Compositions and methods for treating neurological conditions

Abstract

A pharmaceutical composition containing a dopamine reuptake inhibitor or a dopaminergic agonist, an adrenergic receptor antagonist and a pharmaceutically acceptable carrier, and a method for treating neurological diseases by administering the composition.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 426,165, filed on November 17, 2022, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Sponsored Research or Development

[0004] This invention was made with government support under Grant / Contract No. 1R43NS095422 - 01 and 1R43NS103696 - 01A1 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0005] The present invention relates to compositions and methods for treating neurological and neurodevelopmental disorders.

[0006] Background

[0007] One or more effective drugs for treating and improving neurological disease conditions are an unmet medical need. A variety of therapeutic methods have been developed to treat neurological and neurodevelopmental disorders, but these methods provide limited effectiveness. Thus, there continues to be a widespread problem of suffering from these disorders with little hope of effective and durable treatment. The present invention addresses this unmet medical need, and its exemplary compositions and methods are described in more detail below. Summary of the Invention

[0008] In one aspect, the present invention reflects the discovery that these neurological disorders involve excessive adrenergic activity and insufficient dopaminergic activity.

[0009] In one aspect, the present disclosure relates to a pharmaceutical composition comprising a dopamine reuptake inhibitor, an adrenergic receptor antagonist, and a pharmaceutically acceptable carrier. In another aspect, the present disclosure relates to a pharmaceutical composition comprising a dopaminergic agonist, an adrenergic receptor antagonist, and a pharmaceutically acceptable carrier.

[0010] In one aspect, the present disclosure relates to a method for treating neurological and neurodevelopmental disorders by administering these pharmaceutical compositions to a subject in need thereof. Thus, one embodiment of the present invention provides a pharmaceutical composition of a dopamine reuptake inhibitor or a dopaminergic agonist plus an adrenergic receptor (“adrenergic receptor”) antagonist for treating neurological diseases or conditions.

[0011] In another aspect, the method (i.e., the therapeutic use of the composition) further includes the step of measuring the level of a biomarker in a subject in need of treatment. This measurement step can occur before, during, and / or after treatment. In yet another aspect, the present disclosure relates to a drug kit that contains a dopamine reuptake inhibitor or a dopaminergic agonist, an adrenergic receptor antagonist, and a reagent or instrument for evaluating and measuring at least one or more biomarkers.

[0012] Biomarkers for the method or kit can be glucose metabolism and metabolite profiles, and / or lipid metabolism and related metabolite profiles, and / or hormone profiles, and / or cytokine profiles, or and / or cardiac function. Glucose metabolism biomarkers can be, for example, glucose, lactate, pyruvate, the ratio of lactate to glucose, or the glucose disposal rate. Lipid metabolism and related metabolite profiles can include β-hydroxybutyrate or acetoacetate, and / or high-density lipoprotein, cholesterol, triglycerides, or free fatty acids, or the disposal rate of β-hydroxybutyrate or acetoacetate. Hormone profile biomarkers can be, for example, cortisol or leptin. Cytokine profile biomarkers can be, for example, IL-10, Il-4, IL-17, IL-23, or their corresponding ratios. Cardiac function biomarkers can be, for example, heart rate or heart rate variability. Brief Description of the Drawings

[0014] Figure 1A and 1B shows a comparison of tyrosine hydroxylase expression in different postmortem human brain slices (pons / putamen; Figure 1C ). Compared with brain slices from individuals without neurodegenerative disorders, frontotemporal dementia (FTD) brain slices showed increased tyrosine hydroxylase expression in the pons and decreased tyrosine hydroxylase expression in the putamen.

[0015] Figures 2A-2D shows a comparison of lipid and glucose metabolomic activities between healthy controls and patients with frontotemporal dementia. These analyses indicate that the FTD disorder is associated with metabolic dysregulation, namely increased lipid catabolism (β-hydroxybutyrate is a product of lipid β-oxidation) and altered glucose metabolism.

[0016] Figures 3A-3F shows a comparison of the behavioral and lipid metabolism patterns of drug-treated and untreated (vehicle) P301L mice with those of Swiss Webster (SW) mice (the background strain of the transgenic P301L mice and used as healthy controls).

[0017] Figures 4A-4DShows a comparison of the learning and memory behaviors of Fmr1 KO mice treated with different drugs and untreated (vehicle), with FVB mice (the background strain of the transgenic Fmr1 KO mice and used as healthy controls). DETAILED DESCRIPTION OF THE INVENTION

[0019] Those skilled in the art will understand that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth in this application. Instead, these embodiments are provided so that this disclosure will fully convey the present invention to those skilled in the art. Those skilled in the art to which the present invention pertains will, upon benefiting from the teachings provided herein, envision many modifications and other embodiments of the present invention.

[0020] In particular, the neurological and neurodevelopmental disorders can be Fragile X Syndrome (FXS), Neurofibromatosis Type I (NF1), Tuberous Sclerosis (TS), Down Syndrome (DS), or Autism Spectrum Disorder (ASD), Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), Progressive Supranuclear Palsy (PSP), Multiple System Atrophy, Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease, and other related neurological diseases or conditions. The disorders can also include, but are not limited to, neuropsychiatric disorders such as Major Depressive Disorder (MDD), Posttraumatic Stress Disorder (PTSD), and chemical dependence / substance abuse, such as disorders related to cocaine use. Overall, these debilitating neurological conditions have a negative impact on the quality of life of patients, while imposing a burden on the affected families and society as a whole.

[0021] The present invention is based on the surprising results of studies using postmortem human brains and / or animal models of human neurological disease conditions. These results indicate that catecholamine imbalance (such as, but not limited to, adrenergic (activity) excess and dopaminergic activity deficiency) is a component of the pathogenic mechanism. The present invention is also based on the finding that treating animal models of human conditions with a combination of agents - one agent enhancing dopaminergic activity and another agent attenuating adrenergic activity - can normalize the catecholamine imbalance and thereby alleviate disease symptoms.

[0022] The present invention is also based on the discovery that the same brain catecholamine imbalance affects many physiological functions. Physiological functions can include metabolism, such as altered glucose disposal rate (including increased or decreased glucose disposal rate), altered glucose metabolic pathways (gluconeogenesis, glycolysis, and / or oxidative phosphorylation), and dyslipidemia (including increased lipolysis or catabolism). The functions can include alterations in cardiovascular function, such as changes in heart rate (tachycardia and bradycardia) and / or heart rate variability (HRV). And the same neurochemical functions may include the immune status between pro-inflammatory and anti-inflammatory activities (e.g., plasma level of IL-10 or the ratio of IL-10 to IL-17).

[0023] The present invention is also based on the following research results: treating animal models of human diseases simultaneously with combinations of agents (such as agents with pharmacological activity enhancing dopaminergic action and agents weakening adrenergic action) has normalized physiological activities (such as lipid or glucose metabolism). Thus, a given physiological activity, such as metabolism, cardiac and / or immune status, can be used as an objective biomarker for evaluating the brain catecholaminergic balance and therapeutic effects.

[0024] The overall profile of tyrosine hydroxylase expression in different brain regions of FTD patients indicates the pathophysiology of catecholamine imbalance. As Figure 1A and 1B shown, post-mortem studies of neurodegenerative diseases represented by frontotemporal dementia (one of the diseases commonly referred to as tauopathies) show an increased level of tyrosine hydroxylase expression in the pons with the locus coeruleus as the nucleus. Tyrosine hydroxylase is the rate-limiting enzyme for the synthesis of catecholamines (dopamine, norepinephrine, and epinephrine). The locus coeruleus is the main brain region regulating the adrenergic state of the brain. An increased level of tyrosine hydroxylase expression in the pons means an increased adrenergic activity in the brain. These same studies also show a decreased expression of the same enzyme in the putamen, which is part of the dorsal striatum and is the main brain region regulating dopaminergic activity. A decrease in the tyrosine hydroxylase level in this brain region indicates a loss of dopamine synthesis capacity and dopaminergic cells or a general loss of dopaminergic neurons, resulting in insufficient dopaminergic action.

[0025] As Figures 2A-2D shown, metabolomic data analysis of frontotemporal dementia patients indicates metabolic abnormalities compared to healthy individuals, such as increased lipolysis (β-hydroxybutyrate is a product of lipid β-oxidation) and dysregulated glucose metabolism. These metabolic changes imply an altered catecholamine balance consistent with the differential expression of tyrosine hydroxylase described above.

[0026] P301L mice expressing frontotemporal dementia-related mutations of the microtubule-associated protein tau (MAPT) are murine models of human neurological diseases such as frontotemporal dementia, Alzheimer's disease, or diseases collectively referred to as tauopathies. As Figures 3A-3FAs shown, the behavioral and lipid metabolism patterns of drug-treated and untreated P301L mice were compared with those of SW mice (SW = Swiss Webster mice, the background strain of the transgenic P301L mice). Treatment of P301L mice with a combination of propranolol (an agent that attenuates β-adrenergic activity) and 3-(phenylpropyl)-sydnonimine-N-phenylcarbamate (an agent that enhances dopaminergic activity; see Chen et al., U.S. Patent No. 8,415,385) has normalized the behavioral patterns and lipid metabolism activities of these P301L mice that mimic human FTD disorders.

[0027] As Figures 4A-4D shown, studies of the brains of Fmr1-KO mice, a rodent model of human fragile X syndrome and neurodevelopmental disorders, have shown increased expression of dopamine reuptake protein in the mouse striatum and decreased expression of norepinephrine reuptake protein in the mouse brainstem (the locus coeruleus is a brainstem nucleus). Reuptake proteins regulate the extracellular concentration of different catecholamines. These differential expression patterns of the proteins indicate excessive adrenergic activity and insufficient dopaminergic action. Figure 4B .

[0028] The same knockout animals also showed increased glycolytic activity (increased plasma lactate), increased lipid catabolism (increased β-hydroxybutyrate), and dyslipidemia (low HDL and cholesterol), similar to the metabolic disorders in fragile X humans. The altered metabolic activities indicate sympathetic nervous system dysregulation (i.e., catecholaminergic imbalance). Dopaminergic activity regulates sympathetic activation. Treatment of Fmr1 KO mice with ACT01, which specifically activates dopaminergic activity, normalized glucose and lipid metabolism. Figure 4C .

[0029] The learning and memory behaviors of drug-treated and untreated (vehicle) Fmr1 KO mice were compared with those of FVB mice (the background strain of the transgenic Fmr1 KO mice). The studies showed that drug combinations such as 3-(phenylpropyl)-sydnonimine-N-phenylcarbamate (also known as ACT01 or ACT) (25 mg / kg) and propranolol (pro, 3 mg / kg), ACT01 (25 mg / kg), ACT01 (25 mg / kg) and prazosin (Praz, 2 mg / kg), and ACT01 (25 mg / kg) and carvedilol (Carv, 2 mg / kg) significantly (p < 0.05) improved the learning and memory abilities of Fmr1 KO mice (NOR index = novel object recognition index). The effect of the drug combination was superior to that of the individual agents alone.

[0030] That is, treatment of Fmr1-KO mice with a combination of prazosin, carvedilol, or propranolol (agents that attenuate adrenergic activity) and ACT01 (an agent that enhances dopaminergic activity; see Chen et al., U.S. Patent No. 8,415,385) rescued the learning ability and memory function of this animal model and normalized the patterns of lipid and glucose metabolism.

[0031] In another embodiment, the dopamine reuptake inhibitor is selected from, but not limited to, 3-(phenylpropyl)-tropinone-N-phenylcarbamate, 3-(p-methylbenzyl)-tropinone-N-phenylcarbamate, 3-(p-carboxybenzyl)-tropinone-N-phenylcarbamate, 3-phenethyl-tropinone-N-phenylcarbamate, 3-phenethyl-tropinone-N-(3',4'-dichloro-phenyl)-carbamate, 3-(p-nitrophenethyl)-tropinone-N-(3',4'-dinitro-phenyl)-carbamate, 3-(p-fluorobenzyl)-tropinone-N-phenylcarbamate, 3-benzyl-tropinone-N-phenylcarbamate, 3-phenethyl-tropinone-N-(p-chlorophenyl)-carbamate, 3-phenethyl-tropinone-N-(m-trifluoromethyl)-phenylcarbamate, 3-(3',5'-difluorobenzyl)-tropinone-N-phenylcarbamate, 3-(m-fluorobenzyl(benzyp))-tropinone-N-phenylcarbamate, 3-(p-trifluoromethyl-benzyl)-tropinone-N-phenylcarbamate, 3-(p-tert-butylbenzyl)-tropinone-N-phenylcarbamate, 3-(p-methylbenzyl)-tropinone-N-(p'-trifluoromethyl-phenyl)carbamate, and 3-(p-methylbenzyl)-tropinone-N-(p-dimethylamino-phenyl)carbamate, mesocarb, altropane, amfonelic acid, amiphenazole, BTCP, 3C-PEP, DBL-583, difluoropine, GBR-12783, GBR-12935, GBR-13069, GBR-13098, GYKI-52895, ibatropane, modafinil, amobifine, RTI-229, and vanoxerine.

[0032] One embodiment of the invention provides a pharmaceutical composition comprising a dopaminergic agonist and an adrenergic receptor antagonist for treating a neurological disease or disorder.

[0033] In another embodiment, the dopaminergic agonist is selected from, but not limited to, apomorphine, bromocriptine, cabergoline, ciladopa, dihydrexidine, Dinapsoline, Doxanthrine, epinastine, L-dopa, lisuride, pergolide, piribedil, pramipexole, propylnorapomorphine quinagolide, ropinirole, rotigotine, roxindole, Sumanirole.

[0034] In yet another embodiment, the adrenergic receptor antagonist is selected from, but not limited to, propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, metoprolol, nebivolol, esmolol, butoxamine, prazosin, terazosin, doxazosin, silodosin, alfuzosin, and tamsulosin.

[0035] In another embodiment, the dopamine reuptake inhibitor is 3-(phenylpropyl)-spirostane imine-N-phenylcarbamoyl (or mesocarb, or modafinil) and the adrenergic receptor antagonist is propranolol, or carvedilol, or prazosin, or doxazosin, or tamsulosin.

[0036] One embodiment of the present invention relates to a method of adding a combination of an adreno-blocker (such as a β-adrenergic antagonist (e.g., propranolol) and / or a β-α-adrenergic antagonist (e.g., carvedilol or labetalol)) and an agent that enhances dopaminergic activity (such as 3-(phenylpropyl)-spirostane imine-N-phenylcarbamoyl (Chen et al., U.S. Patent No. 8,415,385)) or an agent having similar pharmacological properties (such as modafinil) for controlling a neurological disease state caused by catecholamine imbalance.

[0037] In another embodiment of the present invention, the dopaminergic activity is affected by 3-(phenylpropyl)-spirostane imine-N-phenylcarbamoyl and the adrenergic activity is controlled by a combination of propranolol, or prazosin, or carvedilol.

[0038] In another embodiment of the present invention, the precise composition (and / or dosage) of the dopaminergic enhancer and the adrenergic attenuator is formulated based on the specific biomarker profile of an individual patient and / or the stage of progression of his (her) disease.

[0039] The following examples are used to illustrate certain aspects of the present disclosure and should not be construed as limiting the claims. The contents of all references, pending patent applications, and issued patents cited in this application are expressly incorporated herein by reference. In particular, the specification and associated drawings herein provide exemplary information to show the therapeutic effects of the combination of 3-(phenylpropyl)-spirostan-5-one imine-N-phenylcarbamoyl and propranolol in animal behavior, but are not intended to limit the scope of the invention.

[0040] The methods described above may further include the step of measuring the level of a biomarker in a subject in need of treatment. Also contemplated is the provision of a drug kit that contains a dopamine reuptake inhibitor or dopaminergic agonist, an adrenergic receptor antagonist, and a reagent or instrument for assessing and measuring at least one or more biomarkers.

[0041] The biomarkers for the methods and those contemplated for the kits can be any number of combinations of glucose metabolism (including metabolite profiles), lipid metabolism (including metabolite profiles), hormone profiles, cytokine profiles, or cardiac function. Glucose metabolism biomarkers can be, for example, glucose, lactate, pyruvate, the ratio of lactate to glucose, or glucose disposal rate. Lipid metabolism and profile biomarkers can be, for example, β-hydroxybutyrate, acetoacetate, high-density lipoprotein, or the disposal rate of β-hydroxybutyrate or acetoacetate. Hormone profile biomarkers can be, for example, the basal level of insulin, cortisol (basal and evening cortisol levels), and / or leptin (basal and postprandial leptin levels). Cytokine profile biomarkers can be, for example, IL-10, Il-4, IL-17, IL-23, or their respective ratios. Cardiac function biomarkers can be, for example, heart rate or heart rate variability. Examples

[0042] Example 1: Postmortem study of frontotemporal dementia (FTD)

[0043] Postmortem studies of neurodegenerative disorders, represented by frontotemporal dementia (one of the conditions commonly referred to as tauopathies), have shown that the expression level of tyrosine hydroxylase in the pons of individuals with FTD is increased, and the expression of the same enzyme in the putamen is decreased, compared to individuals without neurodegeneration. The overall profile of tyrosine hydroxylase expression in different brain regions indicates the pathophysiology of catecholamine imbalance.

[0044] Postmortem human brains, pons, and putamen (a part of the dorsal striatum) were stored at -80 °C and used for protein analysis. Tissue sections were homogenized (approx. 100 μg tissue / μL lysis buffer). Protein concentration was determined using a BCA assay (Pierce). The total protein concentration of the denatured lysates from each animal was adjusted to provide the same total protein concentration (e.g., 1 μg / μL), such that the same amount of total protein was loaded onto each lane of a precast gel (e.g., ≥5 μg / lane). Blots were developed using standard techniques and images were captured and analyzed using ImageJ. Data were expressed as relative optical density (“OD”) after normalization to the mean expression level relative to healthy controls.

[0045] As Figure 1A shown, there was a significant increasing trend in the expression of tyrosine hydroxylase in the pons of FTD patients compared to controls (p = 0.0526); conversely, the expression level of the same enzyme in the putamen of FTD patients was significantly decreased (p = 0.0258)( Figure 1B ). The locus coeruleus, a nucleus in the pons, innervates the noradrenergic state of the brain; the putamen, a part of the dorsal striatum, contributes to the dopaminergic state of the brain. The overall results of the protein analysis indicate an altered central catecholamine state, i.e., increased adrenergic activity and decreased dopaminergic activity.

[0046] Example 2: Metabolomics data of FTD patients

[0047] Metabolomic data analysis in patients with frontotemporal dementia indicates metabolic abnormalities such as increased lipid catabolism (β-hydroxybutyrate is a product of lipid β-oxidation) and increased glycolytic activity. The metabolic changes imply an altered catecholamine balance, or excessive sympathetic activity through adrenergic activation and dopaminergic insufficiency.

[0048] Medical records including metabolomic data of FTD patients and age-matched controls were extracted from the UK Biobank (UKBB). Patients were divided into 6 age cohorts (<60, 60 - 64, 65 - 69, 70 - 74, 75 - 79, and 80+). Metabolomic data were used to establish age-related patterns of lipid catabolic activity and the same activity under the influence of FTD was compared.

[0049] As Figure 2A shown, analysis of plasma levels of β-hydroxybutyrate from controls indicates a strong correlation between age and plasma β-hydroxybutyrate levels (R 2= 0.9924, p < 0.0001, Y = 0.0004622*X + 0.02688). This analysis indicates that "normal" metabolic aging involves a predictable process of increased lipolysis. At the same time, the same analysis using data from FTD patients also shows the correlation between age (or disease progression) and β-hydroxybutyrate levels within the FTD cohort (R 2 = 0.5909, p < 0.0094, Y = 0.002753*X - 0.1113).

[0050] Compared with the control, Figure 2B , (slope: F = 4.484, DFn = 1, DFd = 7, P = 0.0720)1), FTD shows accelerated changes in lipolysis related to age or progression, indicating sympathetic overactivation (adrenergic excess and dopaminergic deficiency) under FTD conditions.

[0051] As Figure 2C shown, the analysis of plasma levels of glucose and lactate from the control indicates age-dependent changes in plasma levels of lactate and glucose (R 2 = 0.8453, p = 0.0095, Y = -0.4567*X + 6.004). This analysis indicates that "normal" metabolic aging involves a predictable process of decreased glycolytic activity.

[0052] Different from healthy subjects (control), the same analysis using data from FTD patients ( Figure 2D ) shows no correlation, but the trend indicates increased glycolytic activity or glucose metabolism dysregulation related to age or disease progression.

[0053] Example 3: Study and detection of therapeutic effects in P301L (relative to SW) mice

[0054] This experiment used P301L mice (Taconic Tau-Model 2508, JNPL3 (P301L)) to simulate human FTD, tauopathy, and Alzheimer's disease (see Samaey, C. et al., Early Cognitive and Behavioral Deficits in Mouse Models for Tauopathy and Alzheimer's Disease. Front Aging Neurosci, 2019.11: p. 335. Li, M.Z. et al., Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1-amino acid-mTORC1 signaling. Mil Med Res, 2022.9(1): p. 38; Wenger, K. et al., Common mouse models of tauopathy reflect early but not late human disease. Mol Neurodegener, 2023.18(1): p. 10.). Figure 3A An experimental overview is provided. The mouse model expresses a transgenic human microtubule-associated protein tau (MAPT) P301L mutation on a mixed background (Swiss Webster and C57 / B6). During the 14th to 15th week (age), the metabolite profile and behavioral pattern seem to be different from those of control animals. The occurrence time of these observed differences is similar to the reported occurrence time of previously reported behavioral changes (Samaey et al., 2019).

[0055] First, ( Figure 3B and 3C ) to establish model relevance to human conditions (i.e., dopaminergic insufficiency and noradrenergic hyperactivity), the expression levels of the norepinephrine transporter (NET) in the mouse brainstem (BS-NET) and the dopamine transporter (DAT) in the striatum (ST-DAT) were analyzed, Figure 3A and 3B .

[0056] 15-week-old mice were sacrificed. Brain slices were isolated, frozen, and stored until analysis. The expression levels of DAT in the striatum and NET in the brainstem were analyzed using the same method described previously.

[0057] Overexpression of intracellular Tau causes mTORC1 activation (Li, M.Z. et al., Intracellular accumulation of tau inhibits autophagosome formation by activating TIA1 - amino acid - mTORC1 signaling. Mil Med Res, 2022. 9(1): p. 38). Such activation causes up - regulation of dopamine transporter (DAT) and down - regulation of norepinephrine transporter (NET) expression (Bermingham and Blakely, 2016). Increased DAT expression reduces the extracellular concentration of dopamine, and decreased NET expression increases the extracellular concentration of norepinephrine. As Figure 3B and 3C shown, P301L mice exhibit up - regulation of DAT expression in the striatum and decreased expression in the brainstem; thus, a condition of adrenergic excess and dopaminergic deficiency similar to human FTD disorder is produced.

[0058] P301L mice were divided into 2 cohorts (n = 5 per cohort), and one group of them was treated with a drug combination of ACT01 (@25 mg / kg) and propranolol (@3 mg / kg), while another group of P301L mice and age - matched SW mice were treated with vehicle as disease and healthy controls, respectively.

[0059] Therapeutic effects on animal behavior - Spontaneous activity( Figure 3D ) : Mice were placed in a previously habituated open field (box), and open - field activity was recorded at a fixed pixel resolution. The travel distance (locomotor activity) in the open field for several minutes was evaluated using a free software application (Kinovea) as the total number of pixel points crossed within one minute. Nesting behavior( Figure 3E ) : At the start of the dark cycle, nude mice (housed individually) were provided with 2×2 cotton nesting pads (nestlet) (VWR, 10279 - 140), and the therapeutic effect was evaluated by weighing the "untreated" nesting pad material.

[0060] As Figure 3D and 3E shown, enhancing dopaminergic activity by ACT01 and modulating adrenergic activity by propranolol normalize the animal behavior pattern.

[0061] Therapeutic effect on animal lipid metabolism - Sub - sequent metabolic changes analysis of male P301L mice was also characterized. This study compared the serum levels of β - hydroxybutyric acid in drug - treated, untreated P301L mice and SW mice (SW = Swiss Webster mice, the background strain of transgenic P301L mice), Figure 3FAs shown in the figure, the parallel mediation of lipid catabolic activity by the dopamine reuptake inhibitor ACT01 and the β-blocker propranolol normalizes the catecholaminergic activity.

[0062] Example 4: Study and detection of therapeutic effects in Fmr1KO mice (relative to FVB)

[0063] This study compared the learning and memory behaviors of different drug-treated and untreated (vehicle) Fmr1 KO mice with FVB mice (the background strain of transgenic Fmr1 KO mice; "FVB" refers to the susceptibility of FVB mice to Friend leukemia virus B). Male mice (FVB.129P2-Pde6b<+>Tyr <c-ch>Fmr1 <tm1cgr> / J (Inventory No.: 004624|FMR1 KO)) as an FXS / autism model and using age-matched male mice (FVB.129P2-Pde6b<+>Tyr <c-ch> / AntJ) as a control (Bernardet, M. and W.E. Crusio, Fmr1 KO mice as a possible model of autistic features. ScientificWorldJournal, 2006.6: p. 1164 - 76. Hays, S.A., K.M. Huber, and J.R.Gibson, Altered neocortical rhythmic activity states in Fmr1 KO mice are due to enhanced mGluR5 signaling and involve changes in excitatory circuitry. J Neurosci, 2011.31(40): p. 14223 - 34. Willemsen, R. and R.F. Kooy, Mouse models of fragile X - related disorders. Dis Model Mech, 2023.16(2).). Figures 4A-4D The experimental overview and results are provided.

[0064] Around the age of 5 years, individuals with fragile X present with macrocephaly, such as an enlarged putamen (Shen, M.D., et al., Subcortical Brain Development in Autism and Fragile X Syndrome: Evidence for Dynamic, Age-and Disorder-Specific Trajectories in Infancy. Am J Psychiatry, 2022. 179(8): p. 562-572.; Williams, C.A., A. Dagli, and A. Battaglia, Genetic disorders associated with macrocephaly. Am J Med Genet A, 2008. 146A(15): p. 2023-37.; and Hazlett, H.C., et al., Trajectories of early brain volume development in fragile X syndrome and autism. J Am Acad Child Adolesc Psychiatry, 2012. 51(9): p. 921-33). mTOR is a key mechanism regulating growth. Overgrowth indicates excessive mTOR activity. Tor-related mutations cause macrocephaly in ASD (Yeung, K.S., et al., Identification of mutations in the PI3K-AKT-mTOR signalling pathway in patients with macrocephaly and developmental delay and / or autism. Mol Autism, 2017. 8: p. 66.).

[0065] Activation of TOR activity has also been reported in the Fmr1 KO rodent model (Huang, W.C., Y. Chen, and D.T. Page, Hyperconnectivity of prefrontal cortex to amygdala projections in a mouse model of macrocephaly / autism syndrome. Nat Commun, 2016. 7: p. 13421. Sharma, A., et al., Dysregulation of mTOR signaling in fragile X syndrome. J Neurosci, 2010. 30(2): p. 694 - 702.). Although the relationship between TOR activity and the expression levels of different catecholamine transporters (or reuptake proteins) is well documented (Bermingham, D.P. and R.D. Blakely, Kinase - dependent Regulation of Monoamine Neurotransmitter Transporters. Pharmacol Rev, 2016. 68(4): p. 888 - 953.), to confirm catecholaminergic dysregulation, cortical expression of NET and striatal expression of DAT were analyzed, Figure 4B . Protein analysis (by Western blotting using the method described previously for Figure 1A and 1B ) of brain slices (isolated according to the experimental procedures described previously) from Fmr1 KO mice (postnatal P30) showed increased DAT expression and decreased NET expression (protein expression levels were normalized to β - actin, unlike the brains of humans and animals with neurodegeneration). The upregulated DAT decreased extracellular dopamine, while the downregulated NET increased extracellular norepinephrine. This analysis confirmed the fact that the catecholamine status was altered similarly as previously discussed.

[0066] Therapeutic effects on rodent metabolic activity: As Figure 4C As shown, these animals mimicking human fragile X show altered metabolic activities (e.g., lipids, etc.) similar to those in fragile X humans (Berry-Kravis, E. et al., Cholesterol levels in fragile X syndrome. Am J Med Genet A, 2015. 167A(2): p. 379-84.), patients with FTD, and P301L mice. Such alterations indicate excessive sympathetic activation. Enhancing dopaminergic action through ACT01 seems to attenuate sympathetic activity and normalize the metabolic activities of animals mimicking human fragile X.

[0067] Unanticipated drug combination effects on rodent learning and memory: The novel object recognition test was used to evaluate the therapeutic effects of different catecholaminergic mediations (including drug combinations) on cognitive behaviors, namely learning and memory.

[0068] As shown by the experimental protocol ( Figure 4A ), mice were weaned at P21 (21 days after birth) and then immediately started to be treated daily with the dopamine enhancer reuptake inhibitor 3-(phenylpropyl)-sterone imine-N-phenylcarbamate (or ACT01 or ACT) or the adrenergic blocker, propranolol (Pro), prazosin (Pra), carvedilol (Carv), or their respective combinations with ACT01. ACT01 was provided at 25 mg / kg by oral gavage; propranolol at 2 mg / kg; prazosin at approximately 0.8 mg / kg; and carvedilol at approximately 2 mg / kg. The vehicle was orally administered to the control group. After 2 days of treatment, the animals were subjected to the experimental protocol, with daily drug treatment for 2 hours followed by training / testing for novel object recognition: On day 1 (habituation), each animal was introduced into the middle of a plastic opaque white activity arena and allowed to freely explore for 20 min. On day 2 (sample phase), two identical objects were placed in two opposite corners, and the mice were introduced into the center of the activity arena for a 5-min training phase. Twenty-four hours later, the mice were returned to the activity arena for a 5-min test phase, where one object was replaced with a new object. Object recognition was measured using the preference index (the ratio of the time spent exploring the new object to the total time spent exploring both objects).

[0069] Studies show ( Figure 4D ) that drug combinations, such as 3-(phenylpropyl)-sterone imine-N-phenylcarbamate (ACT01 or ACT) itself have actively enhanced the cognition of animals (Fmr1-KO mice); while adrenergic blockers alone or by themselves may have a suggestive trend but no significant effect. The drug combination effect is significantly better than that of any single agent alone (p < 0.05 is considered statistically significant).

Claims

1. A pharmaceutical composition, the pharmaceutical composition comprising: a. A dopamine reuptake inhibitor; b. An adrenergic receptor antagonist; and c. A pharmaceutically acceptable carrier.

2. The pharmaceutical composition according to claim 1, wherein the dopamine reuptake inhibitor is 3-(phenylpropyl)-sparteine imine-N-phenylcarbamoyl, 3-(p-methylbenzyl)-sparteine imine-N-phenylcarbamoyl, 3-(p-carboxybenzyl)-sparteine imine-N-phenylcarbamoyl, 3-phenethyl-sparteine imine-N-phenylcarbamoyl, 3-phenethyl-sparteine imine-N-(3′,4′-dichloro-phenyl)-carbamoyl 3-(p-nitrophenethyl)-sparteine imine-N-(3′,4′-dinitro-phenyl)-carbamoyl 3-(p-fluorobenzyl)-sparteine imine-N-phenylcarbamoyl, 3-benzyl-sparteine imine-N-phenylcarbamoyl, 3-phenethyl-sparteine imine-N-(p-chlorophenyl)-carbamoyl 3-phenethyl-sparteine imine-N-(m-trifluoromethyl)-phenylcarbamoyl, 3-(3′,5′-difluorobenzyl)-sparteine imine-N-phenylcarbamoyl, 3-(m-fluorobenzyl(benzyp))-sparteine imine-N-phenylcarbamoyl, 3-(p-trifluoromethyl-benzyl)-sparteine imine-N-phenylcarbamoyl, 3-(p-tert-butylbenzyl)-sparteine imine-N-phenylcarbamoyl, 3-(p-methylbenzyl)-sparteine imine-N-(p′-trifluoromethyl-phenyl)carbamoyl and 3-(p-methylbenzyl)-sparteine imine-N-(p-dimethylamino-phenyl)carbamoyl, mesocarb, altropane, amfonelic acid, amiphenazole, BTCP, 3C-PEP, DBL-583, Difluoropine, GBR-12783, GBR-12935, GBR-13069, GBR-13098, GYKI-52895, iodobenzetrine, modafinil, amfepramone, RTI-229 or vanoxerine.

3. The pharmaceutical composition according to claim 2, wherein the dopamine reuptake inhibitor is 3-(phenylpropyl)-sparteine imine-N-phenylcarbamoyl or modafinil or mesocarb.

4. A pharmaceutical composition, the pharmaceutical composition comprising: a. A dopaminergic agonist; b. An adrenergic receptor antagonist; and c. A pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, wherein the dopaminergic agonist is apomorphine, cabergoline, dihydroergocryptine, dinapsoline, doxanthrine, epinastine (β-dihydroergocryptine), L-dopa (levodopa and L-3,4-dihydroxyphenylalanine), lisuride, piribedil, pramipexole, quinagolide, ropinirole, rotigotine, rocindole or sumanirole.

6. The pharmaceutical composition according to claim 5, wherein the dopamine agonist is L-dopa, or apomorphine, or rotigotine.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the adrenergic receptor antagonist is propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, metoprolol, nebivolol, esmolol, butoxamine, prazosin, terazosin, doxazosin, silodosin, alfuzosin or tamsulosin.

8. The pharmaceutical composition according to claim 7, wherein the adrenergic receptor antagonist is propranolol, carvedilol, prazosin, doxazosin, tamsulosin.

9. A method for treating a neurological disease, the method comprising administering to a subject in need thereof a composition according to any one of claims 1 to 8.

10. The method according to claim 9, wherein the neurological disease is Fragile X syndrome (FXS), Neurofibromatosis type I (NF1), Tuberous sclerosis (TS), Down syndrome (DS) or Autism spectrum disorder (ASD), Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD), Progressive supranuclear palsy (PSP), Alzheimer's disease (AD), Parkinson's disease (PD), Major depressive disorder (MDD), Posttraumatic stress disorder (PTSD) or Cocaine use disorder.

11. The method according to claim 10, wherein the neurological disease is Fragile X syndrome, Autism spectrum disorder (ASD), Amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD) or Alzheimer's disease (AD).

12. The method according to any one of claims 9 to 11, the method further comprising measuring a biomarker level in the subject, wherein the biomarker is: a. Glucose metabolism and metabolite profile (e.g., glucose, pyruvate, lactate), or b. Lipid metabolism and profile, or c. Hormone profile, or d. Cytokine profile, or e. Cardiac function.

13. The method according to claim 12, wherein the measuring step comprises measuring the biomarker levels of glucose metabolism and its profile, and / or lipid metabolism and its profile, and / or hormone profile (including dynamic profile), and / or cytokine profile or its ratio), or cardiac function.

14. The method according to claim 12 or 13, wherein the glucose metabolism biomarker is glucose, lactate or pyruvate, or the ratio of lactate to glucose, or glucose disposal rate.

15. The method according to any one of claims 12 to 14, wherein the lipid metabolism and profile biomarker is β-hydroxybutyric acid, or acetoacetic acid, and / or high density lipoprotein, triglyceride, or the ratio of triglyceride to β-hydroxybutyric acid, or the ratio of free fatty acid to β-hydroxybutyric acid, or the disposal rate of β-hydroxybutyric acid or acetoacetic acid.

16. The method according to any one of claims 12 to 15, wherein the metabolite profile consists of β-hydroxybutyric acid, lactate and glucose.

17. The method according to any one of claims 12 to 16, wherein the hormonal profile biomarker is cortisol (basal and evening), leptin (basal and postprandial), or cytokine profile.

18. The method according to any one of claims 12 to 17, wherein the hormonal profile biomarker is basal and / or postprandial leptin.

19. The method according to claim 16, wherein the hormonal profile biomarker is cytokine profile biomarkers IL-10, Il-4, and / or IL-17.

20. The method according to any one of claims 12 to 19, wherein the cardiac function biomarker is heart rate or heart rate variability.

Images