
(56) Stage 2 (score ++): microglial cells show a loss of ramification and increased density.
(57) Stage 3 (score +++): microglial cells show short and think ramifications.
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(59) The comparison between vehicle-treated animals and animals that received HMR before the intra-striatal injection of 6-OHDA showed no differences in the microglial cell count. Both groups showed an average of 750 cells/mm.sup.2.
(60) Morphological evaluation showed moderate microglia activation in both vehicle-treated and HMR-treated animals, but no differences were observed in the density of CD11b+ cells between the two groups. The below table 1 summarizes the morphological evaluation of both groups according to Colburn's scale.
(61) TABLE-US-00001 TABLE 1 Animals Colburn's score HMR-treated PRE-group ++ Control group ++
(62) Surprisingly, animals that received HMR immediately after 6-OHDA infusion showed lower microglia activation than the control group, both in terms of cell count and of morphology.
(63) Table 2 below summarizes the morphological evaluation of both groups according to Colburn's scale.
(64) TABLE-US-00002 TABLE 2 Animals Colburn's score HMR-treated POST-group + Control group ++
(65) This was accompanied by a significant decrease in the density of CD11 b+ cells in the SNc of HMR-treated animals (550 cells/mm.sup.2) with respect to the vehicle-treated group (750 cells/mm.sup.2).
(66) Test 4—Analysis of Microglia Polarization.
(67) The analysis of microglia polarization was performed by evaluating the percentage of CD32+ and CD206+ cells, respectively M1 and M2 phenotypes marker, of the total microglia cells in the lesioned SNc, using respectively CD32 or CD206 (Santa Cruz, 1:300) antibodies.
(68) Since no significant differences in microglial activation were observed between vehicle-treated animals and animals that received HMR before the intra-striatal injection of 6-OHDA, microglia polarization was evaluated only in the groups treated with HMR after 6-OHDA infusion.
(69) Remarkably different patterns of microglia polarization were observed in the HMR-treated animals as compared to the control group.
(70) Microglia polarization was mainly oriented toward the neuroprotective, M2 (CD11b+/CD206+) phenotype in animals treated with HMR, while vehicle-treated animals showed that microglia was mainly polarized toward the M1, neurotoxic (CD11b+/CD32+) phenotype. No evidence of the M2 phenotype was detected in this latter group.
(71) Test 5—Oxidative Stress Evaluation
(72) Plasma levels of reduced (GSH) and oxidized (GSSG) glutathione were estimated by using a biochemical Glutathione assay kit (Cayman Chemical), as an index of antioxidant effect of HMR treatment.
(73) Results were expressed as a GSH/GSSG ratio.
(74) Since no significant difference in neuroinflammation was observed between vehicle-treated animals and animals that received HMR before the intra-striatal injection of 6-OHDA, oxidative stress was evaluated only in the groups treated with HMR after 6-OHDA infusion.
(75) In animals treated with HMR after 6-OHDA infusion, the results, although not reaching statistical significance, showed a trend toward an increased GSH/GSSG ratio, when compared to vehicle-treated animals, expressing greater availability of reduced GSH.
(76)