Inhibition of trehalase to enhance systemic trehalose exposure

Abstract

There is described herein a method of treating a patient with a neurological disorder or a cardiovascular disorder, the method comprising administering to the patient trehalose in combination with a trehalase inhibitor to achieve a plasma Cmax of 1000 - 12500 ng / ml of trehalose in the patient.

Description

[0001] INHIBITION OF TREHALASE

[0002] TO ENHANCE SYSTEMIC TREHALOSE EXPOSURE

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] The application claims priority to U.S. Provisional Patent Application No. 63 / 722,638 filed on November 20, 2024, the contents of which are hereby incorporated by reference in their entirety.

[0005] FIELD OF THE INVENTION

[0006] The invention relates to the treatment of neurological disorders, including Parkinson’s disease, and more particularly to the inhibition of trehalase to enhance trehalose treatment.

[0007] BACKGROUND OF THE INVENTION

[0008] Trehalose, a disaccharide, is designated as a Natural Health Product (NHP) by Health Canada and Generally Regarded As Safe (GRAS) by the FDA, and thus available for clinical use without additional safety or toxicology studies.

[0009] There has been described the therapeutic potential of trehalose and defined plasma exposures associated with efficacy in rodent and primate models of Parkinson’s Disease (1 ,2). Trehalose has also been studied generally in relation to synucleinopathies and proteinopathies, as well as spinocerebellar ataxia 3 (5) and cardiovascular disease (6). WO2017 / 136922 generally describes the use of trehalose for the treatment of neurological diseases. Trehalose has also demonstrated preclinical efficacy in cancer, aging, cardiometabolic disorders, and infectious diseases (7).

[0010] Several groups are studying the therapeutic potential of trehalose; however, they are either investigating intravenous trehalose, which for several reasons is a problematic way of chronically delivering trehalose and may avoid potential gut-mediated benefits of trehalose (3,4), or they are using oral trehalose without knowing if exposures in the clinical study are sufficient to provide benefit.

[0011] Thus, the clinical pharmacokinetics of trehalose remain unknown and it cannot be determined the appropriate clinical doses that provide adequate trehalose exposure.

[0012] SUMMARY OF THE INVENTION

[0013] In an aspect, there is provided a method of treating a patient with a neurological disorder or a cardiovascular disorder, the method comprising administering to the patient trehalose in combination with a trehalase inhibitor to achieve a plasma Cmax of 1000 -12500 ng / ml of trehalose in the patient.

[0014] In an aspect, there is provided trehalose for use in the treatment of a neurological disorder or a cardiovascular disorder in combination with a trehalase inhibitor to achieve a plasma Cmax of 1000-12500 ng / ml of trehalose in the patient.

[0015] In an aspect, there is provided a use of trehalose and a trehalase inhibitor in the manufacture of a medicament for the treatment of a neurological disorder or a cardiovascular disorder, wherein the trehalose and the trehalase inhibitor are in effective amounts to achieve a plasma Cmax of 1000-12500 ng / ml of trehalose in the patient.

[0016] In an aspect, there is provided a kit comprising trehalose and a trehalase inhibitor for the treatment of a neurological disorder or a cardiovascular disorder, wherein the trehalose and the trehalase inhibitor are in effective amounts to achieve a plasma Cmax of 2500 -12500 ng / ml of trehalose in the patient.

[0017] In an aspect, there is provided a pharmaceutical composition comprising trehalose and a trehalase inhibitor in therapeutically effective amounts to achieve a plasma Cmax of 2500 -12500 ng / ml of trehalose in a patient, along with a pharmaceutically acceptable carrier BRIEF DESCRIPTION OF FIGURES

[0018] These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

[0019] Figure 1 : Effect of tannic acid on trehalose plasma levels. Data are mean ± s.e.mean, N=28-29. * = P<0.05, *** = P<0.001 . One way ANOVA followed by Fisher’s LSD.

[0020] Figure 2: Effect of reducing trehalose dose in the presence of tannic acid (150 mg / day) or tannic acid (450 mg / kg) and validamycin A (0.525 mg / kg), N=27-29, mean ± s.e.mean. *** = P<0.001 . One way ANOVA followed by Fisher’s LSD.

[0021] Figure 3: Comparison of trehalase activity in duodenal samples collected from people with Parkinson’s disease and age-matched controls. N = 9 (PD subjects) and 17 (age- matched controls). Mean ± SD.

[0022] Figure 4: Effect of trehalose and the combination of low-dose trehalose + trehalase inhibitors on TH+ve cells in the substantia nigra (TH), striatal dopamine (DA), striatal noradrenaline (NE), striatal DAT, and forelimb asymmetry on Day 42. Mean ± s.e.mean, N=12-18. * / ** / **** = P <0.05 I 0.01 / 0.001 vs. vehicle / EV. + = P <0.05 vs. vehicle / aSyn. One way ANOVA followed by Fisher’s LSD post-hoc test. Tre: trehalose, TA: tannic acid, Vai A: validamycin A.

[0023] Figure 5: Effect of trehalose and the combination of high-dose trehalose + trehalase inhibitors on TH+ve cells in the substantia nigra (TH), striatal dopamine (DA), striatal noradrenaline (NE), striatal DAT, and forelimb asymmetry on Day 42. Mean ± s.e.mean, N=12-18. * / ** / **** = P <0.05 I 0.01 / 0.001 vs. vehicle / EV. + = P <0.05 vs. vehicle / aSyn. One way ANOVA followed by Fisher’s LSD post-hoc test. Tre: trehalose, TA: tannic acid, Vai A: validamycin A.

[0024] Figure 6: Effect of trehalose and the combination of high-dose trehalose + trehalase inhibitors on fecal mass produced in 24 h in 6-OHDA-lesioned rats. Mean ± s.e.mean, N=8-9. Data was analysed by two-way ANOVA followed by Fisher’s LSD post-hoc test. Fecal output was significantly decreased in 6-OHDA rats, compared to unlesioned rats, at all timepoints post-surgery. No treatment (Groups 3-7) significantly altered fecal output compared to 6-OHDA alone. Figure 7: Effect of repeated oral administration of trehalose on striatal dopamine levels in the AAV1 / 2-hA53T-aSyn rat. Trehalose (2.67 g / kg per day) was administered in drinking water [(DW), 2% w / v], as three separate oral administrations 8 hours apart (0.89 g / kg three times a day, t.i.d) or as a single oral administration (2.67 g / kg daily, q.d) for 6 weeks. Mean ± s.e.mean., N = 5-8 rats / group. *** = P <0.001 vs. vehicle / EV vs. AAV1 / 2-EV, #=P <0.05 vs. AAV1 / 2-hA53T-aSyn. One-way ANOVA followed by Fisher’s least-significant difference post hoc test.

[0025] Figure 8: Comparative timecourse of trehalose exposure in the plasma of rats (N=5) and macaques (N=3) following oral administration of trehalose (2.67 g / kg, p.o. administered as a single bolus dose). Mean ± s.e.mean.

[0026] Figure 9. Effect of treatments on forelimb asymmetry (baseline, D22, and D42) in the AAV1 / 2-hA53T-aSyn rat model of Parkinson’s disease. Extent of forelimb asymmetry was assessed via cylinder test before (baseline) and 22 and 42 days following AAV1 / 2 surgery. (A) Baseline. (B) D22. (C) D42. * represents P<0.05 cf. AAV-EV. ++ represents P<0.01 cf. AAV-aSyn. $$$ represents P<0.001 cf. AAV-aSyn + trehalose (0.75 g / day) + tannic acid (150 mg / kg). # represents P<0.05, cf. AAV-aSyn + trehalose (1.0 g / day) + tannic acid (150 mg / kg). Data are mean ± s.e.mean, N-14-16 rats / group. 1-way ANOVA with Fisher’s LSD post hoc test.

[0027] Figure 10. Effect of treatments on striatal dopamine in the AAV1 / 2-hA53T-aSyn rat model of Parkinson’s disease. Striatal dopamine levels were quantified by LC-MS / MS. * represents P<0.05 cf. AAV-EV. + represents P<0.05 cf. AAV-aSyn + trehalose (1 .0 g / day) + tannic acid (150 mg / kg). $ represents P<0.05 cf. AAV-aSyn + trehalose (0.75 g / day) + tannic acid (150 mg / kg). Data are mean ± s.e.mean, N-14-16 rats / group. 1- way ANOVA with Fisher’s LSD post hoc test.

[0028] Figure 11. Effect of treatment on striatal levels of transgene-derived human aSyn in the AAV1 / 2-hA53T-aSyn rat model of Parkinson’s disease. Striatal levels of human aSyn were quantified by ELISA. **** represents P<0.0001 cf. AAV-EV+ vehicle. Data are mean ± s.e.mean, N-14-16 rats / group. 1-way ANOVA with Fisher’s LSD post hoc test for multiple comparisons.

[0029] Figure 12. Effect of test item on trehalose levels in the plasma, 30 minutes after administration of trehalose. Data are mean ± s.e.mean, N=14-16. Figure 13. Effect of test item on trehalose levels in the CSF, 30 minutes after administration of trehalose. Data are mean ± s.e.mean, N=14-16.

[0030] DETAILED DESCRIPTION

[0031] In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.

[0032] We herein define target trehalose exposures for clinical studies. However, the large amounts of trehalose required to achieve them may cause long-term complications. To reduce this risk, we demonstrated that inhibition of trehalase, the enzyme which metabolises trehalose to glucose, allows the trehalose dose to be lowered whilst maintaining trehalose exposure and beneficial effects. One inhibitor, tannic acid, is an NHP / GRAS compound, and the combination of trehalose and tannic acid is described herein. As both trehalose and tannic acid are approved for human use, clinical trials can be rapidly progressed.

[0033] We have demonstrated the following. Combining trehalose with trehalase inhibitors increases systemic and CNS trehalose exposure. Defined doses of trehalose which, in combination with trehalase inhibitors, provide similar exposures to those produced by a dose of trehalose (2.67 g / kg) that is efficacious when given alone. Trehalase activity in the duodenum of people with PD is the same as activity in age-matched controls, thus demonstrating that trehalase is a target that is present and engageable in people with PD. Low dose oral trehalose plus trehalase inhibition protected against aSyn-induced dopaminergic deficits to a level equivalent to, or greater than, high dose trehalose alone. High dose oral trehalose plus trehalase inhibition did not provide additional benefits over high dose oral trehalose alone in a rat model of PD. Treatments based around trehalose and trehalase inhibitors do not worsen Gl dysfunction in a rat model of PD. The rat model of PD utilized is a well known and accepted animal model of PD.

[0034] In an aspect, there is provided a method of treating a patient with a neurological disorder or a cardiovascular disorder, the method comprising administering to the patient trehalose in combination with a trehalase inhibitor to achieve a plasma Cmax of 2500 -12500 ng / ml of trehalose in the patient.

[0035] As uses herein, a “trehalase inhibitor” includes any agent capable of slowing, interfering, downregulating or blocking the activity trehalase. In some embodiments, the trehalase inhibitor is Validamycin A, Trehazolin, Validoxylamine A, Miglustat, 1- deoxynojirimycin, salbostatin, trehazolamine, thiatrehazolin, DNJ, DMJ, MDL 25637, DAB-1 , DMDP, calystegine A3, calystegine B2, calystegine B4, alexine, casuarina, (-)- uniflorineA, 7-deoxycasuarine, 7-deoxyuniflorine A, 7-deoxyuniflorine A glucoside, or tannic acid.

[0036] Preferably, the trehalase inhibitor is tannic acid, further preferably in the form of pentadigalloylglucose.

[0037] As used herein, “therapeutically effective amount refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.

[0038] In some embodiments, the plasma Cmax of trehalose is 1000 - 12500 ng / ml, including all values therebetween. Preferably the plasma Cmax of trehalose is 2500 - 12500ng / ml, preferably 5000-10000 ng / ml. Further preferably, the plasma Cmax is 7500-10000 ng / ml. Further preferably, the plasma Cmax is about 10000 ng / ml. In some embodiments, the plasma Cmax of trehalose is 1000 - 2500 ng / ml.

[0039] In some embodiments, the patient is administered 1-100 g of trehalose. Preferably, the patient is administered 25-75 g of trehalose. Further preferably, the patient is administered 36-65 g of trehalose.

[0040] In some embodiments, the patient is administered 0.1-3 g of tannic acid, preferably in the form of pentadigalloylglucose. Preferably, the patient is administered 0.2-2 g of tannic acid. Further preferably, the patient is administered 0.5-1.5 g of tannic acid. In some embodiments, the trehalose and trehalase inhibitor are administered sequentially, further preferably the trehalase inhibitor before trehalose, further preferably 30 min before.

[0041] In some embodiments, the trehalose and trehalase inhibitor are administered together. Preferably, the trehalose and the trehalase inhibitor are formulated together.

[0042] In some embodiments, the trehalose is for oral administration.

[0043] In some embodiments, the method is for the treatment of a neurological disorder. Preferably, the neurological disorder is any one or combination of synucleinopathies and proteinopathies.

[0044] In an embodiment, the neurological disorder is Parkinson's disease.

[0045] The synucleinopathy can be any of Parkinson's disease with dementia, dementia with Lewy bodies, multiple system atrophy (MSA), essential tremor, Gaucher disease and other lysosomal storage disorders, and neurodegeneration with brain iron accumulation.

[0046] The proteinopathy may be any of Alzheimer's disease, cerebral p-amyloid angiopathy, progressive supranuclear palsy (PSP), retinal ganglion cell degeneration in glaucoma, prion diseases, tauopathies, frontotemporal lobar degeneration, FTLD-FUS, amyotrophic lateral sclerosis (ALS), Huntington's disease and other triplet repeat disorders, familial British dementia, familial Danish dementia, hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander disease, seipinopathies, familial amyloidotic neuropathy, serpinopathies and retinitis pigmentosa with rhodopsin mutations.

[0047] In an aspect, there is provided trehalose for use in the treatment of a neurological disorder or a cardiovascular disorder in combination with a trehalase inhibitor to achieve a plasma Cmax of 1000-12500 ng / ml in the patient.

[0048] In an aspect, there is provided a use of trehalose and a trehalase inhibitor in the manufacture of a medicament for the treatment of a neurological disorder or a cardiovascular disorder, wherein the trehalose and the trehalase inhibitor are in effective amounts to achieve a plasma Cmax of 1000-12500 ng / ml in the patient. In an aspect, there is provided a kit comprising trehalose and a trehalase inhibitor for the treatment of a neurological disorder or a cardiovascular disorder, wherein the trehalose and the trehalase inhibitor are in effective amounts to achieve a plasma Cmax of 1000-12500 ng / ml in the patient.

[0049] In an aspect, there is provided a pharmaceutical composition comprising trehalose and a trehalase inhibitor in therapeutically effective amounts to achieve a plasma Cmax of 1000-12500 ng / ml in a patient, along with a pharmaceutically acceptable carrier

[0050] As used herein, “pharmaceutically acceptable carrier1' means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.

[0051] The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.

[0052] Example 1

[0053] Trehalase inhibition increases systemic trehalose

[0054] This example investigated whether oral administration of trehalase inhibitors (tannic acid and validamycin A) produced an increase in trehalose exposure following oral administration of trehalose (2.67 g / kg) to female Sprague Dawley rats (250-300 g). The trehalase inhibitors were administered 30 min before trehalose was administered.

[0055] Both tannic acid and validamycin A increased trehalose exposure in the plasma and brain (Table 1). However, the two inhibitors appeared to increase exposure in different ways; tannic acid increased the absorption phase whilst validamycin A slowed the elimination phase. Therefore, a combination of tannic acid and validamycin A might provide a greater increase in trehalose exposure than either compound alone.

[0056] Table 1. Pharmacokinetic parameters of plasma trehalose in the absence and presence of tannic acid and validamycin A

[0057] Example 2

[0058] Evaluation of a dose of trehalose + trehalase inhibitor that produces systemic and brain exposure similar to a known efficacious dose of trehalose

[0059] This experiment comprised several pharmacokinetic studies, the aims of which were to

[0060] (i) investigate the dose-response effect of tannic acid in increasing trehalose exposure,

[0061] (ii) investigate lower doses of trehalose, in combination with tannic acid, to find a combination that produces an equivalent trehalose exposure compared to trehalose (2.67 g / kg) alone, and (iii) investigate lower doses of trehalose, in combination with tannic acid and validamycin A to find a combination that produces an equivalent trehalose exposure compared to trehalose (2.67 g / kg) alone. Female Sprague Dawley rats were used in these studies.

[0062] It was found that tannic acid produced a dose-dependent increase in trehalose exposure (Figure 1). Based on these results we decided to continue to investigate the effect of a low dose of tannic acid (150 mg / kg), which translates into a dose that can be quickly evaluated in clinical trials, and a high dose of tannic acid (450 mg / kg) in combination with validamycin A (0.525 mg / kg), which will provide a greater inhibition of trehalase than tannic acid alone, and thus evaluate the maximum potential effect of inhibiting trehalase.

[0063] Based on these results, it was decided to evaluate the following combinations in later experiments:

[0064] 1. Trehalose (2.67 g / kg) + tannic acid (150 mg / kg)

[0065] 2. Trehalose (2.67 g / kg) + tannic acid (450 mg / kg) + validamycin A (0.525 mg / kg)

[0066] 3. Trehalose (1.0 g / kg) + tannic acid (150 mg / kg)

[0067] 4. Trehalose (0.7 g / kg) + tannic acid (450 mg / kg) + validamycin A (0.525 mg / kg)

[0068] Conditions 1 and 2 were aimed at assessing the effect of increasing trehalose exposure above that produced by trehalose (2.67 g / kg) alone. Conditions 3 and 4 were aimed at assessing the effect of reducing the dose of trehalose whilst maintaining trehalose exposure at the level produced by trehalose (2.67 g / kg) alone.

[0069] Example 3

[0070] Determination of duodenal trehalase activity in people with PD and age-matched controls

[0071] The above experiment demonstrated that inhibiting trehalase has the potential to (i) increase trehalose exposure for a given dose of trehalose and (ii) allow a reduction in the dose of trehalose administered whilst maintaining trehalose exposure similar to that produced by 2.67 g / kg. However, it was unknown whether trehalase levels are similar in people with PD compared to age-matched controls (i.e., whether trehalase is a potential target in people with PD). Therefore, we performed a study to investigate whether duodenal trehalase activity was significantly altered in people with PD compared to age-matched controls.

[0072] The study was a prospective, research biopsy collection, clinical study. We collected duodenal mucosal biopsy samples from people with Parkinson’s disease undergoing pre-planned gastrointestinal endoscopy for Duodopa® therapy or investigation of upper Gl symptoms. Duodenal mucosal biopsy from age-matched controls, that show no endoscopic and histological abnormalities, were collected from non-Parkinson’s disease subjects undergoing endoscopic procedure (e.g., asymptomatic individuals for the investigations of iron deficiency anemia, assessment of dyspepsia or gastroesophageal reflux symptoms). Samples were collected from 9 subjects with Parkinson’s disease and 17 age-matched controls, all samples were analysed for trehalase activity. Clinical information including age, sex and smoker status were collected from each subject. A UPDRS assessment (a clinical rating scale used to assess the severity of Parkinson’s disease) was performed on control subjects to ensure that they did not have undiagnosed Parkinson’s disease. Trehalase activity was measured in the duodenal samples using a previously described method based on incubating duodenal samples with known amounts of trehalose and measuring the amount of glucose liberated.

[0073] The demographics of the subjects whose samples were included in the primary analysis are shown in Table 2.

[0074] Table 2. Baseline characteristics of the two cohorts used in the primary analysis

[0075] Data are presented as mean ± SD. *: The MDS-UPDRS score was unable to be collected from one individual and so this score is the average of 8 subjects.

[0076] The average trehalase activity in Parkinson’s disease subjects was 164 ± 95 U / g tissue (Mean ± SD) and the average trehalase activity in age-matched control subjects was 162 ± 94 U / g tissue (Figure 3). In summary, in this study, trehalase activity was not altered by Parkinson’s disease and therefore inhibition of trehalase to increase trehalose exposure is an approach that is of potential benefit in people with PD. Example 4

[0077] Evaluation of trehalose and trehalose + trehalase inhibitor in a rodent model of

[0078] PD

[0079] The aim of this experiment was to evaluate whether, in the presence of trehalase inhibitors: (a) the dose of trehalose can be lowered whilst still providing beneficial effects in a rodent model of PD, and (b) increasing trehalose exposure provides greater beneficial effects in a rodent model of PD.

[0080] The study was performed in female Sprague Dawley rats (250-300g) that virally overexpress mutated human alpha-synuclein (aSyn) in the substantia nigra. The following groups were prepared:

[0081] 1 . empty vector (EV) + vehicle,

[0082] 2. hA53T aSyn + vehicle,

[0083] 3. hA53T aSyn + trehalose (2.67 g / kg / day, once daily, oral gavage),

[0084] 4. hA53T aSyn + trehalose (2.67 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage),

[0085] 5. hA53T aSyn + trehalose (2.67 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage) + validamycin A (0.525 mg / kg / day, once daily, oral gavage)

[0086] 6. hA53T aSyn + trehalose (1 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage)

[0087] 7. hA53T aSyn + trehalose (0.7 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage) + validamycin A (0.525 mg / kg / day, once daily, oral gavage)

[0088] Referring to Figures 4 and 5, the following was demonstrated. aSyn over-expression in rats produced deficits in dopaminergic function as anticipated. High dose oral trehalose prevented these deficits, though not always completely. Low dose oral trehalose plus trehalase inhibition protected against aSyn-induced dopaminergic deficits to a level equivalent, or greater than, high dose trehalose alone. High dose oral trehalose plus trehalase inhibition did not provide additional benefits over high dose oral trehalose alone. Markers of autophagy were elevated in the rodent PD model and normalised following treatment with trehalose.

[0089] These data demonstrate that the use of trehalase inhibitors can dramatically reduce the amount of trehalose that needs to be administered whilst maintaining efficacy in an aSyn-based rat model of PD.

[0090] Example 5

[0091] Evaluation of trehalose and trehalose + trehalase inhibitor on Gl dysfunction in a rodent model of PD

[0092] Gastrointestinal dysfunction occurs in many PD subjects and has a negative impact on quality of life. Most of these dysfunctions result from abnormal motility of the Gl tract and therefore treatments that negatively impact on Gl function are contraindicated. Therefore, we evaluated the effect of trehalose and trehalase inhibitors in the 6-OHDA rat model of PD, a model known to present with Gl dysfunction.

[0093] Sprague Dawley rats (250-300 g) received unilateral injections of 6-OHDA into the medial forebrain bundle. An additional eight rats received a sham injection. Seven groups were prepared as follows:

[0094] 1. Vehicle / vehicle,

[0095] 2. 6-OHDA + vehicle,

[0096] 3. 6-OHDA + trehalose (2.67 g / kg / day, once daily, oral gavage),

[0097] 4. 6-OHDA + trehalose (2.67 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage),

[0098] 5. 6-OHDA + trehalose (2.67 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage) + validamycin A (0.525 mg / kg / day, once daily, oral gavage)

[0099] 6. 6-OHDA + trehalose (1 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage)

[0100] 7. 6-OHDA + trehalose (0.7 g / kg / day) + tannic acid (450 mg / kg / day once daily, oral gavage) + validamycin A (0.525 mg / kg / day, once daily, oral gavage) In 6-OHDA lesioned rats, there was a significant decrease in fecal mass produced over a 24 h period compared to unlesioned animals. This reduction was first noted immediately following surgery and was maintained throughout the experiment (Figure 6), demonstrating that this model of PD is associated with Gl dysfunction. None of the groups receiving trehalose (groups 3-7) exhibited a significant alteration in fecal mass produced over a 24 h period compared to rats receiving 6-OHDA alone (Figure 6). Therefore, we have found no evidence that these potential treatments would worsen gastrointestinal dysfunction that occurs in people with PD. Example 6

[0101] Methods and Materials

[0102] Ninety-two female Sprague-Dawley rats (~275 g at time of surgery) were assigned to the following groups:

[0103] AAV1 / 2-EV = empty aSyn vector, 3.0 x 1012gp / ml AAV1 / 2-hA53T-aSyn = AAV1 / 2 human A53T alpha-synuclein, 3.0 x 1012gp / ml

[0104] N per group refers to number of animals commencing the in-life period On day 1 (D1), AAV1 / 2 human A53T alpha-synuclein (AAV1 / 2-hA53T-aSyn) or empty vector (AAV1 / 2-EV) was administered unilaterally into the right substantia nigra of rats according to stereotaxic techniques.

[0105] On D2, administration (per os, PO) of trehalose, tannic acid and vehicle commenced. Tannic acid was administered 30 min prior to trehalose.

[0106] Behavioural assessment of forelimb asymmetry was conducted using the cylinder test prior to surgery (baseline, D-3) and on D21 (3 weeks following AAV administration) and D42 (6 weeks following AAV administration). D21 and D42 assessments were conducted immediately prior to Test Item administration.

[0107] On D43, animals were killed for postmortem assessments. Samples were collected, processed, and stored as required.

[0108] Trehalose

[0109] Trehalose was formulated weekly by the addition of the appropriate amount of vehicle (ddH20) to weighed trehalose, followed by vortexing until the trehalose was fully dissolved, gentle heating may be used to aid solubilisation. Prepared solutions were stored at 4°C, stable for 7 days. The solution was vortexed daily prior to administration.

[0110] Trehalose was administered at a dose volume of 4 ml / kg. The route of administration was by oral gavage, once daily at doses ranging from 0-1.0 g / kg / day. Trehalose was administered 30 mins after tannic acid administration.

[0111] Tannic acid

[0112] Tannic acid is unstable as an aqueous solution and thus must was formulated daily. Tannic acid was formulated by addition of the appropriate amount of vehicle (ddH20) to weighed tannic acid, followed by vortexing until the tannic acid was fully dissolved.

[0113] Tannic acid was dosed at a dose volume of 1 ml / kg. The route of administration was by oral gavage, once daily at a dose of 150 mg / kg / day. Tannic acid was administered 30 mins before trehalose administration. Animal husbandry

[0114] This study used 84 female Sprague Dawley rats (Charles River, Canada, ~275 g at time of surgery).

[0115] A two-week period of acclimatisation was allowed between delivery of rats and commencement of treatments. Animals were weighed on the first day of acclimatisation and on a weekly basis thereafter.

[0116] Rats were housed in pairs before and after surgeries. All animals were housed at standard temperature (21 ± 2°C) in a light-controlled environment (lights on 6:00 am to 6:00 pm) with access to food (Teklad 7912, Harlan, Madison, Wl) and water ad libitum. The study was conducted according to guidelines established by the CCAC.

[0117] Behaviour assessment - forelimb asymmetry

[0118] Behavioural assessment of forelimb asymmetry was conducted using the cylinder test, prior to surgery (baseline, D-14,) and on D22 (3 weeks following AAV administration) and D42 (6 weeks following AAV administration). Behavioural assessments on D22 and D42 were conducted immediately prior to Test Item administration.

[0119] To examine animal forelimb asymmetry, rats were placed in a clear glass cylinder without a top (15cm diameter x 45cm tall). Animals were deprived of food from 5PM the previous night and testing was conducting the following morning. The number of times each paw touches the side of the cylinder during an individual rear was determined from post hoc analysis of video by an observer blinded to the treatments given. The first limb in any rear to touch the wall was scored a single point. If both limbs contact within 0.4s of each other, then this was scored as a ‘both’. Following a single limb initially contacting the wall, all subsequent exploratory movements about the wall using that limb were scored independently until the other limb contacts the wall with weight support. Alternating stepping motions involving both paws one after the other received a single score for both.

[0120] Administration of viral vectors - stereotaxic surgery

[0121] All surgeries were performed using aseptic technique. On D1 , under isoflurane anesthesia (2% with 2 L / min oxygen flow rate, isoflurane USP 99.9%) and after confirmation of loss of tail-pinch and corneal reflexes, rats were placed in a Kopf small animal stereotaxic frame with the incisor bar set 3.3 mm below the ear bars (interaural line). The animal’s head was shaved, and the skin cleaned thoroughly using disinfectant soap, isopropyl alcohol 70% USP and iodine surgical scrub (7.5% povidone iodine). An incision (~2 cm) was made with a sterile scalpel blade in an anteroposterior direction along the midline. After exposure of Bregma using a cottonbud, a burr hole was drilled in the skull above the right SN at coordinates -5.2 mm AP, and -2 mm ML to Bregma (according to the atlas of Paxinos and Watson, 1986). A customized, 1 inch, 26G Hamilton needle with 45-degree bevel was lowered -7.5 mm below the skull at the injection site. The injection (2.0 pl; 0.5 pl / min) was made (for a total injection time of 4 min) and the needle was left in place for an additional 5 min to ensure complete absorption of the solution. After slow retraction of the injection needle, the incision was closed by means of wound clips and animals were administered saline (50 ml / kg, SC) and analgesic (Ketoprofen, 0.5 mg / kg, SC, ANAFEN®, Boehringer Ingelheim, Canada). Finally, animals were removed from the frame and placed in a recovery cage, positioned atop a thermostatically-controlled pad, and monitored until conscious.

[0122] Necropsy and postmortem sample collection

[0123] On D43, animals were deeply anaesthetised with isoflurane and then killed via exsanguination by way of transcardial perfusion with ice-cold 0.9 % saline containing 0.2 % heparin. The spinal cord was removed from each animal, weighted and fresh frozen. Brains were removed and then placed, ventral up, into an ice-cold stainless steel rat brain matrix and first be cut in the coronal plane at the level of the optic chiasma. A second cut will be made, 1 mm anterior to the initial cut (creating Slice 1). A third cut will be made, 1 mm anterior to the second cut (creating Slice 2). The left and right striata were dissected from Slice 1 (1 mm), placed in separate tubes and immediately frozen on dry ice. The left and right striata were dissected from Slice 2 (1 mm), placed in separate tubes, weighed and immediately frozen on dry ice. The striatal samples from Slice 1 were used to quantify levels of dopamine and metabolites of dopamine (HVA and DOPAC) by LC-MS / MS. The striatal samples from Slice 2 were used to quantify levels of human aSyn via ELISA. The prefrontal cortex was freshly dissected, weighted and immediately frozen. The rostral portion of the brain, including the anterior striatum was immediately frozen in isopentane chilled to -42°C. Tissue was stored in a locked freezer at -80°C. The remaining caudal portion of the brain, including the mesencephalon, was immersed in 4% paraformaldehyde (PFA) for 48 hours for fixation followed by cryoprotection in graded sucrose solutions (15 to 30% sucrose).

[0124] All samples were labelled with Study code, animal ID, collection date, and sample type.

[0125] CSF

[0126] On D43, animals were deeply anaesthetised with isoflurane and after confirmation of loss of tail-pinch reflex a single CSF was collected from the cisterna magna via syringe suction ~30 mins after the last trehalose administration. Total CSF yield (100 pl) was aliquoted into 50 ul aliquots and transferred directly into 0.5 ml sterile Eppendorf polypropylene vials and stored in a locked -80 °C freezer until analysis and / or shipment.

[0127] Plasma

[0128] Terminal plasma was collected from deeply anaesthetised animals via cardiac puncture ~30 mins after the last trehalose administration. Approximately 500 pl of blood will be removed from the heart and transferred to anticoagulant tubes containing K2EDTA additive and stored on wet ice. Within 30 mins of collection, the tubes were centrifuged (2,500 gaveat 4°C for 10 minutes) and the resulting plasma layer removed. The plasma layer will be aliquoted (100 pl / tube) into 2 tubes and transferred into 1.5 ml sterile loBind Eppendorf tubes, frozen on dry-ice in an upright position, and stored in a locked -80°C freezer until analysis.

[0129] LC-MS / MS analysis of striatal biogenic amines

[0130] Left and right striatal tissue samples from all animals were sent on dry ice to the Vanderbilt Neurochemistry Core Facility (Nashville, TN, USA) for determination of dopamine levels and data sent back to Atuka blinded for analysis.

[0131] Tissue processing for biogenic amines

[0132] Left and right striatal samples were homogenized, using a tissue dismembrator, in 100-750 pl of 0.1 M TCA containing 102M sodium acetate, 104M EDTA, and 7.5% methanol (pH 3.8). 10 pl of homogenate was removed for measurement of protein concentration. The samples were then spun in a microcentrifuge at 10,000 RCF for 20 minutes at 4°C. Supernatant was transferred to a new microcentrifuge tube for biogenic amine analysis.

[0133] Biogenic amine analysis

[0134] Dopamine levels were determined by a highly sensitive and specific liquid chromatography / mass spectrometry (LC-MS / MS) methodology following derivatization of analytes with benzoyl chloride (BZC). 5 pl of supernatant were treated with 10 pl each of 500 mM Na2CO3 (aq) and 2% BZC in acetonitrile. After four minutes, the reaction was stopped by the addition of 10 pl internal standard solution (in 20% acetonitrile containing 3% sulfuric acid) containing 200 pg of each13C6-derivatized dopamine-d4. Liquid Chromatography was performed on a 2.0 x 50 mm, 1.7 pm particle Acquity BEH C18 column (Waters Corporation, Milford, MA, USA) using a Waters Acquity UPLC. Mobile phase A is 0.15% aqueous formic acid and mobile phase B is acetonitrile. Samples were separated by a gradient of 98-5% of mobile phase A over 11 min at a flow rate of 600 pl / min prior to delivery to a SCIEX 6500+ QTrap mass spectrometer (AB Sciex, Framingham, MA, USA). The following MRM transitions were monitored for quantitative purposes: 466 - 105, BZC-dopamine; 488 - 111 , 13C6-BZC-dopamine-d4. Automated peak integration was performed using SCIEX Multiquant software version 3.0.2. All peaks were visually inspected to ensure proper integration. Levels of dopamine in samples was calculated using calibration curves constructed on the basis of peak area ratio (Panalyte / Pl.S.) versus concentrations of internal standard by linear regression. Levels were normalized to protein concentration in the tissue extract.

[0135] Protein assay

[0136] Protein concentration in tissue homogenates was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) as described in the provided kit instructions. Absorbance was measured using a POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).

[0137] ELISA for transgene-derived human alpha synuclein

[0138] Striatal tissue from all animals was homogenised in lysis buffer (Biobasic, Cat N. RB4478, Markham, ON) and protease inhibitor cocktail (Roche, Cat N. 11836153001 , Mississauga, ON). Samples were sonicated at 4°C followed by centrifugation (135000 rpm for 15 minutes at 4°C) to produce supernatant. Using a 1 :10 dilution of the supernatant, an aliquot was used to determine total protein levels (BCA assay, Cat N. 23227, Pierce, Rockford, IL). Another aliquot of the sample, 0.001 mg / mL, was used for ELISA procedures according to the manufacturer’s instructions (BioLegend, Cat N. 448607, San Diego, CA). The samples were analysed using CLARIOstar systems quantifying the absorbance counts relative to the amount of aSyn. Levels of aSyn were expressed as pg / mg total.

[0139] Results

[0140] Forelimb asymmetry - Cylinder Test

[0141] Analysis of baseline cylinder data revealed a significant effect of treatment (1-way ANOVA, F (5, 86) = 3.381 , P=0.0078, Error! Reference source not found.A).

[0142] Post hoc Fisher’s LSD analysis revealed that levels of asymmetry in, a) animals in the AAV-aSyn + trehalose (0.5 g / kg) + tannic acid (150 mg / kg) were significantly higher than the AAV-EV group (P=0.0139), the AAV-aSyn group (P=0.0064) and the AAV-aSyn + trehalose (0.75 g / kg) + tannic acid (150 mg / kg) group (P=0.0007). b) animals in the AAV-aSyn + trehalose (0.75 g / kg) + tannic acid (150 mg / kg) group were significantly lower than the AAV-aSyn + trehalose (1.0 g / kg) + tannic acid (150 mg / kg) group (P=0.0129)

[0143] Analysis, of D22, analysis of cylinder data failed to show any significant effect of treatments (1-way ANOVA, F (5, 83) = 1.830, P=0.1159, Error! Reference source not found. B).

[0144] Analysis, of D42, analysis of cylinder data failed to show any significant effect of treatments (1-way ANOVA, F (5, 81) = 1.597, P=0.1703, Error! Reference source not found. C).

[0145] Striatal dopamine levels

[0146] Analysis of striatal dopamine revealed a significant effect of treatment (F (5, 71) = 2.431 , P=0.0431 , Error! Reference source not found.). Post hoc Fisher’s LSD analysis revealed a) a significant decrease in dopamine levels in the AAV-aSyn group (by 33%, P=0.0479), the AAV-aSyn + trehalose (0.5 g / kg) + tannic acid (150 mg / kg) group (by 32%, P=0.0302), and the AAV-aSyn + trehalose (0.25 g / kg) + tannic acid (150 mg / kg) group (by 38%, P=0.0135) compared to the AAV-EV group b) a significant decrease in dopamine in the AAV-aSyn + trehalose (0.25 g / kg) + tannic acid (150 mg / kg) group compared to the AAV-aSyn + trehalose (1.0 g / kg) + tannic acid (150 mg / kg) group (by 32%, P=0.0409) and the AAV-aSyn + trehalose (0.75 g / kg) + tannic acid (150 mg / kg) group (by 33%, P=0.0380)

[0147] Striatal levels of transgene-derived human alpha synuclein

[0148] Analysis of striatal levels of transgene-derived human a-Syn were showed a significant effect of treatment (F (5,81) = 9.263, P<0.0001 , 1-Way ANOVA, Figure ).

[0149] Post hoc Fisher’s LSD analysis revealed that, a) Significant differences (all P<0.0001) were seen between Group 1 (AAV-EV + vehicle) and all other AAV-aSyn-operated groups. Group 1 , as expected, showed very low or undetectable levels of transgene-derived aSyn in contrast with Groups 2 - 6, which showed elevated levels of aSyn due to transgene expression. b) Comparisons between AAV1 / 2-hA53T-aSyn-operated groups were nonsignificant.

[0150] Plasma levels of trehalose

[0151] Terminal plasma samples were collected 30 minutes after the last administration of trehalose and analysed for trehalose levels. Trehalose levels in the plasma were dose-dependent, with high levels seen following trehalose (1 g / kg, 0.75 g / kg) and tannic acid, and lower levels observed when trehalose (0.5 g / kg and 0.25 g / kg) were administered (Figure 12).

[0152] CSF levels of trehalose Terminal CSF samples were collected 30 minutes after the last administration of trehalose and analysed for trehalose levels. Trehalose levels in the CSF were dosedependent, with high levels seen following trehalose (1 g / kg) and tannic acid, lower levels observed following trehalose (0.75 g / kg and 0.5 g / kg) and tannic acid, and no trehalose detected when the lowest dose of trehalose (0.25 g / kg) and tannic acid were administered (Figure 13).

[0153] Study JURA8 was designed to find the minimally effective dose of trehalose when combined with a clinically relevant dose of tannic acid in a AA 1 / 2-hA53T-aSyn rat model of Parkinson’s disease.

[0154] As anticipated, stereotaxic injection of AAV1 / 2-hA53T-aSyn adjacent to the right substantia nigra of the female rat resulted in transgene expression and nigrostriatal degeneration, as demonstrated by a loss of striatal dopamine of 33%. The magnitude of these deficits in study JURA8 were comparable to Atuka’s historical data in vehicle- treated animals in this AAV1 / 2-hA53T-aSyn rat model.

[0155] Assessment of levels of striatal dopamine revealed an effect of trehalose (1 g / kg and 0.75 g / kg) when combined with tannic acid (150 mg / kg) such that dopamine levels in the ipsilateral hemisphere were increased compared to the ipsilateral hemisphere of the AAV1 / 2-hA53T-aSyn + vehicle group (by 36%, and 39% respectively) to levels which were not significantly different from AAV1 / 2-EV levels (92%, and 94% of AAV1 / 2-EV levels). By contrast, trehalose (0.5 g / kg an 0.25 g / kg) when combined with tannic acid (150 mg / kg) did not increase striatal dopamine levels (0%, and -8% difference from AAV1 / 2-hA53T-aSyn levels).

[0156] These results were supported by the bioanalytical results that demonstrated that trehalose (1.0 and 0.75 g / kg) produced broadly similar plasma exposures, with lower doses of trehalose producing substantially lower plasma trehalose exposures. CSF levels of trehalose followed a similar pattern to plasma levels, albeit at a lower level, except that the CSF level decreased between the 1 .0 g / kg and 0.75 g / kg trehalose groups.

[0157] No effect was observed in forelimb asymmetry, as measured by the cylinder test. In this model, forelimb asymmetry can often produce variable results and one possible reason for the absence of a significant forelimb asymmetry could be an imbalance in forelimb asymmetry at baseline. In summary, the combination of trehalose and tannic acid was neuroprotective in this model, and the minimally effective dose of trehalose was 0.75 g / kg in combination with tannic acid (150 mg / kg). For this combination, the plasma level of trehalose, collected 30 minutes after the last trehalose administration (~Tmax), associated with efficacy was 1291 ng / ml. The Cmax in rats may be applicable 1 :1 with humans.

[0158] Example 7 - PROPHETIC

[0159] Phase I clinical study in healthy volunteers

[0160] The study is a Phase I clinical study designed to evaluate the safety, tolerability, and pharmacokinetics of trehalose, in the absence and presence of tannic acid, in healthy participants. The results obtained from this study will enable us to select doses of trehalose and tannic acid to evaluate in Phase II studies. The study will be performed by BioPharma, a Toronto based Contract Research Organization (CRO). The Phase I clinical study will be carried out in 2 parts.

[0161] Part A will be a placebo-controlled, double-blind, sequential, single ascending dose escalation study to determine the safety, tolerability and pharmacokinetics of trehalose following administration of single oral doses in healthy participants. The dose of trehalose that produces trehalose exposures equivalent to those associated with efficacy in preclinical models of PD will be defined.

[0162] Part B will be a placebo-controlled, double-blind, sequential study to determine the safety, tolerability, and pharmacokinetics of a fixed dose of oral trehalose following a single ascending oral dose of tannic acid in healthy male participants. Trehalose will be administered 30 minutes after tannic acid administration.

[0163] The trial protocol is set forth in the below.

[0164]

[0165] Trehalose

[0166] There are at least 9 clinical trials in which large doses of trehalose were orally administered. The highest dose at which trehalose has been administered in these studies is 100 g / day for 6 months. The commonest adverse event following consumption of trehalose are gastrointestinal effects (bloating, flatulence, and loose stools / diarrhea). These effects are more common when the dose of trehalose is greater than 50 g / day. These adverse events have not precluded trehalose being administered at a daily dose of 100 g / day for 6 months. In the Phase I clinical study, the starting dose of trehalose will be 50 g, which is well tolerated by the general population. Higher doses of trehalose, up to 200 g, may be administered, but only to subjects that tolerate lower doses of trehalose. Tannic acid

[0167] Historically, tannic acid was used to treat burns and as an addition to barium enema preparations. Tannic acid is still used today, administered as tannate salts in antihistamines, cough suppressants, decongestants, and anti-diarrhetics. One anti- diarrhetic, Tannalbin, is administered as 500 mg of albumin tannate per tablet, with 1-2 tablets taken every 2 hours. The ratio of albumin I tannic acid in this product is ~50:50, meaning that 3-6 g of tannic acid may be taken daily.

[0168] Clinical trials are also ongoing with Pentarlandir™, ultrapure and potent tannic acid, as a treatment for COVID-19 (NCT04911777) with the high-dose group receiving 1.13 g of tannic acid every 8 h (daily dose of 3.38 g of tannic acid). The results of this study have been announced (July 2022) and Pentarlandir™ was shown to be safe and effective.

[0169] Acute high-dose ingestion and absorption of tannic acid may cause nausea, vomiting, constipation, abdominal pain, and liver damage. The doses of tannic acid that we will investigate (up to 1.5 g / day) are lower than those currently used, or used historically, and so the development of adverse events is very unlikely. However, we will monitor participants receiving tannic acid for 14 days after their last administration to ensure no adverse events develop. We will also exclude participants from the trial if they have a history of Gl dysfunction, or are taking any products that has tannic acid as an active ingredient. We will also require participants not to consume wine or beer (that contain high levels of tannic acid) for the duration of the study.

[0170] In summary, historical tannic acid use, levels of tannic acid in currently used medications, and ongoing clinical development of tannic acid show that daily doses of tannic acid of <2.5 g / day are safe and well tolerated.

[0171] This trial is anticipated to confirm the human doses set forth in this application.

[0172] Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference. Reference List

[0173] 1. He Q, Koprich JB, Wang Y, Yu W bo, Xiao B guo, Brotchie JM, Wang J. Treatment with Trehalose Prevents Behavioral and Neurochemical Deficits Produced in an AAV a-Synuclein Rat Model of Parkinson’s Disease. Mol Neurobiol. 2016 May 1 ;53(4):2258-68. PMID: 25972237. DOI: 10.1007 / sl 2035-015-9173-7.

[0174] 2. Howson PA, Johnston TH, Ravenscroft P, Hill MP, Su J, Brotchie JM, Koprich JB. Beneficial effects of trehalose on striatal dopaminergic deficits in rodent and primate models of synucleinopathy in Parkinson’s disease. Journal of Pharmacology and Experimental Therapeutics. 2019;369(3):364-74. PMID: 30918068. DOI: 10.1124 / jpet.118.255695

[0175] 3. Lee HJ, Yoon YS, Lee SJ. Mechanism of neuroprotection by trehalose: Controversy surrounding autophagy induction. Vol. 9, Cell Death and Disease. Nature Publishing Group; 2018. PMID: 29907758. DOI: 10.1038 / S41419-018-0749-9

[0176] 4. Pupyshev AB, Klyushnik TP, Akopyan AA, Singh SK, Tikhonova MA. Disaccharide trehalose in experimental therapies for neurodegenerative disorders: Molecular targets and translational potential. Vol. 183, Pharmacological Research. Academic Press; 2022. PMID: 35907433. DOI: 10.1016 / j.phrs.2O22.106373

[0177] 5. Noorasyikin MA, Azizan EA, Teh PC, Farah Waheeda T, Siti Hajar MD, Long KC, Norlinah Ml. Oral trehalose maybe helpful for patients with spinocerebellar ataxia 3 and should be better evaluated. Parkinsonism Relat Disord. 2020 Jan;70:42-4. PMID: 31841943. DOI: 10.1016 / j.parkreldis.2O19.12.007

[0178] 6. Kaplon RE, Hill SD, Bispham NZ, Santos-Parker JR, Nowlan MJ, Snyder LL, Chonchol M, LaRocca TJ, McQueen MB, Seals DR. Oral trehalose supplementation improves resistance artery endothelial function in healthy middle-aged and older adults. Aging. 2016 Jun;8(6):1167-83. PMID: 27208415. DOI: 10.18632 / aging.100962

[0179] 7. Chen A, Gibney PA. Dietary Trehalose as a Bioactive Nutrient. Nutrients. 2023 Mar 14; 15(6): 1393. PMID: 36986123. DOI: 10.3390 / nu15061393

[0180] 8. Bergoz R. Trehalose malabsorption causing intolerance to mushrooms. Report of a probable case. Gastroenterology. 1971 May;60(5):909-12. DOI: 10.1016 / S0016- 5085(71)80092-6 9. Bergoz R, Bolte JP, Bueschenfelde K-HM Zum. Trehalose Tolerance Test. Scand J Gastroenterol. 1973 Oct 30;8(7):657-63. DOI: 10.1080 / 00365521.1973.12096765

[0181] 10. Bolte JP, Schonhage F, Forster E, Knolle J, Meyer KH. [Diagnostic significance of the trehalose tolerance test in malabsorption syndromes], Dtsch Med Wochenschr. 1973 Jul;98(28): 1358-62. PMID: 4716791. DOI: 10.1055 / S-0028-1107030

[0182] 11. Ushijima T, Fujisawa T, Kretchmer N. Evaluation of the ability of human and small intestine to absorb trehalose. Shoka to Kushu (Digestion and Absorption). 1995;18:56- 7.

[0183] 12. Oku T, Okazaki M. Transitory laxative threshold of trehalose and lactulose in healthy women. J Nutr Sci Vitaminol (Tokyo). 1998 Dec;44(6):787-98. PMID: 10197310. DOI: 10.3177 / jnsv.44.787

[0184] 13. Arola H, Koivula T, Karvonen AL, Jokela H, Ahola T, Isokoski M. Low trehalase activity is associated with abdominal symptoms caused by edible mushrooms. Scand J Gastroenterol. 1999 Sep;34(9):898-903. PMID: 10522609. DOI:

[0185] 10.1080 / 003655299750025372

[0186] 14. Eshchar J, Friedman G. Acute hepatotoxicity of tannic acid added to barium enemas. Am J Dig Dis. 1974 Sep;19(9):825-9. PMID: 4850412. DOI: 10.1007 / BF01071942

[0187] 15. Esteban Carretero J, Durban Reguera F, Lopez-Argueta Alvarez S, Lopez Montes J. A comparative analysis of response to vs. ORS + gelatin tannate pediatric patients with acute diarrhea. Revista espanola de enfermedades digestivas. 2009 Jan;101 (1):41-8. PMID: 19335032. DOI: 10.4321 / sl 130-01082009000100005

[0188] 16. Allegrini A. Gelatine Tannate for the Treatment of Acute Diarrhoea in Adults. J Gastrointest Dig Syst. 2012;02(03). DOI: 10.4172 / 2161 -069X.1000110

[0189] 17. Koprich JB, Johnston TH, Huot P, Reyes MG, Espinosa M, Brotchie JM. Progressive neurodegeneration or endogenous compensation in an animal model of Parkinson’s disease produced by decreasing doses of alpha-synuclein. PLoS One. 2011 ;6(3). PMID: 21408191. DOI: 10.1371 / journal.pone.0017698

Claims

CLAIMS:

1. A method of treating a patient with a neurological disorder or a cardiovascular disorder, the method comprising administering to the patient trehalose in combination with a trehalase inhibitor to achieve a plasma Cmax of 1000 -12500 ng / ml of trehalose in the patient.

2. The method of claim 1 , wherein the trehalase inhibitor is Validamycin A, Trehazolin, Validoxylamine A, Miglustat, 1-deoxynojirimycin, salbostatin, trehazolamine, thiatrehazolin, DNJ, DMJ, MDL 25637, DAB-1 , DMDP, calystegine A3, calystegine B2, calystegine B4, alexine, casuarina, (-)- uniflorineA, 7-deoxycasuarine, 7-deoxyuniflorine A, 7-deoxyuniflorine A glucoside, or tannic acid.

3. The method of claim 2, wherein the trehalase inhibitor is tannic acid, preferably in the form of pentadigalloylglucose.

4. The method of any one of claims 1-3, wherein the plasma Cmax is 2500 - 12500ng / ml, preferably 5000-10000 ng / ml.

5. The method of claim 4, wherein the plasma Cmax is 7500-10000 ng / ml, preferably about 10000ng / ml.

6. The method of any one of claims 1-3, wherein the plasma Cmax is 1000 - 2500 ng / ml.

7. The method of any one of claims 1-3, wherein the patient is administered 1-100 g of trehalose.

8. The method of claim 7, wherein the patient is administered 25-75 g of trehalose.

9. The method of claim 8, wherein the patient is administered 36-65 g of trehalose.

10. The method of any one of claims 7-9, wherein the patient is administered 0.1-3 g of tannic acid, preferably in the form of pentadigalloylglucose.

11. The method of claim 10, wherein the patient is administered 0.2-2 g of tannic acid, preferably in the form of pentadigalloylglucose.

12. The method of claim 11 , wherein the patient is administered 0.5-1 .5 g of tannic acid, preferably in the form of pentadigalloylglucose.

13. The method of any one of claims 1-12, wherein the trehalose and trehalase inhibitor are administered sequentially, preferably the trehalase inhibitor before trehalose, further preferably 30 min before.

14. The method of any one of claims 1-12, wherein the trehalose and trehalase inhibitor are administered together.

15. The method of claim 14, wherein the trehalose and the trehalase inhibitor are formulated together.

16. The method of any one of claims 1-15, wherein the trehalose is for oral administration.

17. The method of any one of claims 1-16, for the treatment of a neurological disorder.

18. The method according to claim 17, wherein the neurological disorder is any one or combination of synucleinopathies and proteinopathies.

19. The method according to claim 17, wherein the neurological disorder is Parkinson's disease.

20. The method according to claim 18 wherein the synucleinopathy is any of Parkinson's disease with dementia, dementia with Lewy bodies, multiple system atrophy (MSA), essential tremor, Gaucher disease and other lysosomal storage disorders, and neurodegeneration with brain iron accumulation.

21. The method according to claim 18 wherein the proteinopathy is any of Alzheimer's disease, cerebral p-amyloid angiopathy, progressive supranuclear palsy (PSP), retinal ganglion cell degeneration in glaucoma, prion diseases, tauopathies, frontotemporal lobar degeneration, FTLD-FUS, amyotrophic lateral sclerosis (ALS), Huntington's disease and other triplet repeat disorders, familial British dementia, familial Danish dementia, hereditary cerebralhemorrhage with amyloidosis, CADASIL, Alexander disease, seipinopathies, familial amyloidotic neuropathy, serpinopathies and retinitis pigmentosa with rhodopsin mutations.

22. Trehalose for use in the treatment of a neurological disorder or a cardiovascular disorder in combination with a trehalase inhibitor to achieve a plasma Cmax of 1000-12500 ng / ml of trehalose in the patient23. Use of trehalose and a trehalase inhibitor in the manufacture of a medicament for the treatment of a neurological disorder or a cardiovascular disorder, wherein the trehalose and the trehalase inhibitor are in effective amounts to achieve a plasma Cmax of 1000-12500 ng / ml of trehalose in the patient.

24. A kit comprising trehalose and a trehalase inhibitor for the treatment of a neurological disorder or a cardiovascular disorder, wherein the trehalose and the trehalase inhibitor are in effective amounts to achieve a plasma Cmax of 1000-12500 ng / ml of trehalose in the patient.

25. A pharmaceutical composition comprising trehalose and a trehalase inhibitor in therapeutically effective amounts to achieve a plasma Cmax of 1000-12500 ng / ml of trehalose in a patient, along with a pharmaceutically acceptable carrier.

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